T-ce11 receptor associated signals that modulate lymphocyte homeostasis

Louis-martin Boucher

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Medical Biophysics

Univeristy of Toronto

O Copyright by Louis-martin Boucher (1999) National Librafy Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques

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The author retains ownership of the L'auteur conserve la propriété du copyight in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantid extracts îkom it Ni la thèse ai des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Thesis Abstract

Lymphocyte homeostasis is central to the normai functioning of immune responses. Failure of Talls and Bells in either recognizing pathogens, becoming activated, proliferating, destroying pathogens, shutting down, or eventually decreasing in numbers can lead to immune dysfunctions such as the absence of adaptive immune responses, the generation of autoimmune diseases, or the induction of lymphoproliferative diseases. Many cellular receptors function together to regulate lymphocyte homeostasis. Among these, the CD28 CO-stimulatoryreceptor is crucial to T-cell activation and proliferation. Moreover, CD28 has also ben reported to regulate T-ceii survival, a role usuaiiy associated with members of the TNF receptor supergene farnily. Similarly, some TNFR superfamily members, in addition to their role in apoptosis, have also been shown CO influence cellular activation and proliferation. In an attempt to try to understand how these receptors function together to regulate homeostasis, we have studied the activation of intracelluiar signaling pathways downstream of the CD28 and CD30 cellular receptors. The findings, presented in Chapter 2, demonstrate that CD28 activates the sphingornyelinase pathway, a pathway often associated with apoptosis, and that this activation can participate in CD28's CO-stimulatory activity. Chapter 3 presents evidence that CD30 can interact with TRAF-1, -2, and -3 survival factors, and defines the binding specificities of these intracellular signaling effectors with various TNFR superfanily members. The fourth chapter of this thesis is airned at studying the function of a lymphocyte cellular receptor called Toso. The study was done by generating and analyzing a Toso knockout mouse. This receptor is thought to inhibit lymphocyte apoptosis induced by Fas, however, Our results have so far failed to confirm a critical role for Toso in regulating Fas-induced lymphocyte killing. It is our hope that such studies will, one day, further our knowledge of lymphocyte honieostasis enough as to aiiow the design of interventions aimed at regulating specific defects in immune responses. Acknowledgments

à Marie-Françoise

Un de mes plus grand désir est qu'un jour, nos connaissances ne se limitent non seulement à la cellule, mais qu'on puisse aussi comprendre pourquoi certains être humains peuvent être poussés commettre un geste d'apoptose; comme si ce programme celIulaire &taitsi fondamental qu'il pouvait influencer un organisme entier. Si seulement, comme I'apoptose cellulaire, I'apoptose humaine pouvait avoir une raison d'être et si seulement elle était dépourvue de cicatrice. J'espère, du plus profond de mon coeur, que tu as trouve le bonheur que tu cherchais, mais j'aimerais tellement que tu sois encore ici avec nous!!!

1 wish that, one day, our understanding wili not Iimit itself to cells, but that we will also understand why some humans can be pushed to commit apoptosis, as if this ceIlular program was so fundamenta1 that it could influence entire organisrns. If only, as is the case for programmed cell death, human apoptosis could be meaningful and did not leave scars. 1 hope you have found the happiness you were looking for, but 1 wish you were still here with us!!! This thesis wuid not have been accomplished without the help of many. 1 must fmt thank my supervisor Dr. T.W.Mak for his concinuous help, support, and advice that has been pivotal to my wmpleting the work presented in this thesis. 1 am extremely grateful for ttie chance he has given me to work in his laboratory. His support has been unconditional, and for that 1 am ever grateful. I must also thank my cornmittee members Dr. P. Ohashi and Dr. J. Woodgett for their guidance throughout rny Ph.D. studies. The work generated for this thesis has also been supported by a MRC fetlowship for which 1 am very grateful. In addition to these supervisors, members of the Mak lab have ali been pivotal to rny success. Foremost, 1 must thank Drew Wakeharn, not only for his immense help throughout al1 my studies, but also for his attitude of life, which is a source of inspiration to me. I wish him ail the success in everything that iife has to offer him. Another Mak Iab member that has followed me throughout my studies and has never given up on me is Alexandra Ho. 1 owe her so much for aii her support and for her numerous words of encouragement. 1 also want to thank Sue Plyte and Emma Timms for their continuous support and for providing me with chocolate rnilk at hswhere 1 needed a lift. Thanks for everything guys! Of course 1 would not be where 1 am presently if it were not for my family, parents and sisters, to which 1 owe everything. They have supported me during difficult times, and have been at my side during moments of happiness. Never have 1 had a feeling of king let down from any member of my family. They have dways been there to support me in all aspects of my education and 1 cannot thank them enough for ail that they are to me. They have been idols for me throughout my Iife and 1 can only hope to, one day, be as good as they are. Thank you so much! Last, but not least, 1 want to thank my wife Madeleine for her unconditional love, warmth, and support. 1 appreciate her presence in my life so much because it reminds nrr what is really essential in this world. Thanks so much to al1 of you! !! Table of Contents

.. Thesis Abstract ...... u Acknowledgments ...... iv Table of Contents ...... vi List of Tables ...... xi List of Figures ...... xii List of Publications ...... xv List of Abbreviations ...... xvi

Chapter 1: Introduction...... 1 1.1 General overview ...... -2 1.1.1 Lymphocytes and antigenic receptors in adaptive immunity...... -2 1.1 -2 Co-stimulation in peripheral tolerance ...... 5 1.1.3 Lymphocyte homeostasis and AICD ...... -5 1.1.4 Thesis overview ...... 5 1.2 Antigenic signals involved in lymphocyte activation ...... 6 1.2.1 TCR structure and function ...... -6 12.2 TCR signaling ...... -7 1-3 Co-stimulatory signals ...... 17 1.3.1 The two signal hypothesis ...... 17 1.3.2 Co-stimulatory receptors ...... 18 1.3.3 Co-stimulatory signais from CD28 ...... 19 13.3.1 Intracellular signaiing downstrearn of CD28 ...... 21 1 .3.3.2 Recniitment of Itk by CD28 ...... 21 1.3.3.3 Recniitment of Grb2 by CD28...... 24 1.3.3.4 Recruitment of P13K by CD28 ...... 25 1.3.4 Co-stimulatory signals that prevent anergy ...... 25 1.4 Apoptotic sipals involved in lymphocyte homeostasis ...... 29 1.4.1 In vivo and in vitro evidence of AICD ...... 29 1.4.2 The role of the antigen and CO-stimulatoryreceptors in regulating lymphocyte AICD ...... 30 1.4.3 Antigen and CO-stimulationreceptor signaling in AICD ...... 31 1.4.4 The TNFR superfamily and its role in regulating lymphocyte AICD ...... 32 1.4.5 The apoptotic signaling machinery ...... 37 1.4.5.1 The general involvmeat of proteases in apoptosis ...... 38 1.4.5.2 Caspase structure...... 38 1.4.5.3 Caspase substrates ...... 39 1.4.5.4 The specific role of individual caspases in lymphocyte apoptosis ...... 39 1.4.5.5 Caspase regulation ...... 43 1A5.6 Activation of initiator caspases...... 43 1.4.5.7 The death receptors and their Luik to caspases ...... -44 1.4.6 Lipid mediators of apoptosis ...... 47 1A6.1 Generation of ceramide in apoptosis ...... -47 1 .4.6.2 Sphingomyelinase structure and function ...... 47 1.4.6.3 Regulation of sphingomyelinase activity ...... -48 1.4.6.4 Ceramide-induced PKC regulation in apoptosis ...... 49 1.4.6.5 Ceramide-induced PKB inhibition and BAD activation in apoptosis...... 50 1.4.6.6 Ceramide-induced phosphatase activation in apoptosis ...... 51 1.4.6.7 The role of the JNWSAPK pathway in ceramide-induced apoptosis ...... 51 1.4.6.8 Ceramide activation of the mitochondrial apoptotic machinery ... in the actwation of apoptosis ...... 52 1.4.7 The role of mitochondria in apoptosis ...... 53 1.4.7.1 Pro-apoptotic factors released by mitochondria ...... 53 1.4.7.2 Mechanism of release of pro-apoptotic factors by mitochondna ...... 54 1.4.7.3 Regdation of the release of rnitochondrial pro-apoptotic factors ...... 55 1.4.8 Caspase-independent signals in apoptosis ...... 55 1.4.9 Transcription factors involved in apoptosis ...... 56 1.4.10 Integration of the various apoptotic pathways in lymphocyte AICD ...... 57 1.5 Survival signals involved in lymphocyte homeostasis ...... 60 1.5.1 Receptors that regulate lymphocyte survival ...... 60 1.5.2 Inhibitors of DISC formation: FLIPs, ARC and Sentrin ...... 64 1S.2.1 FLIP structure and function ...... 64 1.5.3 Direct inhibitors of caspase activation: cIAPs ...... 65 1.5.3.1 cIAP structure and function ...... 65 1S.3.2 cIAP anti-apoptotic activity ...... 66 1S.3.3 cIAP regulation ...... 66 1 S.4 Proteins that link receptors to survival pathways: TRAFs ...... 67 1 54.1 TRAF structure and function ...... 67 1.5.4.2 TRAF anti-apoptotic activity ...... 69 1S.4.3 TRAF regulation ...... 71 1.5.5 Inhibitors of the mitochondnal apopototic rnehinery: the Bcl-2 family...... 7 1 155.1 Bcl-2 family structure and function ...... 71 1.5.5.2 The Bcl-2 family's anti-apoptotic activity ...... 73 1.5.5.3 Regdation of the Bcl-2 farnily members ...... 75 1.5.6 The PKB/Akt pathway of cellular survival ...... 75 1.5.6.1 PKB stucture and fûnction ...... 76 1S.6.2 PKB anti-apoptotic activity...... 76 1.5.6.3 Regdation of PKB activity ...... 77 1S.7 Transcription factors that regulate ce11 survival: the NFKB family ...... -78

1.5.7.1 NFKB structure and function ...... 79

1S.7.2 NFKB anti-apoptotic activity ...... 81

1S.7.3 NF- regulation ...... 81 1.6 The interplay between co.stimulatory. death. and survival signals ...... 84 1.7 Summary of fmdings presented in the thesis ...... 88

Chapter 2: CD28 signals through acidic sphingomyelinase ...... -90 2.1 Introduction ...... 91 2.2 Materials and Methods ...... 93 2.2.1 Cell culture and reagents ...... 93 2.2.2 Preparation of peripheral T

Chapter 3: Binding sites OC cytoplasmic effectors TRAF1. 2 and 3 on CD30 and other members of the TNF receptor superfamily ... 126 3.1 Introduction ...... -127 3.2 Materials and Methods ...... -129 3.2.1 Cells. libraries and antibodies ...... 129 3.2.2 Yeast two hybrid screen ...... 130 3. 2.3 Transfections ...... -131 3.2.4 Peptides used in binding assays ...... -132 3 .2.5 lmmunoprecipitations and binding assays ...... -132 3.3 Resuits ...... -134 3.3.1 Yeast two hybnd approach using the cytoplasmic tail of CD30 as bait ...... -134 3 .3.2 COS ceii transfections of CD30 with full length TRAF 1, 2 and 3 ...... 139 3.3.3 TRAF binding sites on CD30 ...... -140 3.3.4 Homologous binding sites in other members of the TNF receptor suprfamily ...... -150 3.4 Discussion ...... 154

Chapter 4: Toso deficiency does not alter lymphocyte sensitivity to activation-induced apoptosis ...... 158 4.1 Introduction ...... -159 4.2 Materials and Methods ...... -162 4.2.1 Cell culture and reagents ...... -162 4.2.2 Mouse genomic and cDNA library screening...... 163 4.2.3 Knockout constmct and screening of the modifieci gene ...... -163 4.2.4 Generation of gene targeted mice ...... -164 4.2.5 Northem blotting ...... 165 4.2.6 Jurkat and 7023 infection with retrovims carrying mouse Toso cDNA ...... -165 4.2.7 FACS analysis ...... -165 4.2.8 Ce11 isolation, activation and apoptosis induction ...... -165 4.2.9 Antibody measurements and sheep red blood ce11 stimulation...... 166 4.2.10 NK killing activity...... -167 4.2.1 1 In vivo T-ce11 response to SEB ...... 167 4.2.12 Cellular proliferation assays ...... 168 4.3 Results ...... 169 4.3.1 Mouse Toso cDNA isolation ...... -169 4.3.2 Generation of Toso knockout mice ...... -174 4.3.3 General status of the Toso knockout rnice ...... -179 4.3.4 Activation of the T-lymphocytes in the Toso knockout mouse ...... -179 4.3.5 Activation of B-cells and NK-cells in the Toso knockout mouse ...... 183 4.3.6 Status of various apoptosis stimuli in the Toso knockout T-lymphocytes ...... -190 4.3.7 Thymocyte apoptosis in the absence of Toso...... -200 4.3.8 The effect of the absence of Toso on the apoptotic response of activated B-ceils to Fas ...... 205 4.4 Discussion...... -209

Chapter 5: Discussion ...... -214 5.1 Summary and discussion of results ...... 215 5.1.1 The activation of acidic sphingomyelinase by CD28 ...... -215 5.1.1.1 Acidic sphingomyelinase activity and its role in proliferation versus apoptosis ...... -216 5.1.1.2 Msand sphingomyeiin in signaling ...... 218 5.1.1.3 CD28 and sphingomyelinase: Future Directions ...... 219 5.1.2 Implications of TRAFs in CD30 signaling...... -221 5.1.2.1 Interaction of CD30 with TRAFs as an explanation for CD303 reported activities ...... 221 5.1.2.2 Other CD30 binding proteins ...... 222 5.1 .2.3 Possible TRAF binding proteins ...... -223 5.1 .1.4 CD30 and TRAFs: Future Directions ...... -223 5.1.3 The role of Toso in lymphocyte AICD ...... -225 5.1.3.1 Mechanism of action of Toso ...... -225 5.1 .3.2 Toso and the expression of FLJP ...... -226 5.1.3.3 A possible role for Toso in B-cells?...... -227 5.1.3.4 Toso and FLIP: Future Directions...... -228 5.2 Concluding remarks ...... 230 References ...... -232 Chapter 3 Table 1. Pgalactosidase activity in whole protein extracts from yeast transfected with various baits and various clones of TRAFs 1, 2 and 3...... 138

Chapter 4

Table 1. Effect of Murine Toso overexpression in 7023 and Jurkat ceIl lines...... 173 List of Figures

Chapter 1 Figure 1. After receiving an antigenic signal, the T-ceiis can respond to various

CO-signalsthat will detennine celi fate...... , ...... 4 Figure 2. The TCR complex is composed of the a/p chain and a series of CD3 c hains ...... 1O Figure 3. Kinases phosphorylate ïïAM motifs to initiate the TCR signal...... 12 Figure 4. The multiple signaling pathways that are activated downstream of the TCR lead to the activation of various transcription factors that initiaîe gene expression...... , ...... 14 Figure 5. The CD28 signal activates signaling cascades chat both supplements and complements the TCR signais...... 23 Figure 6. Differences in the signalkg of anergic versus responsive T-cells...... 28 Figure 7. Fas and Fas-L upregulation during T-ceW-ceIl activation initiates the apoptotic proçess ...... 36 Figure 8. Key players in the apoptotic machinery ...... 42 Figure 9. Three pathways that can initiate the apoptotic program downstream of death receptors: direct caspase activation, activation via the rnitochondrial pathway, activation via the sphingomyelinase pathway .. . 59 Figure 10. Various survival factors function together to inhibit multiple pro-apoptotic pathways...... 62 Figure 1 1. kath receptors çan often activate both survival pathways and apoptotic pathways...... 87

Chapter 2 Figure 1. CD28 crosslinking activates acidic SMase in human T-cells...... -... 10 1 Figure 2. A-SMase activity in CD28 crosslinked Jurkat T-cells analyzed by TLC.. 104 Figure 3. SMase activity in CD28 crosslinked Jurkat T-cells analyzed by mass measurernents of ceramide and sphingomyelin...... 106 Figure 4. CD28dependent activation of acidic SMase is inhibited by endolysomotropic agents...... 108 Figure 5. CD3 and CD2 fail to activate A-SMase in resting Talls...... 1 1 1 Figure 6. Cellular response to exogenous A-SMase.. .. -...... - ...... 1 13 Figure 7. Costimulator effects of CD28 and A-SMase on NF- activation in mouse peripheral T-ceils and Jurkat T-celis ...... 116 Figure 8. Expression of human A-SMase cDNA in Jurkat T.ceils ...... 118 Figure 9 . Overexpression of acidic SMase in Jurkat ceiis lads to activation

Chaptcr 3 Figure 1. TRAF clones isolateci in the yeast two hybrid screen using the CD30 iniracellular tail as bait ...... 136 Figure 2 . TRAF1.2 and 3 coprecipitates with CD30 in transfected Cos cells and binds to the carboxyl terminal end of the CD30 tail ...... 142 Figure 3 . Deletion and alanine substitutions within the tail of CD30 binding motif defines the exact binding site for TRAFl ...... 144 Figure 4 . Deletion and alanine substitutions within the tail of CD30 binding motif defmes the exact binding site for TRAF2 ...... 146 Figure 5 . Deletion and alanine substitutions within the tail of CD30 binding motif defmes the exact binding site for TRAM ...... 148 Figure 6 . Similar binding sequences are found in the intraceilular tail of CD30. TNFRp75 and CD40 and defme the specificity of TRAF interactions .... 153

Chapter 4 Figure 1 . Isolation. characterization. and expression pattern of murine Toso ...... 171 Figure 2 . Generation of ES ceUs that have rearranged with the Toso knockout construct ...... 176 Figure 3 . Generation and identification of Toso knockout mice ...... 178 Figure 4 . In vitro T-ceU proliferation is normal in the Toso knockout mouse ...... 182 Figure 5 . In vitro B-ceii proliferation is normal in the Toso knockout mouse ...... 185 Figure 6 . The Toso knockout mouse can generate a T-helper-dependent B-cell humoral immune response ...... 187 Figure 7 . The Toso knockout mouse has nonnai NEC killing activity ...... 189 Figure 8. The T-cell Fas-dependent AICD of the Toso knockout mouse is not significantly different than wild type mice ...... 193 Figure 9 . Various protocols of AlCD fail to demonsuate a difference in the Toso knockout mouse ...... -195 Figure 10. Fas killing of T-ceiis foilowing various activation stimuli is nonnai in the Toso knoçkout mouse...... 197 Figure 1 1. PMA+Ionornycin stimulates normal FLIP expression in the Toso knockout lymphcxytes and sensitizes them equivalentely to Fas killing.. 199 Figure 12. In vivo stimulation of AICD via SEB activation of TxeUs is normal in the Toso knockout mouse...... 202 Figure 13. Induction of thymocyte death by CD3ê and Fas stimulation is not altered in the Toso knockout mice...... 204 Figure 14. Fas expression and killing in activated B-ceils is not affecteci by the lack of Toso...... 208

xiv List of Publications

1. Boucher, L.M.,K. Wiegmann, A. Futterer, K. Pfeffer, T. Machleidt, S. Schutze, T.W. Mak, and M. Kronke. 1995. CD28 Signals through Acidic Sphingornyelinase. J. Exp. Med. 18 1:2059-68.

2. Boucher, L.M., L.E.M. Marengère, Y. Lu, S. Thukral, and T.W. Mak. 1997. Binding Sites of Cytopiasmic Effectors TRAF1,2,and 3 on CD30 and Other Members of the TNF Receptor Superfamily. Biochem. Biophys. Res. Commun. 233592-600. A-SM- Acidic sphhgomyehase a.a. Amino acid Ab An tibody ADP Adenosine diphosphate Ag Antigen AICD Activation-induced ceU death AP-1 Activating protein- 1 APAF- 1 Apoptosis protease activator protein- I APC Antigen presenting ceil AS K-1 Apoptosis signal- regulating kinase ATP Adenosine triphosphate Bci Budl lymphoma BCR Bellreceptor BH domain Bci-2 homology domain BID BH3 interacting domain death agonist BIR repeat Baculovinis IAP repeat BSA Bovine serum aibumin 0 Càspase-activated deoxyribonuclease CAPK Ceramide-activated protein kinase CAPP Ceramide-activated protein phosphatase CARD Caspase recmitment domain CAT Cùloramphenicol acetyl transferase CD Cluster designation CD28R.E CD28 responsive element cdk cyclin-dependent kinase CD-L CD Ligand CED Caenorhabditis elegans ceildeath gene cIAP/HIAP cellular/fiuman inhibitor of apoptosis ConA Concanavilin A cpm counts per minute DAG diacylglycerol DBD DNA binding domain DcR Decoy receptor

xvi DD Death domain DEAE Diethylaminoethy1 DED Death effector domain DISC Death-inducing signaling complex DMEM Dulbecco's muiimal essential media DNA Deoxyribonucleic acid DR Death rezeptor EBV Epstein-Barr virus EDTA Ethylenediaminetetraacetic Acid ES cell Embryonic stem cell FACS Fluorescence-activated ceil sorter FADD FAS-associated protein with a death domain FAF FAS-associated factor FAP FAS-associated phosphatase Fas-L Fas ligand FCS Fetal calf senun FLASH FLICE-associateci huge protein F'LICE FADD-like interleukin- 1beta

IKK II& kinase

xvii IL Interleukin ILXR Lnterleukin receptor InsP3 Inositol triphosphate I.P. Intrapentoneal lTAM Immunoreceptor tyrosine-based activation motif JNK Jun N-terminal kinase kDa kiloclalton LAT Linker for activated T-cells ku kucine LIF Leukemia inhibitory factor LIM lin- i 1 isl- 1 mec-3 motif mi Latent membrane protein 1 LPS Lipopol ysaccharide rnAb monoclonal an tibody MAPK Mitogen-activated protein kinase MEKK MAP kinasdERK kinase kinase MHC Major histocompatibility complex mui minutes MKK MAP kinase kinase N-SMase Neutra1 sphingomyehase Neo Neornycin NFAT Nuclear factor of activated T-cells NFKB Nuclear factor kappa43 NIK NF-kappaB-inducing kinase NK ce11 Natwal killer ceii O.D. Optical density PBL Peripheral blood Iymp hoc ytes PBS Phosphate-buffered saline PC Phosphatidy lcholine PCR Polymerase chah teaction PH domain Plecksuin hornotogy domain PHA Phytohernaggluthin PI3 K Phosphatidylinositol3' phosphate kinase Pm Protein kinase B PKC Protein kinase C PLC Phospholipase C PMA Phorbol 12-myristate 13-acetate PMSF Phenylmethylsulfonyl fluoride PT Penneability transition PtdIns Phosphatidylinositol RAIDD RIP associated ICE/CED3 homologous protein with a Qeath domain RNA Ribonucleic acid rPm rotation per minute SAPK Stress-ac tivated protein hase SDS Sodium dodecyl sulfate SEAISEB Staphylococcus enterotoxin AB Ser Serine SHU3 domains Src homology domain 2/3 SM Sphingomyeline SMase Sphingomyelinase STREP Strep tavidin TCR T-celi receptor TF Transcription factor Th cell T-helper celi TLC Thin layer chromatography TNF Tumor necrosis factor TNFR TNF receptor TRADD TNF-R 1 -associatecl death domain protein TRAIL TNF-related apoptosis-inducing ligand Tryptophan Tyr Tyrosine UBC Ubiquitin-conjugating enzyme w Uitraviolet VDAC Voltagedependent anion channel of the rnitochondria X-gal 5-Bromo4-chloro-3- indolyl-beta-D-Galactopyranoside Chapter 1: Intraduction 1. General overview

1.1.1 Lymphocytes and antigenic receptors in adaptive immunity The immune system's activity can be divided into two major types of response: an innate and an adaptive response. Innate immunity is the fmt line of defense and is not

strictly pathogen specific. It's main function is probably to control the infection until the

adaptive immune response, mediated by T and B-lymphocytes, cm corne into play to elirninate the pathogen [l, 21. This second response is pathogen specific and is regulated by an antigen specific receptor on the surface of these lymphocytes. The specificity of these receptors is defined by a variable region in the protein that is proàuced by genomic rearratlgement of the receptor gene. Because of this, the dogrna has been that single ceiis could only express fùnctional receptors containing the same variable regions and, hence, with the same antigen binding specificity (31. Although the antigenic recognition capability of single ceUs is iimited, the enormous diversity of T-ceii and Bell receptors (TCRBCR) found among the entire population of T- and Btells allows for the specific recognition of millions of antigens. However, in order to have enough lymphocytes to mount a strong immune reaction, a selection and amplification of the appropriate clones is necessary. The recognition of the antigen by the TCWBCR is essential for T- and Bell activation and selection. However, this signa1 is insufficient for the clona1 amplification. Various studies have shown that CO-stimulatorysignals originating from other receptors on the surface of Lymphocytes, which are antigen-independent, influence the cellular response to antigen recognition (Figure 1). Figure 1. After receiving an antigenic signal, the T-cells can respond to various CO-signals that will determine cell fate. (see text for details) APC

1) "ON/OFF" antigenic signal

signals

\ "ON" Anergy 2) Functional costirnulatory signal / \ Fas + 112 cytokines

optosi

Proliferation+------Differentiatiow ------AlCD 1.1.2 Co-stimulation in peripheral tolerance The regulaîion of Tell activity by CO-stimulatory signais is crucial to the prevention of autoimmune reactions. Since genornic rearrangement of the TCR and BCR is nos directed, ceils recognizing self-antigen must be eliminated or controlied to avoid sucn reactions. It is thought that much of the elimlliaîion of autoreactive T-ceils and B-celis occurs during their development in the thymus and bone marrow, mspectively, as these immature ceUs recognize self-antigen present in these tissues. However, this mode1 does not explain the regdation of lymphocytes recognizing peripheral tissue specific antigen. Because of this, CO-stimulatory models have been proposed to explain this periphed tolerance. These models suggest that mature T-cells can only be activated by "professional" antigen presenting cells that can give a CO-stimulatorysignal. Because of this, lymphocytes would not react against antigen presented on the surface of "non- professional" peripheral tissue ceiis that do not provide this CO-stimulatorysignal.

1.1.3 Lymphocyte homeostasis and AICD In addition to the antigenic receptor signai and to the CO-stimulatorysignal, other signals are necessary to regulate lymphocyte homeostasis. Because of the clonai expansion of lymphocytes that occurs during an immune reaction, receptors must regulate the inactivation and elirnination of the.se lymphocytes in order for the immune reaction to resolve. This elirnination of activateù lymphocytes occurs by an apoptotic process caiied "activation-induced ceii death" (AICD). Abnormalities in such a processes have been attributed to various diseased States that cause autoimmunity or abnormai lyrnphoproliferation.

1.1.4 Thesis overview Of aii the receptors that have been found to regulate pathways that control lymphocyte homeostasis, two i'lieshave been shown to be pivotal. The CD28 family of receptors plays a criticai dein CO-stimulatorysignaiing and the TNFR superfamiiy is involved in lymphocyte AICD. However, definhg such definitive roles to these receptors is imprudent. In fact, CD28 has been shown to affect AICD and some TNFR family

members cm have CO-stimulatoryactivity. In an attempt to further understand common pathways that are used by various receptors to reguiate lymphocyte homeostasis, 1 have studied signaling pathways downsîream of CD28 and the TNFR superfamily member, CD30, that could explain their homeostatic properties. in addition, 1 have studied a novel protein called Toso that is thought to regulate the activity of receptors invoived in AICD. 1.2 Antigenic signals involved in lymphocyte activation

The necessity of an antigenic signal, given by the TCRBCR, in dowing lymphocytes to become responsive to various stimuli has become a dogma of the regulation of the adaptive immune system. Because of the pivotal role of such receptors, many studies have looked at signaling pathways that are activated downstrearn of these antigenic receptors. Although the TCR recognizes cognate Ag processed and exposed on antigen presenting cells whereas the BCR reçognizes soluble Ag, these studies have revealed that many of the signaiing pathways activated by the TCR have much similarities to those activated by the BCR [4].'

1.2.1 TCR structure and function

TCR signaling is a sine qua non condition to the activation of T-cell. This activation leads to the synthesis of various cytokines that regulate T-celi function and proliferation. Among these cytokines, the most studied is clearly IL-2. In fact, it has been shown that, in tem of IL-2, mature TaIl activation proceeds in orderly steps starting with the passage from the Goto the G, stage of the ceIl cycle, the secretion of IL-2, and the expression of the high affinity heterotrimeric forrn of the IL-2 receptors. The binding of

' This thesis is airned primarily at the coiitrol of T-ceIl homcostasis. Although the fourth chapter also looks at some aspects of B-cell proliferation and AICD, BCR signaling will not bc extcnsively mentioncd in this thesis. A revicw of BCR signaling can be found in [SI. IL-2 to the IL-SR then stimulates tbe passage of ce& fiom Gl into the S phase of the ceil cycle and their proliferation [6]. Such events are al1 initiated and regulated by the antigenic signals given by the TCR.

There are two types of TCRs, each composed of huo chains: either an ap or a y6 heterodimer 17-91.2 These TCR proteins have an immunoglobulin-like extracellular domain, a transmembrane dornain, and a short 5-12 a.a. intracellular tail. The TCR recognizes cognate antigen processed into peptides and presented on specific proteins belonging to the major histocornpatibility complex (MHC) [4]. The antigenic specificity of the TCR recognition is mediated by variable regions in the Tg like extracellular dornain of the TCR that contact both the specifk MHC molecule as well as the peptide containhg groove of the MHC [IO-121. Presentation of peptides, to T-cells, via the MHC molecules is done by Ag presenting celis (APCs). Although the recognition of the cognate antigen on APCs is critical to tbe antigenic signai, other CO-receptors,that do not modify antigenic specificity, also appear to play a role in generating the TCR signal [ 13, 141. Of these, the CD4 and CD8 coreceptors have been show to defme the class of MHC molecules that the T-ce11 will recognize and to be essential for TCR signaling.

1.2.2 TCR signaling It is the non-covalent interaction of the TCR receptors with a family of polypeptide chains that comprise the invariant CD3 complex and include CD~E,CD3-y. CD36, CD35 and the CD36 splice variant CD3q [15, 161, that ailows the TCR signal to activate downstream signaling pathways [17, 181. In fact, the TCR can be viewed as a complex consisting of four dirners, an aB disulfide-linked TCR heterodimer, two non-lovalent CD3 dimers composed of and 6ê, and a disuifide linked homodimer of 5 or a heterodimer

Thc information and studies that arc given in this thesis are restrictcd to ap TCR expressing T-cclls. [19, 201 (Figure 2). It has been demonstrated that the TCR signal is Iliitiated by the phosphorylation of immunoreceptor tyrosine-based activation motifs (ïïAM) m22] found in the CD3 intracellular tails [23]. The phosphorylation of iTAMs is mediated by intraceMar tyrosine kinases of which Lck and Fyn appear to be centra1 1241. Localization of these kinases to the TCR is thought to be mediated by interaction with proteins that fonn the TCR complex. Fyn cm interact with the CD3r chain [25] and Lck interacts with the CO-mptors CW and CD8

[26]. These CD4 and CD8 coreceptors are found to be mutually exclusive on mature T- cells, and, by interacting with class II or class I MHC molecules [27] respectively, are thought to recruit Lck to the TCR complex 126,281 (Figure 3).

Upon phosphorylation of the CD3c chains. a kinase dedZAP-70 is recruited to the phosphorylated tyrosine residues in the ïïAMs via SH2 domains [22, 29, 301. The importance of ZN-70 in TCR signaling is underlined by naturally occuring mutations of

ZAP-70 in hurnans which 1eads to severe defects in TCR signaling [31-331. A similar defect in TCR signaling was identified in the ZAP-70 knockout mouse [34]. Upon interacting with the TCR complex, ZAP-70is activated by tyrosine phosphorylation events, probably also mediated by the Lck kinase [35] (Figure 3). Active ZAP-70 then proceeds to phosphorylate various adapter proteins, such as LAT and SLP-76, which act as docking proteins for the various pathway initiators that are activated by the TCR signal (Figure 4). Figure 2. The TCR complex is composed of the a@ chah and a series of CD3 chahs. The CD3 chah contain lTAM motifs (identifiai by 0) that need to be phosphorylated to initiate the signaling cascade. Kinases such as Lck and Fyn are thought to participate in this phosphorylation (see text for details). APC Figure 3. Kinases phosphorylate ITAM motifs to initiate the TCR signal. The activation of the the TCR signal is initiated by the phosphorylation ( P ) of iTAM motifs ( 1) by kinases such as Lck and Fyn. These phosphorylated KAM motifs then recruit SH2 containing proteins such as the Zap7O kinase (see text for details). APC Figure 4. The multiple signaling pathways that are activated downstream of the TCR lead to the activation of various transcription factors that initiate gene expression. Multiple phosphorylations ( P ) lead to the recniitment of adaptor proteins thaî activate various signaling pathways incluàing the MAPK, the SAPKIJNK, the NF-, and the NFAT pathway. These signaling pathways uitimately regulate the transcription of genes important in T-ce11 response such as IL2 (see text for details).

Phosphorylated LAT has been shown to recruit pathways that regdate intracellular ca2+and mitogen-activated protein kinase (MAPK) activity by interacting with PLCp This regulation is due to PLCy's ability to hyrolyze phosphatidylinositol into diacylglycerol

@AG) and inositoi phosphate (InsP3) [36]. DAG is responsible for the activation of the

PKC family of kinases [24] which can activate the MAPK pathway via the regulation of both Ras and Raf [25, 37, 381. InsP3 is responsible for the rise in intraceiiular Ca2'[24,

391 which induces the activation of the caicineurin phosphatase by caimodlin [40, 4 11. Active caicineurin dephosphorylates the nuclear factor of activated Tcells (N'FAT) fady of transcription factors which can then rnigrate to the nucleus and promote the transcription of genes such as IL-2 [42,43] (Figure 4). Phosphorylated LAT also binds to the p8S subunit of the class 1A of PI3Ks [44, 453. This regulatory p85 subunit then recruits the catalytic pl 10 subunit of PI3K to the membrane where it cm be activated by Lck [46] to phosphory!ate its lipid substrates PtdIns(4)P and PtdIns(4,5)P2 into PtdIns(3,4)P, and PtdIns(3,4,5)P, [47] (Figure 4). In addition, PI3K cm be activated by the direct interaction of GTP bound Ras, involveci in the MAPK pathway, with the catalytic pl 10 subunit [48,49]. The generation of phosphorylated Lipids by PI3K allows the recruitment of various PH domain containing proteins to the cytoplasmic membrane. Such proteins include the Rac-guanine nucieotide exchange factor (GEF) proteins: Vav and TIAM-1 150-531. These

GEFs promotes the formation of the active GTP bound form of Rac that can activate the

MEKK proteins. The MEKKs are the upstrearn kinases that start the phosphorylation cascade which utimately leads to INK/SAPK phosphorylation and activation [SO, 54-57]. Regulation of Vav GEF activity can be mediated by Lck [57] or Fyn [57, 581, as well as the Itk kinase [59] which contains a PH domain and is also recruited to P13K products

[GO]. In addition to regulating the JNK pathway, P13K can induce the activation of îhe MAPK pathway via recruitment of the SOSGEF and its activation of Ras [61]. The MAPK pathway can also be activated by PI3K via riecniitment of PLCy [62] leading to the activation of PKC, as mentioned above. Altematively, PUK products might participate in the pro-survivai activity of the TCR signal by activating the PKB protein kinase (see section 1.5.6).

Further activation of the MAPK pathway by the TCR signal can be accomplished by the recruitment of the SOS GEF via binding to Grb2 which interacts with the phosphorylated LAT protein [44, 633. Upon binding to Grb2, SOS can stimulate the modification of GDP bound Ras to its active GTP bound form which functions to âctivate the Raf kinase by translocating it to the membrane. Raf is an upstream kinase that initiates the cascade of kinase phosphorylation events that ultimately regulate MAPK activity [64,

651. In addition to recruiting the MAPK pathway, Grb2, and the Grb2 associated protein Gads, can recruit the SLP-76 adapter protein [66-701. SLP-76 is phosphorylated by ZAP-

70 and is thought to function in TCR signaling by participating in the activation of PLCy

17 1, 721 as well as Vav [57,73-751(Figure 4).

The activation of these pathways by the TCR signals appears to converge towards the regdation of various transcription factors (TFs) that are important in the production of cytokines such as IL-2. The MAPK pathway ultirnately leads to the phosphorylation and activation of the Eikl transcription factor which upregulates the expression of the c-Fos TF [6,76,77].The JNWSAPK pathway induces the phosphorylation and activation of the c-

Jun TF [78, 791. ca2' mobilization leads to activation of the NFAT TF (see above). NFAT and the AP-1 TFs, composed of c-Jun and c-Fos heterodimers [80], bind to the IL- 2 promoter and function cooperatively to upregulate L-2 expression 181-831. However, the signals generated by the TCR are insufficient by themselves to generate an appropnate IL-2 transcriptional response in T-cells. For an appropriate IL2 production response to occur, CO-stimulatorysignais have been shown to be necessary. Lymphocytes can respond ic various ways to antigenic stimulation. They can get activated and either go on to proliferate, to differentiate, to becorne non-responsive, or to undergo ceil death (Figure 1). Although the TCRs/BCRs give the antigenic specifkity to the activation of the lymphocytes, the different responses that lymphocytes can have following antigenic stimulation appear to be reguiated by CO-stimulatoryreceptors. The importance of both tbe initial TCR/BCR signal, as weU as the cestimulatory signals, is now a dogma of lymphocyte regulation.

1.3.1 The two signal hypothesis The modulation of the response of lymphocytes to antigen recognition was long thought to be crucial in preventing the auto-immune reaction of Tells against self antigen. In 1970, Bretscher and Cohn suggested that T-ceils needed two signais to become activated, as a mechanism to prevent auto-immune reactivity 1841. This dogma of T-ceU activation has now been extensively demonstrated by stimulating the TCR/CD3 complex in the absence of APCs' CO-stimulatorysignals 185, 861. This stimulation was shown to induce a state of T£eU non-responsiveness termed anergy [86]. The frnding that addition of APCs that could not stimulate the TCR into the culture rescued the anergy [87], suggested that CO-stimulation by other receptors was independent of TCR signals.

Furthemore it was found that the co-stimulatory signais could only be given by a select group of ceils including activated B-cells, dendritic cells and macrophages, cded

"professionai" APCs [14]. Such a mode1 offered an explanation for the lack of reactivity of T-cells to self-antigen presented by peripheral tissues, which are composed mostiy of non- professional APCs and would therefore stimulate T

Further support for the two signal mode1 was obtained by the discovery of the CD28 receptor. This receptor is unable to activaîe T-cells on its own, but can prevent

TCR-induced anergy and can enhance the TCR-induced proliferation and cytokine production [89]. Such characteristics satisfy the criteria for a CO-stimulatorysignal. Very recently, a novel receptor that is stmcturaliy similar to CD28 has been identified. This inducible costimulator (TCOS) protein also appears to have CO-stimulatoryactivity in fhat it can enhance antigenic-induced Tell proliferation and secretion of lymphokines. However, whereas CD28 promotes pnmarily the secretion of IL-2, which plays a role in T- ceil proliferation, ICOS appears to stimulate the expresion of IL-IO, which plays a role in B-ce11 differentiation [go]. Although CD28 is the prototypic costimulator receptor of T-cells, there is evidence that members of the TNF receptor superfamily cm also function in CO-stimulatory pathways. This family of receptors is defined by a series of specific repetitive cysteine motifs in their extracellular region which are crucial for Ligand-receptor interactions [91]. Although some rnembers contain a death domain in thek intraceilular tail (see section 1.4.5.7), most of the CO-stimulatory properties of this family of receptors have been associated with proteins that do not contain such a domain. This group include NGFR

(nerve growth factor receptor) p75, ATAR, TR2, RANK, CD27, CD30, CD40, 4- lB8,

0x40, and GITR [92]. Co-stimulatory properties have been assigned to the 4- 1BB protein in T-cells and to the CD40 protein in B-ceils. In fact, 4-1BB receptor binds to a ligand found on "professional" APC and appears to mediate Teil CO-stimulation, especially of CDS' T-cells, since it can upregulate proliferation and IL-2 production in the absence of CD28 activation [93-961. In addition, 4-1BB may function to lower the threshold of CD28 signaling that is necessary to sustain IL2 production and proliferation [97].However, its role in CO-stimulationof resting T-ceiis is uncertain since its expression is limited to activated T-cells [93,98]. In B-cells, CD40 is considered to be a pNnary CO-stimulatory receptor. In fact the joint activation of the BCR with CD40 cm inhibit Beli AICD [99, 1001. CD40 has also been shown to promote proliferation, differentiation, generation of rnemory B-cells, and isotype switching, depending on the stage of Bail developrnent. CD40 is also necessary for the appropriate T-ceU-dependent Bali activation, since mice lacking expression of either the CD40 on B-cells or the CWLon Tells faü to develop a TeMependent B- ce11 immune responses [10 I]. It is likely that there are links between the CD28 and TNFR superfamily pathways. in fact, CD40 stimulation of B-cells has been shown to mediate the upregulation of the

CD28 ligands [IOl]. In support of this, mice carrying nuil mutations in the CD28 gece have a defect in germinal center formation that is similar to that found in the CD40 knockout mice [102]. Although TNFR superfamiiy mernbers can have CO-stimulatcry activity, it is not clear that their activity is always tnily that of a CO-stimulatoryreceptor. In fact, CD40 cm activate B-ce11 proliferation in the absence of the BCR signal [103]. Instead, the TNFR superfamily mernbers probably have a more important role in controling homeostasis by promoting cellular differentiation, cellular survival, or cellular apoptosis.

1.3.3 Co-stimulatory signais from CD28

CD28 is a cellular receptor, belonging to the Ig supergene family, that is present as a homodimer on the surface of immature and mahue Taells [ 1043. It is also upregulated foIlowing TCR/CD3 aggregation [105]. The CO-stimulatoryeffect of CD28 appears to be mediated by its interaction with two ligands, B7-1 (CD80) [106, 1071 and B7-2 (CD86)

[log, 1091, that are expressed on "professional" APCs 1141. The finding that both the B7-

1/B7-2double knockout mice and the CD28 deficient mice faii to mount a proper Teil response to antigen, causing a defect in B-cell differentiation to IgG secreting cells, underlines the importance of these Ligands in mediating the co-stimulatory signal [1 10-

1131. It is thought that most of the pro-survival and CO-stimulatory effects of CD28 are attributable to its enhancement of the production of various cytokines such as L-3, iL-4,

IL-5, IL-9,IL- 13, TNFa, GM-CSF (granulocyte/macrophage colony stimulahg factor),

IFNy, and especiaily IL-2 186, 1 14- 1201. In fact, one of the primary findings following antigenic CO-stimulationby CD28 is the enhancement of IL-2 production [12 11. The upregulation of IL-2 is thought to occur via the activation of various transcription factors (TFs) that bind to the IL-2 promoter and drive the transcription of the IL2 rnRNA. At least one TF binding site in the CD28 promoter, the CD28 responsive eiement (CD28R.E)- has been found to be activated in a CD284ependent manner. This transcription factor appears to upregulate E-2 mRNA transcription by interacting with the

NF- family memkr c-Re1 11221. Other TFs, such as NFKB, NFAT, c-Fos, and c-Jun, that are also activated by the TCR signals, can be further activated by CD28 [ID-1251.

This may be achieved by CD28's activation of simiiar pathways to the TCR, such as the MAPK and the SAPK pathways [122, 126-1291, as well as through different pathways such as CD28's cyclosporin-A-independent NFAT activation [ 1 301 (Figure 5). In addition to increasing the transcription of IL-2 mRNA, one of the main mechanisms by which CD28 might increase IL-2 production is via the stabilization of the

IL-2 RNA message [ 1201. Furthemore, CD28 can increase the cellular response to IL-2 by upregulating the expression of the a and P chahs of the IL-2 receptor 113 1- 1331.

Because there is evidence that L-2 can promote T-ce11 proliferation as well as protect naive or recently activated Tells from apoptosis 1134- 1361, the reguiation of the L-2 response by CD28 appears to be a primary mechanism of action by which this coreceptor can mediate its CO-stimulatory effect on T-cells. However, this rnight not be the only mechanism of action of CD28. In fact, CD28 can also upregulate the expression of several adhesion molecules that strengthen the T4:APC interaction 11371 ant promote œll survival [ 138, 1391. In addition, although the upregulation of IL2 by CD28 might explain its proliferative activity, it does not explain the anti-anergic effect of CD28 which has been shown to be IL-2-independent. 1.3.3.1 Intracellular signaling downstream of CD28

Signaling by CD28 is at least partly mediated by its tyrosine phosphorylation. in fact, aggregation of CD28 induces its phosphorylation and the subsequent phosphorylation of intracelluiar proteins including LAT, Dok, Vav, and Itk (59, 140-1451. 1.3.3.2 Recruitment of Itk by CD28 The Itk kinase can bind directly to CD28 via interaction of its SH3 domain with a proline nch sequence in the CD28 intraceilular tail [141, 1461. This leads to the phosphorylation and activation of Itk, possibly by L.ck [146-1481. in addition, Itk can be activated by its recruitment to PI3K phosphorylated lipid products via its PH domain (se section 1.2.2) [6O]. It is thought that Itk is at least partly responsible for the phosphorylation and activation of Vav by the CD28 protein [59, 149, 1501. Such a signal could complement the TCR signals (see section 1.2.2) (Figure 5). In addition to Itk, the WW domain of YAP (Yes-associated protein) can bind to CD28's proline rich domains, however, the role of such interaction is still unclear [ 15 11. Figure S. The CD28 signal activates signaling cascades that both supplements and complernents the TCR signals. The CD28 signai adds to the stimuli of the TCR by Meractivaihg the MAPK, SAPK, NFAT and NFKB pathways, but also adds unique signals such as those leading to the activation of the CD28RE transcription factor. These CD28 signals allow efficient IL2 production by T-ceiis (see text for details).

1.3.3.3 Recruitment of Grb2 by CD28 Other than proline rich regions in the CD28 tail, the phosphorylation of CD28 also appears to enable the recniitment of signaling effectors. CD28 is tyrosine phosphorylated on a YMNM motif in its iniracellular tail and this allows the binding of the Grb2 adapter protein [152-1541. The importance of this interaction has been suggested by the expression of CD28 proteins that carry mutations of the N residue in the YMNM motif and fails to

bind Grb2, but binds the p85 subunit of PI3K eficiently. These mutants have reduced IL- 2 production in response to CO-stimulation [149]. However, it is not clear whether the inhibition of IL2 production by these mutants is only due to the abolition of Grb2 binding to the phosphorylated tyrosine residue of CD28 since mutations of the Y residue in the YMNM motif do not dramaticaiiy affect the increase in IL-2 production induced by CD28 [155]. This suggests that another protein might interact with this site in a phosphotyrosine- independent manner. Alternatively, the Grb2 protein has been shown to bind to CD28's proline rich regions via its SH3 domain and to recruit the adapter protein Shc via its SH2 domain 115 11. The recruitment of Shc suggests a mechanism by which CD28 could activate the MAPK

pathway by binding of Shc to the SOS GEF which functions to activate Ras (se section

1-2.2). However, it is unclear whether such a signaling pathway is active since, although the anti-CD28 antibody can stimulate Ras activation 1149, 1531, CD28's natural ligand B7-

1 cannot 11561. A more physiologicai role of Grb2 recmitment to the CD28 receptor is probably to activate of the JNWSAPK pathway via Vav recruitment and phosphorylation [149](Figure 5). In fact, it fias been suggested that during antigenic stimulation the TCR signal mainly activates the MAPK pathways whereas the CD28 signal mainly activates the INWSAPK pathway 11291. 1.3.3.4 Recmitment of PI3K by CD28 In addition to Grb2, the phosphorylated YMNM motif of CD28 has ken found to associate with the SH2 domain of the p85 regdatory subunit of PI3K (Figure 5) [152- 1541. Interestuigly the binding of PI3K to this motif has been shown to occur only in CWT-cells. Because CD28 activation offI3K is only additive to that of the TCR signal [157], it is thought that the role of CD28 signaling in PI3K activation might rather be to prolong the PI3K activation rather than to increase its activity dramatidy [158]. This is consistent with various reports demotutrathg the importance of sustained signaiing in T- ce11 activation (1591. Although CD28 activation of PI3K is thought to play a role in CD283 functions, it is unclear whether it plays a role in the CO-stimulatory increase in K-2 production. Some reports have shown that mutaiecl fonns of CD28, that can no longer bind PI3K, block antigenic-induced IL-2 secretion in Tadl hybrïdornas [ 160, 16 11. However, other investigators have shown opposite results in Jurkat ceils 1155, 158, 162, 1631. Furtherrnore, the transfection of dominant negative forms of PI3K have been shown to enhance NFAT-dependen t transcription in Jurkat cells, suggesting an inhibitory role for PI3K [164]. Because of such controversy, it is difficult to derive a final conclusion about the importance of P13K in CO-stimulatory-induced IL-2 response. Alternatively, it is possible that the role of PI3K activation by CD28 is to regulate ceU survival via the activation of PKi3 (se section 1.5.6), rather than the regdation of IL-2 production.

1.3.4 Co-stimulatory signals that prevent anergy One of the key role of CO-sumulatory signals is to prevent T-ceU anergy. It is unclear why this occurs, however it is known that anergic cells have a block in MAPK and JNWSAPK activation [126, 1271. Initial evidence suggested that the difference between anergic cells and responsive cells might be that anergic cells express an IL-2 promoter repressor 11651, or that they preferentially express the Fyn kinase, instead of Lck and ZAP-70, bound to their CD3c chah [166]. However these results have fded to My explain why anergy occurs.

Recent advanes in understanding anergy has corne from the study of CD28's regulation of Cbl phosphorylation. In aaergic cells, Cbl is constitutively phosphorylated and there is no increase in phosphorylation following TCR stimulation [167]. Phosphorylated Cbl has been shown to bind to the CrkL adapter [168- 1711 which can recmit the C3G GEF that acts as a guanine exchange factor to activate the Rap 1 GTPase

[ 172, 1731. This GTPase is found in an activated GTP bound state in anergic T-cells, but is only poorly activated in responsive ceUs [167]. Furthemore, its overexpression in Txelis inhibits the antigenic- and co-stimulatoty-induced IL2 production [167]. In addition, anti- CD28 can inhibit the activation of Rap 1 by preventing constitutive phosphorylation of CbI

[ 1741. it is thought that Rap 1 functions by sequestering Raf which would inhibit MAPK activation. Alternatively, Cbl rnight direc tly inhibit TCR signaling by sequestering multiple pathway activators such as Grb2, Src, Fyn, PLCy, Nck, PI3K and 3BP2 [15 1, 169, 170,

175- 1803 (Figure 6). Figure 6. Differences in the signaling of anergic versus responsive T- cells. Anergic cells have multiple signals that inhibit TCR signaling. Among these, Cbl appears to play a role in preventing activation signals and the activation of an IL2 repressor may also function to block IL2 expression. The CD28 signal can block these inhibitory pathways (see text for deuils).

1.4 Apoptotic signals involved in lymphocyte homeosfasis

As mentioned previously, both the antigenic and co-stimulatory signals are crucial to the clonal expansion chat is necessary to mount an immune response. However, to maintain lymphocyte homeostasis and prevent the accumulaiion of reactive ceiis, these ceUs must eventuaiiy be inactivated and elirninated. It was not uatd the late 1980's that researchers started to realize that in certain circumstances, stimulating the TCR/BCR could induce the lymphocytes to die. This phenomenon was caiied "activation-induced ceil deatii"

(AICD). Ashwell et al. were the fmt to describe this phenomenon in T-ceil hybridomas expressing a specific TCR 118 11. This ceIl death was found to be apoptotic in nature 1182- 1851 and could be inhibited by various treatments including cyclosporin-A, the ca2+ ionophore A23 187, the phorbol ester PMA, anti-CD3fïCR antibodies, anti-Thy- 1 antibodies, and anti-Ly-6 antibodies 1182- 1853. Simîlar findings were obtained in some B- cells foiiowing ligation of their BCR with antibodies [186-1891.

1.4.1 In vivo and in vitro evidence of AICD

Various fonns of AICD have been shown to occur in vivo. It has been demonstrated that viruses, by activating T4lresponses, can increase their sensitivity to AICD [190]. In fact such a rnechanism has been evoked as an explanation for the TuAl depletion that is seen in AIDS [191, 1921. More specific experiments ai& at studying in vivo AICD have been done by injecting miœ with StaphyIococcal enterotoxins. S~aphylococcalenterotoxins are superantigens that can bind the MHC proteins to specific vp chains of the TCR complex independent of the antigenic specificity of the TCR. This process lads to activation and proliferation followed by a gradua1 apoptotic deletion of the vp specific Tcells [193]. SirniIar cddeletion occurs in TCR and BCR transgenic mice injected with the TCiUBCR specific antigen [LW-1961. Considerable information about the mechankm by which mature lymphocytes are

eliminated has also been gaineci from in vitro studies of AICD. Most protocols of in vitro AlCD consist of a primary antigenic activation of the lymphocyte followed by a second antigenic stimulation which induces the apoptosis. However, various methods have yielded different concIusions. Some reports have suggested that the Fas protein is crucial

[ 197-2001 whereas others have irnplicated TNF [ 195, 20 11 and perforin 12021 (see section

1.4.4). Similar Bell activation protocols have also pointed to a role for Fas in Bell AICD [203]. These types of studies have been pivotai in the discovery of various receptors that are important in detemiining the fate of lymphocytes.

The balance between ceIl survival signals and death inducing signais probably

determines such regulation of cdfate. Although survival signals can be constitutively present in ceUs, death signais, as weii as further pro-survival signds, can be upregulated or downregulated upon cellular stimulation by various receptors including TCR/BCR, CO- stimulatory receptors, and receptors belonging to the TNFR superfamdy.

1.4.2 The role of the antigen and CO-stimulatoryreceptors in regulating lymphocyte AICD Although the prototypicai receptors that regdate AICD are thought to belong to the TNFR superfamily, TCR signals have aiso been directly associated with AICD. This is especidy true of immature thymocytes, but also of mature lymphocytes. Research in the last few years has led to the dogma that the decision of a thyrnocyte to die (negative selection) or to survive (positive selection) is determined by the avidity of the TCR for the

Ag-MHC complex [204]. Immature B-cells in the bone marrow have also been shown to be sensitive to excessive IgM stimulation [205,2û6]. Furthemore, CO-stimulatorysignals, such as CD28, can also prornote AICD when ceLls are activated. In this respect, CD28 has ben shown to render mature T-cells more sensitive to Fas-induced apoptosis [207], possibly by upregulating the expression of the pro-apoptotic proteins Bcl-Xs and BAD (see section 1.5.5.1) 12071. CD28 rnight also mediate its effect indirectiy by uptegulating the expression of IL-2. In fact, although L-2 has been associated with the survival of naive and recentIy activate T-cells, it has been shown to mediate the increased sensitivity to AICD of prolonged activated T-celis [13+

136, 2081. Consistent with this finding, IL-2 and IL-2 receptor-a icnockout mice have defective T-lymphocyte AICD and suffer from a lymphoproliferative disease [209,210] .

1.4.3 Antigen and co-stimulation receptor signaling in AICD Death signals can be viewed to act in one of two fashions: the promotion and activation of apoptotic pathways or the inhibition of pathways that block apoptosis. Evidence is mounting that both processes are probably stimulateci during death signaling that promotes AICD. Although no pathway has yet been clearly shown to directly link the antigenic rece p tors to the apoptotic mac hinery , some possibilities have been proposed including the activation of kinases. One report has shown that the CD3 chahs are less phosphorylated in cells that undergo apoptosis [211]. In addition, B-celi lymphomas susceptible to IgM- stimulateci growth arrest and apoptosis were shown to have higher activity levels of a member of the src-family of kinases called p5~B'k[212], which, when inhibited, could block AICD [2 131. In T-cells, the ZAP-70 and Lck kinases have been found to promote Fas-L expression and to be important in mediating AICD [2 L4,2151.

In addition to kinases, AICD has been associated with increases in intraceliular Ca2+

[184, 2 16, 2 171. In support of such a link, the NFAT transcription factor, activated by Ca2+ signaling, has been shown to induce the expression of genes that can promote apoptosis such as Fas-L [218] and IL2 [208, 2191. Also, a recent report dernonstrated that activation of calcineurin by Ca2+leads to the dephosphorylation of BAD and its subsequent interaction with Bcl-XI, thereby promoting apoptosis (see section 1.5.5.3) [220]. 1.4.4 The TNFR superfamily and its role in regulating lymphocyte AICD Among alI the cellular receptors that have been shown to influence lymphocyte survival and death, none have been more extensively associated with lymphocyte homeostasis than the TNFR superfamily. This superfady can be divided into two groups baseci on the presence of a death domain in their intracellular tail. Death domain containhg proteins have al1 been associated with some fom of apoptotic activity. They include Fas, TNFRp55, DR3, DR4, and DM. The second group does not have a d& domain and has been associated with a variety of cellular functions such as CO-stimulation,proliferation, and differentiation, as well as apoptosis. Consistent with the role of TNFR superfamily members in modulating AICD, TCR antigenic stimulation has been shown to upregulate some of these receptors including Fas, Fas-L, TNF,CD40L, CD30L and CD30. Among these, Fas appears to be essential to the process of AICD [197-2001. The lymphoproliferative and autoimmune diseases that develop in humans and mice that cary mutations in the fas gene have offered support to this view 192, 22 1-2261. More specifically, T-lymphocytes from rnice carrying Fas signaling mutations have been shown to be partly resistant to AICD in vivo and in vitro

[197, 227, 2281. However, the role of Fas in T-lymphocyte AICD may not be universal since some reports have shown that CD8' T-cells activated in vivo by a viral infection are not regulated by Fas but are efficiently clonally down-regulated 1229,2301. in this respect, some studies have showed that while Fas is crucial for CWT-ceIl AICD 1198-201, 23 1, 2321, TNF rnight be more important in CD8' MCD [201]. In fact, the remaining AICD sensitivity that is found in the CDS' T-cells of Fas mutant mice can be abolished by the use of anti-TNFa antibodies [20 11. Surprisingly, although TNF-induced apoptosis as been mostly attributed to the death domain containhg TNFRp55, it appears that this deletion of CD8' ceiis occurs primarily via TNFRp75. Furthemore, consistent with the fmding that some deletions of mature lymphocytes are independent of Fas or TNF [194,230,233], CD30 has also been shown to induce Fas- independent AICD of some T-ceil hybndomas [234]. In addition to these receptors, DR4 and DR5 have also been associated with AICD [235, 2361. However the lack of a iymphoproliferative disease in both the TNFRs 1237-2391 and the CD30 knockout mice

[240], such as is seen in the Fas knockout mouse, suggests that there may be redundancy between the lymphocytic apoptotic responses induced by the various members of the TNFR superfamily. So far, only Fas signals appear to be poorly compensated by other family members. This is not to say that the other receptors do not play a role in AICD, especially in cases where Fas kiiiing is more limited, such as in CDS+ cells. In fact the breeding of the lpr mouse into a TNFR~~S"background leads to an enhanced

1yphoproli ferative disease [24 11. In addition, the importance of these receptors in AICD might be ceii specific. In fact, although Fas is important in mature T-lymphocytes AICD, it does not appear to play a critical role in the AICD of negatively selecting thymocytes [242, 2431. This is however somewhat still in dispute due to the publication of some reports suggesting a role for Fas in thymocyte development when these celis are stùnulated with high dose antigen [244]. In contrast, the CD30 and CD40L knockout miœ have shown a clear defect in negative selection using TCR transgenic models that failed to reveal a role for Fas in thymocyte negative selection, suggesting a more important role for these receptors in activation- induced negative selection of thymocytes [240, 2451. However, the role of TNFR superfamily members in regulating immature lymphocyte selection is still very controversial and rnight only be involved in a srnaii proportion of selection since none of the knockouts of the TNFR superfamily rnembers have shown a gross defect in lymphoid development 12461. In B-cells, both CD4û and Fas appears to contribute in controUing AICD [203]. In fact, self-reactive B-ceiis stimulated by their cognate antigen undergo ceii death upon contact with peripheral CD4' Tceiis in a Fas- and CD40Ldependent fashion [247]. Since

B-ceiis don't express Fas-L [248], the induction of Fas mediated Bddeath is probably mediateà by the expression of Fas-ligand on the surface of tbe activated CD4' ceils [249]. Hence Bell survival also appears to be tightly regdated to their activation as weii as to the activation of T-ceils (Figure 7). In addition to being effectors of AICD, some of the TNFR superfamily members have aiso ben associateci with a regdation of the ceii sensitivity to the apoptotic response stimulated by other members. In fact both CD40 and CD30 have been shown to increase sensitivity to Fas and to TNFR-induced apoptosis [247, 2501. Interestingly, for CD40, this might be dependent on the. TCR signaling since this effect has been shown to occur only in the absence of the BCR signal [2511. The importance of the TNFR superfamily, and especially of the death receptors, in regulating lymphocyte apoptosis has been clearly established in the last few years by the identification of signaling pathways that lin.these receptors directly to the proteases that constitute the core of the apoptotic machinery. Figure 7. Fas and Fas-L upregulation during T-cetUB-cell activation activate the apoptotic process. Upon activation of Tells by APCs, such as B-cells, and upon activation of Beils via antigen binding to the IgM receptor, Fas is upregulated on their surface. Fas-L upregulation on the T-ceiis cm induce both T-ce11 and Bceli apoptosis (see text for details).

1.4.5 The apoptotic signaling machinery Apoptosis is believed to be an innate cellular program thai dows the efficient rlPath and rernoval of =lis from a multicellular organism without eliciting a damaging inflarnmatory or immune response. The features of an apoptotic celi were fmt described by morphological changes in its ce1 structures including nuclear and cytoplasmic condensation, foiiowed by nuclear DNA fragmentation and packaging into apoptotic bodies that are quicidy resorbed by surrounding phagocytic ceils [252]. The ceils also show biochemical modifications such as a reduction in the rnitochondriai transmembrane potential, intracellular acidification, production of reactive 0, radicals, translocation of phosphatidylserine to the outer leaflet of the cellular membrane bilayer, proteolysis of DNA into intemucleosomal fragment and enzymatic cleavage of vzuious cellular proteins 12531. These features are relatively weii conserved in many cells that undergo apoptosis, suggesting that a comrnon pathway of apoptosis is activated by various apoptotic stimuli.

Such a cornmon pathway was originaiiy identified by mutational studies done in the nematode Caenorrhabdiris elegans. These studies identified three key proteins that were involved in apoptosis [254]. The fmt was called CED-3 and is an enzyme that cleaves proteins after specific aspartic acid residues. Because the activity of the catalytic domain of these enzymes is dependent on a cysteine residue, this type of "cysteine driven aspartase*' protease has been called caspase. Such enzymes are also found in mammalian cells, and form a farnily for which there are now 13 known members. The autolytic cleavage and activation of CED-3 is promoted by a second protein called CED-4, for which there are now three putative mammalian homologues: the apoptotic protease-activating factor - 1 (Apaf- 1) 12551, FLASH 12561, and CARD4Nod- 1 1257, 2581. Both CED-3 and CED4 are pro-apoptotic, but their activity is controiled by another protein caiied CED-9, which binds to and inhibits the activation of the CED-

3:CED-4 complexes [259-26 11. The mammalian homologue of CED-9 is the Bcl-2 protein that defmes a large family of mnmmalisn proteins for which there are over 20 known members. Some of these mmbers are pro-apoptotic and others are anti-apoptotic [262]

and they will be discussed later conceming their important dein ce11 survival (see section 1-5.5). Similar rnechanisms of apoptotic pathway regulation found in the Celegans have also been identified among the mammalian counterparts. in this respect, the Bcl-2 family member, Bcl-XL. has been shown to inhibit the activation of wpase-9 by the Apafl protein [263]. 1.4.5.1 The general involvment of proteases in apoptosis Proteases are key effectors of the apoptotic machinery. By cleaving crucial intracellular proteins, these enzymes cm lead to a breakdown in cellular integrity and to the characteristic features of apoptosis. Although other proteases might play a role in apoptosis, there is no doubt that the key proteolytic enzymes that have been shown to be cornerstones in the induction of apoptosis are the caspases 12641. In fact, caspases have been shown to be extensively involved in many forms of apoptosis, including in AICD [265]. There are presently 13 known human caspases that are divided into three classes based on their substrate specificity [266,267]. 1.4.5.2 Caspase structure Caspases are produced as large inactive proteins containing an amino terminal prodomain of variable length (O to 20 kD), a large subunit (-20 kD), and small subunit (-10 kD) [253,268]. Their activation occurs by the removal of the prodomain followed by cleavage and association of the large and small subunits. in the case of caspase-l and caspase-3, one heterodimer of large and small subunits have been shown to interact with a

second to forrn an active tetrameric enzyme with dual catalytic sites [269-27 11. The active enzymatic site of these proteases is centered around a cysteine residue containd within a conserved QACXG motif. The caspases cleave substrates carboxy-terminal to an aspartic acid residue and the preferred specificity of a caspase for a specifk sequence is determineci by at lest four amino acids amino terminal to the aspaxtic acid 1266, 2671 as weli as by the tertiary stnicture of the proteins. 1.4.5.3 Caspase substrates The dopathat has evolved fiom the discovery of the caspases is that by cleaving various proteins they can promote the ceiiular phenotypes that are seen during apoptosis. Based on the previously posnilated assumption that apoptosis occurs due to an imbalance between pro-apoptotic signals and pro-survival signals, it is not surprising that some of the targets of caspases are survival factors, such as Bcl-2 (see section 1.5.5) which when cleaved by caspase-3, releases a fragment that actuaiiy promotes apoptosis 12721.

Caspases also act to inhibit various other enzymes that are involved in maintaining the nuclear structure, such as lamins 12731; that are involved in DNA repair , such as DNA-PK @NA protein kinase) and PARP (poly(ADP-ribose) polymerase) [274-2761; and that are involved in cell cycle regdation, such as the retinoblastoma gene [277]. In addition to inactivating proteins, caspases can also activate enzymes responsible for the phenotypic changes seen during apoptosis. In this respect, by cleaving the inhibitor of a caspase-activated deoxyribonuclease (I-CAD), caspase-3 induces the translocation of the I-CADCAD heterodimer to the nucleus where it can exert its nuclease activity and induce the internuclmsornai DNA degradation that is seen in apoptosis 1278-2801. As well, by removing an inhibitory dornain in gelsolin, an actin filament cleaving enzyme, caspase-3 produces a constitutively active protein that can breakdown the actin superstructure and induce the morphological changes seen in apoptosis. 128 11 (see Figure

8). 1.4.5.4 The specific role of individual caspases in lymphocyte apoptosis Much effort has been spent to define spific roles of individual caspases in various apoptotic processes. Caspases -2, -3, -6, -7, -8, -9, and -10 are thought to be key mediators of apoptosis. Most of the information on the role of caspases has corne from the use of inhibitors that preferentidy block tbe activity of caspases that have similar substrate specficity. Such studies have suggested thaî caspase-3 like activity is important in lymphocyte AKD [265]. This fïnding bas been supported by the generation of caspase-3 knockout rnice which have a defect in Fas-induced apoptosis [2823. The caspase-8 knockout mouse bas aiso been shown to have defect in Fas killing, but also in TNF- induced apoptosis [283]. In contrast, caspase-9 and Apaf- 1 knockout miœ do not have a defect in Fas-induced apoptosis, but rather in other apoptotic pathways such as induced by glucocorticoids, y-irradiation, UV-irradiation and DNA damaging agent-induced thymocyte death [284-2871. The caspase-2 knockout miGe do not appr to have a major defect in lymphocyte regulation, however the death of B-celu lymphoblasts induced by perforin and granzyme B is reduced [288]. The fmding that caspase-3 is important in Fas signaling, but that caspase-9 and Apaf-1 are dispensible agrees with the known signaling pathways downstream of Fas (see section 1.4.5.7). Figure 8. Key players in the apoptotic machinery. Activation of the apoptotic machinery by death receptors le& to the activation of initiator caspases, such as caspase-8, whkh regdate effector caspases, such as caspase-3. The effector caspases either activate or inactivate proteins necessary for regulating dular integrity, leading to ceii deatb by apoptosis. @ISC=death inducing signahg complex) (see text for details). DlSC TNF/Apo3L

DcR2

suwival factors

Breakdown of 1.4.5.5 Caspase regulation Interestingly, one of the main substrates for caspases are other caspases. This cleaving of other caspases activates them and allows rapid activation of the caspase cascade and recruitment of the apoptotic machinery [253]. It is a gemral fmding that the initiator caspases of this cascade contain longer prodomains than the "downstream" caspases. Caspases containing long prodomains include caspase-2, -8, -9, and -10. The activation of these initiator caspases occws very early following apoptotic signahg and appears to be the detennining factors in initiating the apoptotic cascade.

However, these initiatm caspases may not be mediating the regulation of the proteins that produce the various apoptotic changes. Rather, the primary fùnction of the initiator caspases is probably to activate the downstream effector caspases, such as caspase -3, -6, and -7. It is these caspases that appear to be responsible for cleaving the intraceiiular death targets (see Figure 8).

It is thought that the activation of upstream initiator caspases is dependent on specific protein-protein interactions with their prodomain that increase their proximity to each other and promotes their autocatalytic cleavage into highly active enzymes 1289, 2901.

In the case of caspase-8 and - 10, the presence of a death effector domain (DED) in their prodomain is necessary for their activation by adapter proteins downstream of death receptors (see section 1.4.5.7) 1291, 2921. In the case of caspases-2 and -9, a caspase recruitment domain (CARD) in their prodomain is found to be necessary for theu activaiion

[293]. At Ieast caspases -2, -8 and - 10 are directly recruited to death receptors via these domains and they appear to be at the top of the caspase cascade activateci by these receptors.

1.4.5.6 Activation of initiator caspases The activation of ini tiator caspases occurs via various intracellular and extraceilular signals. Most extracellular signals activating lymphocyte apoptosis are linked to the stimulation of the TNFR superfamily death receptors [264] (see section 1.4.5.7). Similarly many intraceliular signais, such as DNA damaging agents, appear to converge on disrupting the rnitochondrial integrity [294].

However, such a distinction is far from being clear cut, and there appears to be much cross-talk between intracellular and extracellular inducers of apoptosis. In this respect, aiany death receptors have been shown to activate both a mitochondriai- independent and -dependent apopcotic program 1295, 2961. In addition, various signals

that directly activate the caspases have been found to recruit the mitochondrial apoptotic machinery via a caspasedependent mechanism [297-3013. In view of this, it is most probable that direct activation of initiator caspases by death receptors can work independently or in a concerted effort with mitochondrial-dependent apoptotic pathways to eff~cientlyinduce a complete apoptotic response. 1.4.5-7 The death receptors and their link to caspases The last few years have presented us with the ehcidation of various signaling cascades downstream of deah receptors that directly links them to the caspases. The death receptors contain a conserveci intraceilular dornain caüed the death domain that is necessary for their apoptotic inducing activity 12231. Among these receptors, the best characterized are Fas (CD95) and TNFRp55 (CD120a) [302, 3031. Since their identification, many other death domain containhg receptors have been discovered. These include the death receptor 3 (DR3, Apo3, WSL-1,TRAMP, or LARD) 1304-3081, DR4 13091, and DR5 (Apo2, TRAIL-R2, TRICK2, or KILLER) [309-3 161. These receptors are activated by interacting as trimers with trimeric ligands belonging to the TNF superfamiiy. Fas-ligand binds to Fas [302,303], TNF and lymphotoxin-a binding to TNFRp55 [302, 3031, Apo3 ligand (Apo3L, TWEAK) binds to DR3 [3 17, 3181, and Apo2 ligand (Apo2L, TRAIL) binds to DR4 and DR5 [23S, 3 19, 3201. In addition, DR4 and DR5 can be inhibited by decoy receptors called decoy receptor 1 (DcR 1, TRID, TRALR3, or LIT) [3 10, 3 15, 3 16, 32 1-3231) and decoy receptor 2 (DcR2, TRALR4, TRUNDD) [324-3261. These decoy receptors have an extracellular region that mimicks DR4 and DR5 and binds to Apo2L, but lacks a functional intraceilular tail. (Figure 8)

The death domain was the first motif discovered thai gave a due as to how these death receptors could induce apoptosis. It was found that, upon ciustering of the receptor, death domains multimerize with thetuselves and with de& domains present in other intraceliuiar signaling effectors [327]. The key intmceiidar mediator that interacts with Fas via this death domain is the Fas associated protein with a death domain (FADD, Mort 1j [292,328]. This protein can also bind indirectiy to the TNFRp55 [329] and DR3-receptors [304] by interacting with the TNFR p5S associated protein with a death domain OD).

TRADD directly interacts with the death domain of TNFRp55 [330] (see Figure 8). In both cases, FADD appears to be the iink between these receptors and the caspases by the interaction of its "death effector domain" (DED) with one of the two DED found in the prodomain of the initiator caspase-8 (FLICE, MACH) [33 1, 3321. The efficient formation of this Fas-FADDcaspase-8 complex, which forms a "death inducing signaiing complex" (DIX), appears to be key to the direct activation of caspases by the fas death receptor [295].

A cmcial role for linking Fas to the apoptotic machinery through FADD has been clearly shown in the FADD knockout mice and FADD dominant negative transgenic mice which have a defect in Fas-induced AICD of lymphocytes 1333-3361. As weli these mice have a defect in TNF-induced apoptosis suggesting that the link to apoptosis through the TNFRp55-TRADD-FADD-caspase8pathway is crucial for TNF-induced apoptosis. These findings suggest that this pathway is non-rdundant in Fas- and TNF-induced apoptosis, however, it is not clear that it is the only pathway that is activated following the stimulation of these receptors.

in addition to these pathways, the TNF receptor interacting TRADD protein can also bind to the death domain of the receptor interacting protein RIP. Although a role for

RIP in ce11 survival has been suggested by its cmcial role in NFKB activation by the TNFR 13373 (see section 1-57), it can also recmit a death domain containing protein ded RAIDD (CRADD) which can interact with the CARD domain of caspase-2 and activate this caspase to induce apoptosis . This is potentially another pathway by which TNFRp55 can activate caspases directly, however the crucial defor FADD in TNF-induced apoptosis suggests that tbe TNFR~SS-TRADD-RIP-RAIDD-C~S~~S~-~pathway might not be so crucial in the apoptotic function of TNF within the immune system. Although FADD is critical to TNF and Fas apoptotic signaling, there are many conflicting reports as to whether DR4 or DR5 can also recniit this death adapter [309, 3 1 1- 3 13, 3 16, 32 11. However, the fmding that the FADD knockout cells, that are no longer responsive to TNF, Fas, and Apo3, are still Myresponsive to DR4-induced cell death points to the use of an altenate pathway of caspase activation by this receptor [333].

It is also of interest to note that although these pathways ünk directly to the apoptotic rnachinery, they may also regulate other cellular functions. In this respect, FADD mutant mice also show a defect in T-lymphocyte proliferation and in Bell development suggesting that FADD can also play a role in proliferative responses by a mechanism as yet still unknown [333, 3341. It is to be notice. that, in addition to recruïting proteins that activate the caspases, the death domain of Fas can also recmit a deah domain containing protein called Daxx [338], which can interact with the ASKl kinase. ASKI can Merphosphorylate and activate MEKKl which is an upstream kinase that can initiate the JNWSAPK pathway

[339]. ASKl has also been linked to the activation of JNKISAPK by TNFa. However, the importance of the INK/SAPK pathway in the apoptotic signal generated by the death receptors is still unclear especially in view of the fact that the FADD knockout T-cells, which can stiii activate the INKISAPK pathway in response to Fas, do not undergo apoptosis [333, 3341. These finding suggest that Daxx may not be a key player in Fas- induced apoptosis within the immune system. 1.4.6 Lipid mediators of apoptosis Although direct activation of caspases appears to be criticai to the apoptotic response induced by death receptor signaling, other pathways have also been shown to induce lymphocyte apoptosis. Of these, sphingolipid metabolites such as ceramides can promote apoptosis [340, 3411. In fact, the endogenous generation of ceramides has been found to occur foiiowing the activation of rnany stress pathways that induce apoptosis, such as oxidative stress, W, ionizing radiation, heat, and chemotherapeutic agents [342,

3431. Stimulation of various cellular receptors such as IFNyR, TNFRpSS, IL- IPR, Fas, and NGFRp75 have also ken shown to promote the generation of ceramides [340, 34 11. Furthemore, ceramides have been implïcated in IgM-induced B-ce11 AICD [344]. 1.4.6.1 Generation of ceramide in apoptosis

Ceramides can be produced by the activity of ceramide synthase that cataiyzes the fusion of a sphinganine sphingoid base with Acyl-CoA to form dihydroceramide. This compound is then fiirther oWdized to cefamide 1345). Such a mechanism for the generation of ceramide has been suggested to play a role in the induction of apoptosis by the dmg daunorubicin [346, 3471 as well as by TNF stimulation [347-3501. However, in most apoptotic stimuli studied, the generatioa of ceramides appears to rather be mediated by an increase in the activity of sphingomyelinases on sphingomyelin [35 11. The catabolism of sphingornyelin by these enzymes leads to the production of ceramide and phosphatidylcholine (PC).

1.4.6.2 Sphingornyelinase structure and function Three main s phingomyelinases (SMases) have so far been characterized [3521. The first two SMases have an optimal activity at a neutral pH. One is cytoplasrnic membrane bound and is ~g2'-dependentand the other is cytosolic and is Me-independent. The third SMase functions at an acidic pH. AIthough the SMases may be involved in the normai turnover of sphingomyelin, both the acidic SMase and the neutral SMase have also been shown to participate in various apoptotic stimuli. The localization of the ~9"-dependent mmbraae bound neutral SMase towards the outer leaflet of the ceiiuiar membrane makes it an ideal candidate for SM breakdown since most of the SM accumulates in this outer leaûet 13531. In the case of the acidic SMase, aithough its housekeeping tùnctions have been associated with its presence in lysosomes, its apoptotic activity is probably better atÉributed to a presence in caveulae which are transiently for& in the plasma membrane in response to various stresses 1354,

3551. In fact, these transient vesicular compartments appear to form in response to the generation of cerarnide by the acidic sphingomyelinase [356, 3571. Furthemore, a secreted isofom of acidic SMase may also play a role in the apoptotic response 1358-3601. There is still much controversy on the precise role of sphingomyelinase in signahg pathways. The difficulty of studying such proteins is compounded by the fact that only the gene for the acidic sphingomyelinase protein has yet been discovered and thai few laboratories specialize in such lipid signaling. Hopefully the discovery of the genes for the neutral sphingomyelinase and its genetic manipulation will help remove much of the controversy surrounding the role of cerarnide generation in the process of lymphocyte AICD. 1.4.6.3 Regulation of sphingomyelinase activity

Both acidic and neutral SMases have been found to be activated by TNFa and Fas

[361-3643. It is thought that the release of ceramide by these receptors could, at least partiy, be responsible for their apoptotic activity. Aithough very little is known conceming signaling mechanisms chat link the Fas and TNFR death receptors to SMase activation, one study has identified a protein ded FAN that can interact with TNFRpSS and appears to prornote the activation of the neuîral sphingornyelinase [365]. However this protein bas not yet been shown to induce apoptosis. It has also been suggested that a slower peak of neutral sphingomyelinase activation might be mdiated by a the slow depletion of glutathione and the appearance of free radicals that occurs following oxidative stress or TNFmediated signaling [366]. PKCs might offer another mechanism of SMase regdation since PKC activation has been shown to block the activation of neutral sphingomyelinase and PKC inhibitors are capable of upregulating neutral sphingomyebase activity 13671. Activation of the acidic sphingomyelinase is also relatively unknown. It has been proposecl that a PC specific phospholipase C (PLC)is involved in the geaeration of diacylglycerol @AG) following TNF stimulation [368]and that this rise in DAG can activate the acidic sphingomyeiinase [369]. However, it is unclear how this pathway would be regulated since other stimuli of

DAG production, such as anti-CD3~stimulation, do not activate âcidic SMase [370]. In this respect, it is possible that the lucalization of the pools of DAG and ceramide produced is critical in determining the ceiiular outcome. Alternatively, la2S-dihydroxyvitamin D, has been shown to activate sphingomyelinases [371]. In addition, sphingomyelinases might also be controUed via kinases and phosphatases, since a tyrosine phosphatase ded hernatopoietic ceil phosphatase (HCP) has been shown to augment Fas-induced ceramide production [372]. Furthemore, the FADD and TR4DD proteins, when overexpressed, have been shown to upregulate acidic sphingomyelinase activation by TNF, suggesting a possible link with these proteins f373J.However this link must be before the activation of caspases since the generation of ceramide by the death receptors appears to be caspase- independent 1374-376 1. 1.4.6.4 Ceramide-induced PKC regulation in apoptosis The role of PKCs in the apoptotic response induced by ceramide was first suggested by the finding that ceramides could inhibit PKC activity and that PKC had been shown to possess anti-apoptotic activity 1377, 3781. In fact, PMA stimulation, which activates PKC activity, has been shown to inhibit apoptosis induced by exogenous ceramides 13671. Recently, it has been suggested that this anti-apoptotic activity of PKC rnight be mediateci by its influence on tbe PKB pro-survival pathway (see section 1.5.6) 1379, 3801. The inhibition of PKCs by ceramide rnay not be valid for ail isoforms of PKC. In

fact, researchers have shown thaî ceramides can activate PKCG [38 1, 3821 and that this

activation can negatively regulate the PKB pathway p83]. However such a process has

also been shown to induce the activation of the NFKB transcription factor (381, 3841.

How such an activation could link cetamide to apoptosis is very controversid since most

research suggests that NF- activation has anti-apoptotic rather than pro-apoptotic activicy

(see section 1.5.7). In addition, the exact role of PKC in inhibiting apoptosis is further obscured by the fmding that caspases cm cleave the 6 and 0 isoform of PKCs into constitutively active products that appear to promote apoptosis [385,386]. 1.4.6.5 Ceramide-induced PKB inhibition and BAD activation in apoptosis Ceramides have also bexm shown to inactivate the PKB pro-survivd pathway. It has been suggested that this inactivation is a consequence of the activation of the MAPK pathway by a ceramide activateci protein kinase, CAPK, which can phosphorylate the Raf-1 kinase upstream of the MAPK pathway [387]. Alternativeiy, it has been suggested that the ceramides can activate the MAPK pathway by stimulating the Vav exchange factor to regdate Ras activity and to recruit the Raf-1 kinase 1362, 3881. The implications of such activation has been studied in cells expressing smaü arnounts of the Bcl-2 family pro- apoptotic protein BAD. In these cells, ceramide-induced MAPK activation stimulated a prolonged inactivation of the Akt/PKB which led to the dephosphorylation of BAD and an increase in its apoptotic activity (see section 1.5.6.2) [389-3911. In support of a critical role of BAD in ceramide-induced apoptosis, inhibition of BAD expression, or mutation of the regulatory phosphorylation sites of BAD can block the ceramide-induced apoptosis. Alternatively, ceramides might interfere with PKB activation by direcly influencing PI3K activity or by activating PKCC [383, 390, 39 11. These fmdings suggests that when BAD is expressed within cells, cetamide signaling might recniit this pathway to induce apoptosis. 1.4.6.6 Ceramide-induced phosphatase activation in apoptosis Ceramides production has been associateci with an increase in the activity of the ceramide activated protein phosphatase (CAPP) [392]. However, the role of rhis phosphatase appears to be in regulating ceramide-induced ceii cycie arrest instead of regulating apoptosis. In contrast, another phosphatase of the protein phosphatase 1 (PP 1) famiiy bas ben shown to be activated by ceramides and to play a role in ceramide-induced caspase activation [393], but this protein remains to be identified. 1.4.6.7 The role of the JNKBAPK pathway in ceramide-induced apoptosis

In addition to the activation of the MAPK pathway, ceramides can also activate the c-Jun kinase- 1 (JNK- l)/stress activated protein kinase (S APK) pathway . This pathway is activated by a cascade of kinases similar to the MAPK pathway, but involving a MEK kinase dedMEKKl which can phosphorylate the SEKl/MKK4 kinase upstream of the JNK- l/S APK kinase [339,394-3971. Ultimately. J'NWSAPK activity phosphorylates and activates the c-Jun transcription factor. It is thought chat the JNWSAPK pathway might play a role in the apoptotic response to ceramide since overexpression of a dominant negative MKK4 or c-Jun has been shown to block ceramide-induced apoptosis [397]. The activation of the JNWSAPK pathway by ceramides is at least partly due to the stimulation of the Rac 1 G-protein, since a dominant negative Rac 1 can block ceramide- induced JNWS APK activation [398]. Alternative1y, ceramides cm activate the MAPKKK TAKl which has been shown to regulate MKK4/SEKl phosphorylation as well as to activate the NF- inducing kinase NIK [399,400]. The activation of the JNK/SAPK pathway by ceramides can lead to tbe upregulation of Fas-L, TRAIL, and TNF, suggesing a mechanism by which ceramides might promote apoptosis [401, 4021. However, the frnding thai caspare-8 deficient mice, which are resistent to Fas- and TNFR-induced apoptosis, are still sensitive to ceramide-induced apoptosis demonstrates that upregdation of Fas and TNF cannot be the only mechanism by which ceramides can promote apoptosis 12831. In fact, the importance of MK/SAPK activation in the ceramide-induced apoptotic response has becorne very controversial due to various reports showing opposite eRects of activation of the JNWSAPK pathway on apoptosis. In addition, Neimann Pick Disease cells, that have mutations in the acidic SMase gene, do not generate ceramide in response to daunorubicin treatment, and ttiis failure to generate ceramide leads to a defect in Fas-ligand upregulation and apoptosis but not to a defect in JNK/SAPK activation 13941. Furthemore, the knockout mouse of

MKK4/SEK 1, actuaUy shows an increased sensitivity of lymphocytes to Fas-induced apoptosis, suggesting a role for the JNWSAPK pathway in protecthg lymphocytes from apoptosis [403]. Because of such contradictory data, it is still unclear what role the JNWSAPK pathway actually plays during ceramide-induced lymphocyte apoptosis. 1.4.6.8 Cetamide activation of the mitochondrial apoptotic machinery in the activation of apoptosis The finding that Apaf-1 knockout mice are resistant to ceramide-induced apoptosis and that in hibitors of the mi tochondrial perrneability transition can block ceramide-induced apoptosis, has led to the suggestion that the apoptotic machinery induced by cerarnides rnight involve cytochrornec and the mitochondnal pathway (see section 1.4.7) [287, 4041. In support of this, a study has shown that ceramides can, in fact, stimulate the release of cytoc hrome-c from rnitochondfia [NS,4061. However the involvement of mitochondria in cerarnide-induced apo ptosis migh t not be universai since there are example of ceramide- induced apoptosis that is independent of rnitochondrial permeability transitions [407,408]. 1.4.7 The role of mitochondria in apoptosis The importance of mitchondria in apoptosis was initiaily suggested by the hding that in ceil free systems, nuclear condensation and DNA &gradation was dependent on the presence of mitochondria [409]. In fact, even the inhibition of caspases is insufficient to completely block celi death induced by many stimuli, and it has been shown that, in these cases, the apoptosis is ultimately activated by an altemate pathway involving the rnitochondria 1410-4121. Furthetmore, although de& receptors can directly recruit the caspases via adapter proteins such as FADD, it has been mported that lymphocytes that are inefficient in aggregating the DISC foliowing Fas stimulation cm die anyway via an apoptotic process that is also dependent on mitochondria [295, 4 131. Such fmdings have ken further supported by the discovery that mitochondria cm, in fact, release a variety of pro-apoptotic proteins [296]. 1.4.7.1 Pro-apoptotic factors released by mitochondria

One mitochondrial protein that was found to have pro-apoptotic activity was cytochrome-c, an important compownt of the electron transport chain. The release of cytochromec from the mitochondria has been reporteci following many pro-apoptotic stimuli such as UV irradiation, staurosporine, y-irradiation, ceramides, and Fas signahg

[297, 300, 407,4 14-4 161. Foiiowing its release, cytochrome-c, in the presence of dATP, has been shown to interact with the Apaf-1 and caspase-9 proteins [417] and to promote the activation of caspase-9 which can then activate the effector caspase-3. In support of such a finding, caspase-3 -1- celis do not undergo apoptosis following microinjection of cytochromex [417]. Alternatively, it has been suggested that even if the caspase cascade is not activated by cytochrome-c, the loss of this protein €rom the rnitochondria would decrease the capacity of the electron transport chain to produce ATP w hich would result in ceii death due to the incapability of maintaining cell homeostasis. Such a form of non- apoptotic death migth serve as a backup mechanism for inducing ce11 death. 0thpro-apoptotic proteins alsa appear to be released by the mitochondria. These include the procaspase 2 and 3 14181, as well as a caspase-like protein calied apoptosis inducing factor (Ail?)which has procaspase-3 activating capabilites [298,419,420].

1.4.7.2 Mechanism of release of pro-apoptotic factors by mitochondria The mechaaism by which pro-apoptotic factors are released by mitochondria is still poorly understood. Many stuclies have fmussed on the opening of the permeability transition pore, in the inner mitochondrial membrane, to explain this release. Opening of the PT pore often accompanies apoptosis [421, 4223 and inhibitors of these pores, including chernical agents [423] and the Bcl-2 family of anti-apoptotic proteins (see section 1.5.5) [423-4261, can often block apoptosis. The PT pore is a polyprotein channel fonned of the voltagedependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and cyclophilin D 1427, 4281.

Many hypotheses have be postulateci to explain bow the PT pore could pmmote the release of pro-apoptotic factors from the mitochondria. It has ben suggested that due to the hyperosmolarity of the matrix of the mitochondria, opening of the PT pore would cause the swelling of the mitochondria and, uitimately, could cause the rupture of the outer membrane and the leaking out of the apoptotic mediators [296, 2971. Ln support of this, the anti-apoptotic protein Bcl-XI has ben shown to prevent mitochondrial swelling and membrane disruption [297]. However, this hypothesis is still controversial since most apoptotic stimuli do not cause mitochondrial membranes swelling until very Iate in the process of apoptosis 14291. In fact, it has been reported that cytochrome-c release and caspase activation can occur in the absence of signifiant loss of rnitochondrial membrane potential 1297, 299-30 1,4301.

Altematively, the hyperpolarization of the rnitochondriai inner membrane has recently ken suggested to be another mitochondrial change that wcurs following an apoptotic stimulation. This process can also be suppressed by members of the Bcl-2 family that block apoptosis (see section 1.5.5) [297]. However, how this hypetpolarization might participate in apoptosis is sawiknown. Finally, a recent report might give the answer to this question since it has shown that the VDAC unit of the PT pore is modified by Bax and Bak to aUow the cytochromec's passage through the pore [431]. In contrat, Bcl-Xi was shown to close the VDAC by direcùy binding to it. Hence the PT pore rnay be directly responsible for cytochromei: release. 1.4.7.3 Regulation of the reiease of mitochondrial pro-apoptotic factors The regulation of the mitochondna release of pro-apoptotic factors is still relatively unknown. However, a recent protein cded BID has been identifid and shown to be cleaved by caspase-8 resulting in a fragment that can stimulate the release of cytochr0rne-c from the mitochondria [432]. It is thought chat BID, induces the release of cytochrome-c by inducing a conformational change in Bax 14331. Such a pathway of activation of rnitochondrial apoptosis might explain the recniitment of this apoptotic machinery by death receptors [295], but stiU leaves many questions concerning the caspase-independent activation of apoptosis by the mitochondria.

1.4.8 Caspase-independent signals in apoptosis The importance of caspases in apoptosis is unquestionabIe, however they might not be the only mediators of apoptosis. In fact, Bax-induced apoptosis is caspase-independent

[4 104 12, 426,4341. Furthemore, a protein dedALG-2 has been identified [435] as a ca2+ binding protein necessary for AICD induced by anti-TCR/CD3. Fas, and Glucocorticoids stimulation and the inhibition of ALG-2 expression blocks AICD without affecting the activation of the caspases [436]. This suggests that activation of caspases might not be the only mechanism by which celi death occurs. 1.4.9 Transcription factors involved in apoptosis The importance of gene transcription in AICD, such as traacription of death receptors, is demonstrated by the fact that inhibitors of protein synthesis can also inhibit the

AICD of lymphocytes [ 184, 185,4371. Hence the regulation of transcription factors (TFs)

is criticai in controlling the apoptotic pmxss. The search for TFs that have a unique role of inducing iymphcyte death has led to the discovery of a protein calleci Nu77 (NGFI-B) [438]. This transcription factor, and its family homologue Nor-1 [439], have been shown to be potent mediators of thymocyte

[44û, 44 11 and mature T-celi [442, 4431 AICD. Their activity is independent of the IL-2 response to TCR stimulation and is thought to involve the upregulation of Fas-Ligand. Support for the important role of this family of TFs in AICD has come fom transgenic mice that express a dominant negative form of Nur77 in their thymocytes. These mice have a severe defect in thymocyte negative selection [444, 4453. Surprisingly, mice carrying a null mutation for Nur77 do not show such a defect nor do they have a defect in AICD of peripherai Telis 14461. This suggests that Nor-1, or another unknown family member, rnight compensate for the absence of Nur77. The Nurr77 family of transcription factors are not unique in mediating lymphocyte apoptosis. Another transcription factor cailed TDAGS 1 has also been shown to induce the upregulation of Fas following TCR stimulation 1447, 4481 and a forkhead transcription factor, FKHRL 1, which is inhibited by the PKB pro-survivai pathway (see section 1 .5.6), can upregulate the expression of Fas ligand 14491. 1A.10 Integration of the various apoptotic pathways in lymphocyte AICD The multiple studies on apoptotic signahg have shown thai many signaliag pathways can be activated by a single apoptotic stimuli. It is likely that al1 these pathways integrate togettier to overcome pro-swivai factors and efficiently induce apoptosis. However some pathways rnay be more dominant than others. The generation of FADD (33 31, caspase-8 [28 31, caspase-9 1284, 2851, and acidic sphingomyelinase [360] knockouts indicate that the FADDIcaspase-8 pathway is probably the strongest pathway activated during Fas-induced lymphocyte AKD and cannot be compensateci by the other pathways. However in ceils that contain many pro-survival factors or in cells that fail to adequately remit the DISC components, the FADD/caspase-8 pathway might not be strong enough to promote apoptosis and might need accessory help via the mitochondrial CS951 and the sphingomyelinase pathways to efficiently induce AICD (Figure 9). Figure 9. Three pathways thal cm initiate the apoptotic program downstream of death receptors: direct caspase activation, activation via the mitochondrial pathway, and activation via the sphingomyelinase pathway. Activation of the effector caspases by the death receptors depends on the recniitment of the DISC complex. Ln cells where this recmitment is efficient, the receptors cm directly activate the caspase cascade (1). However in ceiis where this recruitment is inefficient, other pathways involving the mitochondria (2) or the sphingomyelinases (3) may be necessary (see text for details).

1.5 Survival signals involved in lymphocyte homeostasis

As has been previously meationed, the final decision of a ceil to undergo AICD or to survive and proLiferate is probably detennineû by the balance betweea apoptotic and survival stimuli. It is therefore not surpnsing that Teils have evolved many anti-

apoptotic factors tbt are aimed at inhibithg the various apoptotic pathways (Figure 10).

Furthemore, because of this delicate balance, it is dso not surprishg that many receptors, by activating both swival and apoptotic signais, can sensitize cells towards apoptosis as well as towards çell survivaVproliferatioa. This is the case of rnany members of the TNFR superfarnily for which the cellular environment is criticai in detennining whether the pro- apoptotic or pro-sumival signals of these receptors will prevail[450].

1.5.1 Receptors that regulate lymphocyte survival Both antigenic stimulation and CO-stimulationhave been associatecl with lymphocyte survival. In fact, antigenic stimulation of the TCR alone can decrease Fas-induced AICD.

It is unclear how exactly this anti-apoptotic activity is mediateci, however, recent studies have suggested that this might occur either because of the activation of the PKB survivai pathway by the CaM-K (Ca2+/calmodulin-dependent protein kinase) kinase king activated due to smali rises in intracellular calcium (see section 1.5.6) [451], or because of the upreguiation of the mRNA transcription of the anti-apoptotic protein FLIP induced by the

MAPK pathway (see section 1.5.2) [452]. Figure 10. Various survival factors iunction together to inhibit multiple pro-apoptotic pathways. Survival factors (in boid) act on various aspects of the apoptotic machinery to inhibit apoptosis. Some factors, such as FLIPs, inhibit formation of the DISC, the Bcl-2 family functions to inhibit the mitochondrial pathway, and IAPs function to block caspase activity (see text for details).

Co-stimulatory signais dso appear to potentiate this anti-apoptotic activity. In fact, celis stimulated with both anti-CD3&and anti-CD= are even less sensitive to AICD [453].

The importance of CD28 signalhg in promoting cellular survival in response to continuous antigenic stimulation appears to be especially tme of CDS+ ceiis [454] and, consistent with this, CD28 has been shown to reduce TNFa-induced Teli apoptosis [455]. The anti- apoptotic activity of CD28 has been associated with the expression of the pro-sumival gene Bcl-M (see section 1.5.5) [453] and the downregulation of Fas-L expression [456].

However, CD28's pro-survival activity is probably dependent on the state of activation of the T-cells since, in some cells, CD28 can sensitize cells to AICD (see section 1A.2). This Merentiai effect of CD28 is possibly mediated by the induction of IL-2 secretion, since

IL-2has been shown to promote survival of recenîiy activated T-cells, via the upregulation of both Bcl-2 and Bcl-Xi (see section 1.5.5) [135, 1361, but to promote AICD of prolonged activated Tqells [208].

In view of the finding that the CD28 stimulation cm lower the threshold for activation and cytokine production without lowering the Fas-induced apoptosis threshold [457], it bas been suggested that part of the CO-stimulatoryactivity of CD28 might be the result of a shift in the ceil's sensitivity towards survival and proiiferation rather than apoptosis. In addition to pro-swival signds ongïnating from the antigenic and CO-stimulatory pathways, some TNFR superfarnily members have also been shown to promote lymphocyte survivai and to protect from AKD. This is the case of CD40 which, when co- stimulated with the BCR, can promote expression of the ad-apoptotic protein Bcl-XI and ce11 survival (see section 1.5.5) [458,459]. CD30 has also been indirectly associated with pro-survival activity because of its almost universal expression on Hodgkin lymphoma cells [460]. Other receptors have also been reported to inhibit AICD. Among these, a novel receptor, called Toso, has recently been identifieci as an inhibitor of Fas-induced apopcosis of lymphocytes. AU the anti-apoptotic activity of these receptors appear to block various elements of the apoptotic signaling machinery and their joint effort in inhibiting apoptosis is probably crucial to Iymphocyte survival.

1.5.2 Inhibitors of DISC formation: FLIPs, ARC and Sentrin One of the earliest steps available in inhibiting caspase activity consists of inhibiting the death receptor signaling at its source. Prevention of the recniitment of proteins that form the DISC downstream of death receptors is, in fact, one rnechanism that both ceUs and vimses have evolved to promote ce11 survival [46 11 (Figure 10). 1.5.2.1 FLIP structure and function Two such inhibitors have been isolated fiom mamrnalian cells and compose the FLIP family of inhibitors 14621. Both these homologues contain the DED domains of caspase-8, but they either lack the caspase domain or have a mutated inactive caspase domain. It is thought that FLPs inactivate the Fas signal 14621 by competing with caspase-8 for its interaction with the DED domain of FADD. in doing so they effectively inhibit the recruitment of caspase-8 to the DISC and prevent its activation [462]. Such a proposed mec hanism of action of FLIPs suggest that these proteins are anti-apoptotic, however conflicting reports have brought much controversy concerning the role of these proteins in inhibiting lymphocyte AICD. In fact, while FLIP is able to inhibit Fas kiiiing, in some cells, lymphocytes rnight not be dependent on FLIP to regulate AICD. The generation of a FLIP knockout mouse should help define the importance of FLIPs in regdating lymphocyte AICD. Inhibition of DISC formation is not unique to FLIPs. Another protein called Sentrin has also been isolated as an ad-apoptotic cellular rnediator that contains a death domain which binds to the Fas death domain and inhibit the recniitment of FADD to the DISC 14631. In addition, the apoptosis repressor with a caspase recniitment domain protein (ARC), has also been identified as a negative regplator of caspases by competing with the recruitrnent of CARD domain containing caspases 14641. However the physiological role of these proteins in lymphocyte AICD remains to be cleariy established.

1.5.3 Direct inhibitors of caspase activation: cIAPs The initial discovery of direct caspase inhibitors stems fmm the study of vital proteins. Two such viral anti-apoptotic proteins were identifie- and are dedp35 [465, 4661 and serpin [467]. These proteins function by acting as uncleavable caspase substrates. By interacting with the caspases, they prevent the caspases from cleaving their target proteins. (Figure 10) 1.5.3.1 cIAP structure and function

A family of proteins that can act as caspase inhibitors has also been isoled from rnammalian celis and have been shown to protect T-cells from various apoptotic stimuli [468, 4691. These rnammalian inhibitors of apoptosis (cIAPs) indude NAIP 14701, XIAP(h1LP) [471, 4721, Wl(cIAP~/MM.C/~ITA) 1472-4751, HIAPS (cIAPl/MIHB) [472-4741, and Survivin [476]. The spectnim of anti-apoptotic activity of IAPs is surprisingly large. In fact individual IAPs, or various combinations of IAPs have been shown to block almost every

type of apoptotic stimuli known [477]. In the case of lymphocytes, XIAP appears to be the most potent inhibitor of Fas-induced apoptosis. However, ail these studies have involved overexpression of these proteins and it is difficult to conclude the defmitive physiological activity of these proteins fiom such results. The iAP family members are characterized by the presence of multiple BIR (Baculoviral Inhibitor of apoptosis Repeat) domains and a C-terminai Ring Zn finger 14771. Both these domains are thought to participate in the anti-apoptotic activity. In addition to

these domains, the HIAPl and HIAP2 proteins also contain a CARD motif (see section

1.4.5.5). 1.5.3.2 cIAP anti-apoptotic activity The presence of a CARD motif in HIAPl and HiAP2 suggested that these proteins might act by bhding to and inhibiting caspases [293]. Although there is still some controversy, caspase -3 and -7 have, in fat, been shown to be inhibited by XIAP [478-

4801. As weU IAPs have been shown to inhibit the activation of the upstream CARD domain containing initiator procaspase-9 [48 11. Inhibition of the caspases by IAPs appears to be mediated by their BIR domains [482]. Other pathways that could explain the pmtective activiîy of IAPs have aiso been proposed. The discovery of a murine IAP, caiied BRUCE, which contains a BIR domain and a UBC (ubiquitin-congugating enzyme) motif suggests that some IAPs may target apoptotic mediators for proteosomal degradation via the u biquitination pathway 14831, dthough this remains to be proven. Alternatively, it is also possible that IAPs fimction by inhibiting upstream factors involved in death receptor signaling. This has been suggested by the ment finding that

HIAPl can bind to and inhibit a novel kinase, RIP2, that can associate with the TNFRp55 and with CD40, and that has pro-apoptotic activity 1484, 4851. In addition, HIAPl and

HIAP2 have been found to activate the NFKB pro-survival transcription factors (see section

1S.7) [486, 4871. The activity of KAPs in inhibiting apoptosis is most iikely multidimensional and much study remains to be done to understand the main anti-apoptotic pathways that these protein utilize. In this respect, the analysis of mice carrying targeted genomic disruption of the various iAPs should help in further defining this.

1.5.3.3 cIAP regulation Very little is known concerning the regulation of cIAPs. Most studies have looked at the upregulation of IAP RNA transcription by various stimuli. Arnong these, TNFa has been shown to upregulate HIAP1, HIAP2. and IaAP [488] and CD40L stimulation of B- celis can upregulate the expression of HIAPl [4891. Recent evidenœ has suggested that this upregulation of cIAPs mRNA might be mediated through the pro-survival transcription factor NF- (see section 1.5.7). In support of this, the HIAP1 -induceci protection fiom TNFa-induced apoptosis is dependent on NFKB activation [487] and TNFa-induced upregulation of HIAP1, HIAPS, and XIAP is inhibited by the overexpression of IKB [488].

Aithough modifying the IAP expression is one mechanisrn by which regulation of IAP can occur, it is unlikely to be the only one. It seems more plausible that LAPS are also regulated by various pst-translational mechanisms. In this context, the Wshave been found to interact with TNFR associated proteins (TRAFs) (see section 1-54), and it is thought that such interactions might serve to recruit the IAPs to the cellular receptors [477]. In fact overexpression of TRAFs and ïAPs potentiate the anti-apoptotic effect of these proteins. However, the exact role of this interaction between IAPs and TRAFs still remains to be clearly established.

1.5.4 Proteins that link receptors to survival pathways: TRAFs Many of the TNFR superfamily mernbers that can regdate ce11 survival do not contain a death domain in their intraceliular tail. Even though the homology in the intraceliular taiI between such family mernbers is very lirnited, one common iature that appears to link rnany of these receptors is the recruitment of a unique family of TNF receptor associated proteins (TRAFs). 1.5.4.1 TRAF structure and function TWs were originaliy discovered as proteins interacting with the TNFR p75 intracellular tait. Six members of the TRAF family have now been isolated [490494].

TRAF-1 can bind to CD30 14951 and 4-1BB [4%]; TRAF-2 to TNFRp75 [493], CD27 [497, 4981, CD30 [495], CD40 [499], 4-IBB [500], OX-40 [Sol], ATAU [502], and indirectly to the TNFR p55 via an interaction with TRADD [329]; TRAF-3 to CD30 14951, CD40 [49 1, 4921, ad0x40 [496]; TRAF-5 to CD27 14981, CD30 [503], CD40 15041, the LTP receptor [494], 0x40 [Sol], and ATAR 15021; and TRAF-6 binds to CD40 [490] and is downstrearn of the IL-IR signai [SOS]. in contrast, TRAF4 does not appear to bind to TNFR superfamily members. In addition, heterodimerization of TRAFl to TRAF2 allows TRAFl recniitment to receptors that bind TRAFS, such as the TNFRp75 [4933.

The TRAF proteins contain a con~e~edC-tenninal TRAF domain which is important in mediating homodimerization and heterodimerization of TRAFs as weii as their interaction with the receptors [49 1, 499, 506, 5071. Ali TRAFs, with the exception of ml,also contain a N-teminal ring fmger. In addition, TRAF3 and 5 also contain a unique isoleucine zipper domain of unknown function [492,494]. With the exception of TRAF-4, which is a nuclear protein that might function as a transcription factor [SOS, 5091, all TRAFs have been found to localize to the cytoplasm. It is thought that most of the TRAF effects are mediated in the cytoplasrn by interactions with the TNFR superfamily members.

The activation of the NF- transcription factors by TRAFs has led to the suggestion that these proteins can stimulate cellular survival (see section 1.5.7) [5 10-5 131. This hypothesis has been strengthened by the finding that thymocytes from mice carrying nul1 mutations in the TRAM gene have an increased sensitivity to TNFa-induced apoptosis

[5 14, 5151. TRAF6 also appears to have survival activity as well as lymphocyte regdatory activity, possibly due to a critid role in CD40 signaling since TRAF6 deficient mice fail to develop a proper T-helperdependent Bali activation and B-cells from these rnice don? proliferate in response to CM. A similar role for TRAM in CD40 signaling has also been dernonstrated in mutant rnice [5 16, 5 171. In contrast to such fmdings, TRAFs have also been associated with pro-apoptotic activity. In this respect, the TRAF6 knockout mice have a similar phenotype in the brain to the Apaf- 1 and caspase-9 knockouts, suggesting that at ieast TRAF6 might be controiling a pro-apoptotic protein in the nervous system. Such an apoptotic promoting activity for other TRAFs remains to be clearly established, however, various TNFR superfamiiy members that recruit TRAFs have been shown to have some pro-apoptotic activity. In this respect, the CD30 receptor knockout rnice have a defect in thymocyte negative selection [240]. Furthennore, T-ceil hybndomas that express mutated CD30 receptors that fail to bind TRAFs have been shown to have a defect in CD30 rnediated-TCRdependent AICD 12341. Since no pro-apoptotic proteins have yet been found to interact with the CD30 receptor, it has ben suggested that this pro-apototic activity might be rnediated by its regdation of TRAF2 sequestration and degradation [250]. Such activity might serve to remove the pro- survival TRAFs, and, in so doing, to tilt the cellular balance towards an apoptotic response rather than a survival response. 1.5.4.2 TRAF anti-apoptotic activity A cornmon mechanism by which TRAFs appear to mediate their effects is through the activation of the NF- transcription factors. Although TRAM has been associated with an NFKB inhibitory activity [491, 4991, the ring finger domain TRAFs 2, 5, and 6 have aii been shown to be potent activators of the NFKB transcription factors [5 181. In fact, The TRAF6 knockout rnice have demonstrated that TRAF6 is necessary for the activation of NFKB by IL- 1 [5 191. Surprisingly, TRAF2 deficient thymocytes, which are more sensitive to TNFa-induced apoptosis, do not have a defect in NFKB activation but rather a defect in JNWSAPK activation [5 14,s151. In accordance with this, TRAFs 2, 5, and 6 have al1 kenshown to activate the JNWSAPK pathway [520]. The activation of NFuB by TRAFs appears to be mediated by tbeir interaction with a kinase called NM. This kinase is a component of a kinase complex which regulated tbe phosphorylation and degradation of the NFicB inhibitory proteins (IrBs - see section

1.5.7.3) [S2 11.

Alternatively, TRAFs have been shown to interact with the ASK- 1 kinase which can phosphorylate the MEKKl proteh [522-5241. This suggests a direct link between JNWSAPK activation and TRAFs 1523, 5241. There is aiso recent evidence that

MEKKI, cm activate the IKB kinase complex and induce NF& activation [525]. A dominant negative fonn of ASKl has been shown to inhibit TFWF2-induced JNWSAPK pathway activation, suggesting the importance of this protein in this activation [522]. However. this might not be the only pathway used to activate the JNWSAPK activation since the activation of JNWSAPK by TRAFZ has been also indirectly associateci with the activation of the germinal center kinase related protein (GCKR) which can also phosphorylate MEKKl [526]. However the role of CCKR in the TRAF signaiing pathways still remains to be clearly estabiished.

The fnâing of a hypersensitivity to ma-induced apoptosis concomitant with a block in activation of the MWSAPK pathway, in the TMknockout mouse, suggests that the major anti-apoptotic pathway activated by TRAF;! might be via the JNWSAPK pathway. There is however SUmuch controversy on the role of lNK/SAPK as an anti- apoptotic pathway and in view of this, one cmot preclude the possibility that TRAF2's activation of a survival pathway is independent of its activation of the JNWSAPK pathway . Other mechanisms have also been proposed to explain TRAFs anti-apoptotic activity. It is thought that T'RAFTSinteraction with the anti-apoptotic A20 protein could be partly responsible for its anti-apoptotic activity [527]. Other proteins such as MnSOD (manganese superoxide dismutase) [528-5321 and EX-IL [533] have also been implicated.

The hding that the A20 and MnSOD proteins inbibit NFKB activation by TRAF2 further

suggests that NFKB might not be the main mechanisrn by which TEUW2 inhibits apoptosis.

Furthemore, TRAFl and TRAFî have also been found to interact with cIAP-I and cW-2 suggesting another mechanisrn by which TRAF2 can inhibit apoptosis [474] (see section 1.5.3). 1.5.4.3 TRAF regulation The fmding that TRAFs can bind either directly to the intraceliular tail of the TNFR superfatnily or indirectiy via adapter proteins such as TRADD, suggests that part of their regulation is probably via their recruifment to these receptors. Interestingly, binding to receptors dght not only mediate TRAF activation, but in some cases, may promote TRAF degradation. In fact, CD30 has knshown to promote the degradation of TUF2 12501. TRAF activity also appears to be modified via ktinteraction with intraceiiular regulators, such as the 1-TRAF protein (inhibitor-TRAFmANK), which has been sho wn to both inhibit and activate TRAFs depending on the cells studied 1534,5351, and the TRIP protein, which is a component of the TNFR and CD30 signaling pathways and has been shown to inhibit TRAF activity [536]. Furthermore, aithough not formally provzn, the finding that TRAF6 can bind to the IRAK (IL-IR-associated kinase) serindthreonine kinase suggests that TRAFs regulation rnight aiso involve phosphorylation events [505].

1.5.5 Inhibitors of the rnitochondrial apopototic machinery: the Bcl-2 family

Since apoptotic signals are not only generated directly from death receptors, but can also be mediated by the rnitochondrial pathway, it is not surprising that a large farnily of anti-apoptotic proteins, typified by the Bcl-2 protein, have been isolated that can regulate this apoptotic pathway. 1.5.5.1 Bcl-2 family structure and function

More than 15 members of the Bcl-2 superfamily have been identifiai so far [262,

5371. Among these, some bave been identified as inhibitors of apoptosis, such as Bcl-2 and Bcl-XI [262, 5371, and others as promoters of apoptosis, such as Bax [538] and Bik [539, 5401. These family members are defined by the presence of up to four distinct Bcl-2 homology domains (BH1-Q) that are crucial to their activity [262]. The BEI domains appear to mediate homo and heteto-dimerization between BH-containing proteins [541-

543). It is thought that the pro-apoptotic activity of certain fdymembers is, at least partly, due to their interaction with the anti-apoptotic family members. Furthemore, the ratio of anti- to pro-apoptotic faniily members Ui such interactions has been shown to be important in determinhg oell fate [538]. In addition to this mechanism of activity, some pro-apoptotic farnily members can also induce apoptosis independently of their interaction with the anti-apoptotic family members [544].

The importance of the Bcl-2 family members in regulating lymphocyte survival was onginaily suggested by the initial idenMmtion of the Bcl-2 gene as one of the genes involved in the t(14: 18) chromosomal translocation found in human BuAl foilicular lymphomas [545-5471. Following this, many studies demonstrateci the anti-apoptotic activity of this protein. In fact, overexpression of Bcl-2 can protect lymphocyte celi lines from withdrawal of cytokines, such as IL-3, CL-4, GM-CSF,IL-2, and IL-6 [548-5531.

The hypothesis that the Bcl-2 family is important in modulating lymphocytic homeostasis has been further supported by the generation of transgenic mice overexpressing such proteins and of rnice carrying nuii mutations in the various gens coding for these proteins.

Transgenic mice expressing Bcl-2 in thymocytes have a defect in y-irradiation, UV irradiation, cytokine withdrawal, dexamethasone, staurosporine, and cytotoxic dmg- induced apoptosis [537]. Furthermore, Bcl-2 and Bcl-XI transgenic mice appear to have increased thymocyte survival in the absence of a positive selecting signal, especiaily of the CDS' lineage [554-5573. However, the role of these proteins in thyrnocyte selection is probably not unique since overexpression of Bcl-2 or Bcl-Xi fails to completely block negative selection in TCR transgenic mice [554, 557,558J. The role of Bcl-Wcl-Xl in mahm lymphocyte development has also been demonstrated in these mice. Transgenic mice overexpressing Bcl-2 in peripheral lymphocytes develop Bcell and T-ceil lymphomas [559-5611. The mle of Bcl-2 in mature lymphoid sumival is also supported by the fact that Bcl-2 knockout mice exhibit marked lymphocyte apoptosis [562-5641. However the reason for this sensitivity to apoptosis is unclear since these celis do not appear to have an increase in Fas-induced AICD [565-5671. The absence of a Bcl-2 effect on Fas-induced apoptosis is not unreasonable since the death receptors can activate the caspase cascade directly (see section 1.4.5.7). Bcl-2 might, however, have a function in preventing Fas-induced apoptosis in cells that fail to adequately initiate the caspase cascade ktlyand necessitate the mitochondrial pathway to do so. Pro-apoptotic family mernbers of the Bcl-2 family also appear to play a role in controlling lymphocyte homeostasis. The generation of Bax knockout miœ and of Bax transgenic rnice has shown that Bax activity can play a critical role in lymphocytes apoptosis [568-5701. The fact that Bax 4- knockouts can partidy rescue the increased lymphoid apoptosis found in the BcI-2 -/- mice when crossed together suggests that the Bax:Bcl-2 ratio is probably important for determining lymphocyte apoptosis [57 11. 1.5.5.2 The Bcl-2 family's anti-apoptotic activity The mechanism of action of the Bcl-2 anti-apoptotic famiiy members is samostly unresolved. Structural studies have, however, suggested a role for Bcl-2 family members in pore formation since the BHl and BH2 dornains of Bcl-M contain a-helices that share sirnilarity with the pore fonning domains of bacterial toxins [542]. In fact Bcl-2, Bcl-XI, and Bax cmform pores in lipid biiayers and, at least for Bcl-2 and Bax, these pores have ken shown to have selectivity for channeling specific ions [572-5761. One mechanism proposed for the Bcl-2 anti-apoptotic activity is its ability to prevent the pore formation by Bax 15741. However the exact invotvement of these small pores in apoptosis is still unclear. Furthemore, recent &ta suggests that Bcl-XI has an anti-apoptotic activity that is dependent on both its pore-forming abiiity as weil as on its capability to heterodimerize with other proteins 15771. Because of the IocaIization of these proteins to the mitochondria [578, 5791, it is thought that, at least part of their activity, is to inhibit rnitochondrial apoptotic pathways. In support of this, Bcl-2 and Bcl-Xl have been shown to inhibit the release of cytochrome-c from the mitoçhondria [255, 414, 417, 578, 5791. This hypothesis has fuaher been supported by the frnding that the BH4 dornain of Bcl-XI, which is required for its pro- survival activity, can bind to Apaf-1 and inhibit the activation of caspase-9 1263, 5801. Furthemore, pro-apoptotic family members such as Bik, Bax and Bak have been shown to bind to Bcl-XI and, in doing so, to free Apaf- 1 so that it can activate caspase-9 [259, 263,

5801.

Although the inhibition of cytochromec release and Apaf- 1 activity is appeaiing to explain the mitochondrial anti-apoptotic function of the Bcl-2 farnily members, the definitive explanation might not be so simple. A recent report suggests that the anti- apoptotic activity of Bcl-XI in response to growth factor withdrawal is better explained by its facilitation of ATPIADP exchange in the mitochondria [581]. In support for a Link between apoptotic regulation and the maintenance of appropriate ATP homeostasis, Bax-

induced apoptosis has been shown to be dependent on the presence of a functional FP,- ATPase proton pump in the inner membrane of the mitochondrial [582]. Such a

mechanism might be important in cases where pro-apoptotic proteins, such as Bax and Bak, induce a caspase-independent apoptosis [583-5851. Although the addition of caspase inhibitors fails to block membrane alterations and DNA condensation induced by overexpression of Bax-like proteins 1410,426, 5861 they can inhibit the DNA degradation induced by Bax, suggesting that some apoptotic phenotypic changes could be entirely dependent on the activity of caspases and other couid also be induced by other rnechanisrns. Although inhibiton of the rnitochondrial apoptotic pathway is a likely mechanism of action of the Bcl-2 family members, evidence also exists that it is not their only activity. One alternative mechanism of action has been proposed, during the process of AICD of T- lymphocytes, from the fmding that Bcl-2 overexpressing T-lymphocytes secrete less IL2 in response to activation [587]. This is thought to be caused by a defect in NFAT activation due to the sequatering of calcineUrin to the piasrna membrane by Bcl-2 [588]. Since IL-2 has been shown to be important in making activated lymphocytes sensitive to AICD [208], the decreased IL-2 production induced by Bcl-2 codd be a factor influencing AICD. Another mechanism of anti-apoptotic activity has been shown for Bcl-XI which can inhibit the ceramide generation that follows TNFa stimuiation [589].

1.5.5.3 Regulation of the Bc1-2 tamily members Many reports have shown that various stimuli can regulate the RNA levels of Bcl-2 family members [262, 5371, such as the upreguiation of Bax by the p53 gene [590]. However the mgdation of members of the Bcl-2 farnily also appears to occur at a pst- translational level. In support of this, the Jun kinases (JNKsISAPKs) have been shown to phosphorylate and inactivate Bcl-2 1591-5943. Ln addition, the phosphorylation of Bcl-2 on Ser7O can upregulate its activity 15931. It is thought that such pst-translational modification can regulate these proteins by inducing conformational changes, however, it also appears that they can serve to recruit other regulatory proteins, as is often seen in signaling networks. This is the case of BAD phosphorylation by PKB (see section 1.5.6) which induces its interaction with 14-3-3 proteins resulting in its sequesteration and inactivation.

1 S.6 The PKB/Akt pathway of cellular survival Another pathway that can regdate the activity of various survival factors involves the PKB/Akt protein t&at was originaiiy discovered as the murine retroviral oncogene, v- Akt which couid induce the formation of spontaneous thymornas [595-5971.

1.5.6.1 PEU3 stucture and function Three madanhomologues of the v-Akt gene were cloned and were found to be composed of a central serine/hreonine kinase domain [598-W] flanked by a plekstrin homology (PH) domain at the N-tenninus and a regdatory domain at the C-terminus 1598- 60 11. The PH domain of PKBs mediates the formation of PKB homo-trimeric complexes in celis and has been shown to be crucial to the activation of these proteins 16021. PKBs have been found to be activated by various celiula.signals including cytokines (IL-2, -3, - 4, and -9,chernokines (IL-8 and RANTES), various stress signals, and TCR signaling 11603-6 121. It has ben shown that PKBs are multifimctional proteins, that, among other things, can promote cellular survival. In support of this, overexpression of PKB protects hematopoietic ceils from IL-3 withdrawal-induced apoptosis and PKB is rapidly activated by leukocyte survival factors 1604, o07,608,6 IO]. The importance of the regulation of the PKB pathway in lymphocyte homeostasis has recently been evidenced by the discovery of an inhibitor of this pathway called PTEN

(see section 1S.6.3). This protein is a phosphatase that is mutated in various tumors [6131 and PTEN knockout mice have been shown to develop spontaneous thymornas 16 141. Such findings suggest that the PKB pathway is critical in reguîating homeostasis of thymocytes and other cells. 1.5.6.2 PKB anti-apoptotic activity Various survival pathways have been associated with PKB activi ty . Among these, the induction of Bcl-2 expression by PKB has been reported 16151. However, at least in fibroblas ts, this upregulation is not necessary for the PKB 's pro-survival effects [6 161. Nonetheless, modifying the activity of other Bcl-2 family members may be a primary pathway by which PKB inhibits apoptosis. In support of such a hypothesis, active PKB has been shown to phosphorylate BAD at a serine residue which promotes its interaction with 14-3-3 proteins [617-6201. C-Raf has also been shown to phosphorylate this residue as well as other residues on BAD [621]. It is thought that by phosphorylating BAD, PKB and c-Raf can induce its sequestration by 14-3-3 proteins and, in doing so, prevent the pro- apoptotic activity of BAD. Such a mechanism of regulatim of Bcl-2 family members by PKB is an appealmg anti-apoptotic pathway, however it is probably not the only one since IL4 can activate PKB and ceil survival without inducing BAD phosphorylation [622]. In this context, a report has shown that overexpressed PKB can phosphotylate and inactiva. caspase-9 directiy in human ceiis [623]. However, it remains unclear whether this is a normal physiologicai activity of PKB and whether this can occur in cells other than of human origin.

In addition, another PKB anti-apoptotic pathway has recently been suggested by the finding that PKB can phosphorylate and induce the cytoplasmic retention of a forkhead transcription factor, FKHRLl [449]. This transcription factor, when allowed to enter the nucleus, can promote the transcription of pro-apoptotic genes such as Fas Iigand [449]. 1.5.6.3 Reguiation of PKB activity Mechanisrns of regulaîion of PKB were originaliy suggested by the fmding that v- Akt, which consists of a fusion of the retroviral gag protein with the PKB gene, is abnorrnally targetted to the cellular membranes [624]. Furthemore, the finding that this protein was constitutively phosphorylated [625-6271 Ied to the hypothesis that PKB, once targeted to the cellular membrane, might be activated by a phosphorylation process.

Support for this relocalization of PKB in its activation was supported by the finding that PKBYsPH domain can interact with the phosphorylated lipids generated by PI3K [628]. AIthough this pathway of PKB recruitment has been well established, there is evidencç that PKB may also be activated by some PI3K-independent stimuli, such as heat shock 16051. However ttie importance of such an alternate pathway in lymphocyte regulation remains obscure.

Upon translocation of PKB to the membrane, the protein is phosphoylated on a serine and threonine residue 16291. The phosphorylation of the serine and threonine residues releases an inbibitory interaction of one PKB molecule over the other members of the homo-uimenc PKB complex [ml] and this results in an active PKB complex. The importance of phosphoryiations in the regdation of PKB xtivity has been further supported by the discovery of a kinase that can phosphorylate the regulatory threonine residue of PKB [630]. This kinase contains a PH domain that also targets it to the PI3K lipid products [628, 63 1, 6321. Because of this, the protein was ded PtdIns(3,4,5)P3- dependent protein kinase- 1 (PDK- 1). The activity of PDK-1 appears to be constitutive and its regulation is probably mediated by the generation of the ptiosphorylated iipids by PI3 K.

The search for a good universai candidate kinase that can phosphorylate the regulatory serine residue of PKB is stiil on.

An inhibitor of the PKB pathway has also recently been identified as the PTEN phosphatase. This protein can dephosphorylate the PI3K phosphorylated iipids [633]. In doing so, PTEN effectively inhibits the recruitment to the membrane of the mediators of PKB activation and downregulates PKB activity. Hence the regulation of the PKB survival pathway is dependent prirnarily on the phosphorylation and dephosphorylation of lipids that recmit the PKB and its kinases to the cellular membrane.

1 S.7 Transcription factors that regulate ce11 survival: the NF'KB family The transcription of various pro-survival genes is critical in detennining to what extent pro-survival factors will prevail in promoting ceii survival. Although many transcription factors probably participate in generating the swvival signais, the NFKB family of transcription factors have been shown to be pivotal in determining ceil survivai.

1.5.7.1 NFKB stmcture and function

NF- transcription factor complexes are composeci of a dimer of proteins belonging to the Re1 famiiy [634]. This family of proteins is charactecimi by a Rel- hornology domain in their N-teminus which mediates nuclear localization, DNA binding, dimerkation with other Re1 family mernbers, and dimerrization with inhibitors belonging to the IKB family. The Re1 family of proteins include p65 (RelA), the pSO protein, c-ml, rel-

B and the p52 protein. Each dimer has unique erwisactivating activity and althmgh most appear to act as transcription activators, the p50 and p52 homodimers have been shown to be transcriptional repressors 16341.

NF- is activated by various stimuli that can infiuence ce11 survival. Among these are TCR stimulation, CD28 CO-stimulation,TNFa, IL- 1, LPS, viruses, ionizing radiation, and oxidative stress 1635-6381. Although some controversy on the role of NFKB as a pro- survival or a pro-apoptotic factor still remains, there is now growing evidence that NFK8's primary function is to promote cell survival. In support of this, cells transfected with a constitutively active inhibitor of NFKB, have a defect in TNFa and Fas-induced celi death

1510-513, 639, 6401. in addition, p65lRelA knockout mice die embryonicaly due to massive hepatocyte apoptosis [641,6421-

However, to attribute strictly a pro-survival role to NFKB would be unwise since some studies have found that, in some hybridoma cells, NFKB is necessary for AICD to occur 16431. This contradictory function of NF* is exemplified in transgenic rnice expressing a constitutively âctive inhibitor of NFKB (IKBa). These mice have both a defect in anti-CD3e mediated thymocyte deletion as weii as a decrease in Live CDS' Tceii

[Ml,suggesting that NFKB's activity is probably dependent on the ce11 type as weii as its status of activation.

Much of our understanding on the physiological role of NFKB in Iymphocyte homeostasis has corne from the generation of mice carxying nul1 mutations in NFKB genes.

NF- 1 (p50) deficient mice, fd to activate NF- and to induce Bcell proliferation in response to LPS. However IgM-induced protiferation does occur as a result of the activation of a p65-cRel NFKB dimer [645, 6461. In addition, these rniœ have a selective defect in Ig class switching and maturation. RelB deficient rnice appear normal until 10 days pst-partum, afier which they deveIop a Telldependent phenotype involving thymic atrophy, splenomegaiy, bone marrow hyperplasia, and an abnormal immune response that leads to multiorgan inflammation [647-6491. Furthemore, this phenotype can be exacerbated by crossing the

ReiB deficient mouse into the NF@ l/pSO deficient mouse suggesting that some family members can, at Ieast partly, complement the absence of another [650]. C-Re1 deficient rnice displays defective T and B ceii proliferation in response to mitogens, defective humoral responses, and defective production of IL-2 by T-cells [65 11. AIthough p65melA knockout mice exhibit massive hepatocyte apoptosis [64 1, 6421, RelA, by itself, does not appear to play an essential role in lymphocyte development

[641, 6521. However, a combined role of RelA and the p50 protein in the induction of lymphocyte differentiation factors secretion by bone marrow cells has been reported [652]. Results hmaii these knockout mice suggest thai NFKB complexes have multiple

activities that can reguiak immune responses by influencing lymphocyte homeostasis.

However there appears to be partial functionai complementation between NFKB members.

1.5.7.2 NFKB anti-apoptotic activity

It is thought that NF- exerts its activities by promoting the transcription of

various pro-survival factors. Among these, NFKB has been found to upregulate the

transcription of HIAP 1 (see section 1-5.3) [486488,653] and of the Bcl2 homologue Bfl-

1/Al which can block TNFa-induced apoptosis [654]. Furthemore, NF- can induce the expression of the anti-apoptotic proteins A20 [527], MnSOD 1528-5321, and EX-IL

[533]. The influence of NFKB on the expression of various cytokines that influence lymphocyte homeostasis, such as IL-6 and IL-2, could be another mechanism to explain

NFKB's anti-apoptotic activity [655].

In contrast to such findings, studies suggesting a pro-apoptotic role for NF- have

linked NFKB activation with the downregulation of the Bcl-Xi in thymocytes [644] as weil as with the upregulation of both Fas and Fas-ligand [644, 656, 6571. In view of these contradictory effects, it is likely that the final outcome of NF- signaling will be determined by the sum of these effects as weU as the state that the ceil is in. Hence a ceil

that is preferentially ready for apoptosis might be encouraged to do so by NF*, whereas

in other cetls, NFKB could tilt the balance toward a survival outcome. 1.5.7.3 NFKB regulation

The activity of the NFicB complexes is controiled by their interaction with

regdatory factors that bind to their rel homology domains. Most of the NFKB proteins are

normdy inhibited by an interaction with members of the cytoplasmic inhibitors of NFKB

(IKB) family. These IuBs contain ankyrin repeats that are responsible for their binding to the Re1 homology domain [658]. This interaction masks the nuclear localization signal so that NFKB fails to enter the nucleus. In addition, there is evidence that IicBs can directly inhibit the interaction of NFKB with DNA [659,660].

The farnily of IKBS include IKBa, IieBB, IKBê, IKB~,1-6, and Bcl-3. These members have some specificity for the NFicB complex they inhibit. IKBa and 1-B can regulate p50:p65 and p5O:cRel dimers [658]. The importance of these proteins in regulating NFKB activity has been shown in IKB~knockout mice that have elevated constitutive activity of NF- and die within 9 days of birth due to thymic atrophy, splenic atrophy, and runting [661, 6621. In contrast to IKBa and IKBP, Bcl-3 is found in the nucleus of cells and can bind p52 proteins, however, this interaction appears to promote p52-induced transcription rather than inhibit it (663). The finding that Bcl-3 is implicated in a chromosornal breakpoint from a B-cell chronic lymphocytic leukemia celi line and that Bcl-3 knockout mice have a reduction in B-cell numbers as welI a defect in germinal center formation and in T-cell mediated immunity, further underlines the importance of this protein in regulating lymphocyte homeostasis [664]. In addition to IKBa@ and Bcl-3,

IKBE has been shown to specifically inhibits p65:p65 and P65:cRel complexes and IKBy, which is unique to mature B-cells, is thought to inhibit p50 and p52 homodimers. However the role of these proteins in regulating lymphocyte homeostasis is stiil unclear.

The activation of the NF- complexes, by various ceMar signals, appears to occur via the phosphorylation and degradation of the IKB proteins. This regdatory phosphorylation event occurs on two serine residues in the N-terminai portion of the IKB protein (Ser 32 and 36 of IKBa) [665-6681 which then promote the ubiquitination and ultimately the degradation of the IKB protein by the 26s proteosorne [669-6711. By removing the IKBs, NF- can translocate to the nucleus and activate gene transcription.

The importance of IKB phosphorylation in the activation of NFKB has recently been further supportai by the discovery of two kinases, IKKa and IKKB which form a complex that phosphorylates IKE~on the two regulatory serine residues 1672-6741. Gene targeting of both the IKKcx [675] and KI@ [676]have shown that IKKQ is in fact crucial to hepatocyte survival and to the activation of NFKB by cytokines such as TNFa and IL- 1.

Recent evidenœ suggests that the activation of the IKKs by TNFa or IL-1 is mediated via a third kinase ded IKKyfNEMO (NF-kappaB Essential MOdulator) [677,

6781. Tt is thought that this factor might play a role as a scaffold in linking IKKa and

IKKP to upstrearn proteins that can regdate their activity, such as MEKK 1, a kinase dso shown to activate the JNKISAPK pathway [677, 679-6811. Such kinases might explain the observation that the JNWSAPK pathway and the NFKB pathway are often activated by similar stimuli. Another scaffold protein, sirnilar to NEMO has been identifïed and cded MAP (IK.K-complex- associated protein) [682]. This protein cm mediaie the interaction of ZKKs with the NIK kinase. As has been mentioned previously, NIK appears to reguiate

NFKB activation by the TNF receptor through its interaction with TRAFî [521].

Apparently by increasing the proximity of aU these kinases together, such scaffold proteins

stimuIate the phosphorylation and degradation of IiciBa leaving NF- in its active fonn.

In addition to the regulation by the IKKs, &Ba might also be regulated via the

phosphorylation of a tyrosine at position 42, possibly by the hkkinase [683]. However

the role of this phosphorylation is unclear since one group has suggested that in can prevent

the degradation of IKB [684], while another has suggested that it promotes the activation

of NF- by inducing the dissociation of the IuB:NFuF3 complex [683]. These conflicring

reports demonstrate the need for further research in order to elucidate the role of other

phosphorylation events in the regulation of IKB activity.

Furtherrnore, NFKB regulation cm also occur independently of the IKBs, by

modifying its transcriptional activity. in this respect, PKA has been shown to

phosphorylate the p65 subunit of NF- and, by doing so, to promote NFKB's

transcriptional activity [685]. In addition, the glucocorticoid receptor has been shown to

suppress the transcriptional activîty of nuclear NFKB by an IKB-independent mechanism

[686]. 1.6 The interplay between CO-stimulatory,death, and survival signais

Viewing lymphocytic responses to antigenic stimulation into either activation and proliferation, or survival, or apoptosis is much to simplistic and these processes are not so distinct. As has been discussed in this introduction, similar signaling pathways are often used by various ceceptors that axe tbought to mediate different cellular responses (see

Figure 1 1). In addition, some cellular outcomes have been tightly associated with other cellular processes, such as the association of proliferation with apoptosis. This is also the

case of lymphocyte dflerentiation which can influence the sensitivity to AICD. An example of this is in the differentiation of Th type celis. CD4+ Tcells, upon repeated antigenic stimulation, can differentiate into a Th 1 type

ce11 that secretes mainly IF'-yand IL-2, or into a Th2 type ceil that secretes rnainly IL-4,

IL-5,and IL- l O [687,688]. Various stimuli can influence this differentation including co- stimulation [689]. Interestingiy, Th1 cells have recently been shown to be more sensitive to Fas-induced AICD as compared to Th2 type celis 1690, 6911, possibly due to the expression of Fas signaling inhibitory proteins, such as FAP, by the Th2 celis or due to different outcomes that originate directiy from the TCR signaling [690-692]. Through such findings, we cm begin to understand how various receptors that are involved in co- stimulation, such as CD28, can also sensitize cells to AICD by promoting a Th1 type of differentiation [693, 6941. Similarly, TNFR superfamily proteins, such as 4- 1BB and CD30, which are found to be expressed mainly on Th 1 and Th2 type cells, respectively, rnight also influence lymphocyte AICD by either promoting such a differentiation or by directly participating in Th specific AICD [97,695,696]. The challenge we have trieci to undertake in the studies presented in this thesis was to try to further understand how various receptors involved in lymphocyte CO-stimulation and homeostasis interplay with each other. Our approach to studying this has been to analyze signaling pathway used by these receptors and to study the role of regdatory proteins that influence the outcorne of these signaling pathways in iymphoçyte homeostasis. Figure 11. Death receptors can &en activate both survival pathways and apoptotic pathways. Although death receptors are thought to activate apoptosis, some, such as the TNFRp55, can activate both survivai and apoptotic signahg pathways. The outcome of stimulation of these receptors probably depends on the balance between survival and pro-apoptotic factors in the cell. By modifying this balance other receptors, such as CD30, cm influence the cell's sensitivity to an apoptotic stimuli (see text for detaiis). cascade 1.7 Summary of findings presented in the thesis

The introduction to this thesis has presented various signaling pathways and their use by receptors that are crucial in the regdation of lymphocyte homeostasis. The results presented in this thesis attempts to further understand the mechanisms by which three differen t lymphocyte specific cellular receptors participate in lymphocyte homeostasis.

Although CD28 has been shown to act as a CO-stimulatory receptor, it can ah influence lymphocyte survival and AICD. The second chapter of this thesis attempts to defme a signahg pathway downstream of the CD28 co-stimulatory protein that could explain its infiuence on cellular homeostasis. The data presented in this chapter provides evidence that the sphingomyelinase pathway, known to play a role in promoting apoptotic responses, is also activated by CD28 signaling. CD30 is another lymphocytic receptor belonging to the TNFR superfamily. It has been shown to play a role in thymocyte negative selection as well as in the AICD of mature T-celis. The third chapter of this thesis identifies the interaction of TNF teceptor associateci factors (TRAFs) 1,2 and 3 with the intraceiiular tail of CD30 and defines the consensus binding sequences that are suficient for such interactions to take place. Since TRAFs have ahbeen shown to be important in regulating cellular survival, these findings suggest a mechanism by which CD30 can influence lymphocyte homeostasis by disturbing the balance between survival and death signals.

Finally the last data chapter of this thesis analyzes the physiological function of a novel lymphocyte receptor called Toso. There is evidence that suggests that Toso might be involved in modulating Fas apoptosis and, by doing so, controlling the sensitivity of lymphocytes to AICD. in this chapter, the study of the role of Toso in AICD was done by generating mice carrying nul1 mutations in the Toso gene. However, Our studies bave not, to this date, been able to confirm an in vivo role for Toso in regulating lymphocyte AICD. The ultimate goal of such studies is to try to understand how various receptors that modulate lymphocyte homeostasis can infiuence each other and the outcome of a lymphocytic responses to antigenic stimulation. The hope is ihat some day, such information couid heip design therapeutic strategies aimed at modifying distinct variable involved in lymphocytic homeostasis. Chapter 2: CD2û signals through acidic sphingomyelinase

' The results from this chapter have been published in the Jouniol of Erperimental Medicine 18 1:2059-68 (1995). This publication was a joint effort between our laboratory and that of Dr. M. Kronke. 1 dd sphingornyelinase assays on murine cells and on Jurkat T-celis (Figure 1 E.F) as well as those using endolysomotropic agents mg 4). In addition, 1 did the NFKB band shifts (Figure 7 4B)and the assays on sphingomyelinase CO-stimulatoryactivity (Figure 6). The remaining experiments were pcrformed by people from Dr. Konke's laboratory. 2.1 Introduction

The generation of an adaptive immune response requins the activation of naive lymphocytes that recognize antigens with their specific receptors. However, triggering of the antigen-specific receptor (WR)done appears to be insufficient to activate effector cells and only provides an incomplete stimulatory signal 188, 6971. In vitro and in vivo studies of Tell activation have demonstrateci that additional celi-associated ligands play a key function during the initial phase. Some of these Ligands function to stabilize the physical interaction of the APC and the Tali 1698-7001, but others provide important co- stimulatory signais that are crucial for complete activation [88, 114,701,702J. Co-stimuiatory molecules induce separate signals rhat complement TCR function. TCR complex-initiated signal cascades have been extensively studied. They comprise cytoplasmic protein tyrosine kinases such as members of the src famiIy (Lck, Fyn) and the syk/ZAP-70 family as well as protein tyrosine phosphatases such as CD45 (see section

1.2.2) [25, 26, 3 1, 703-709). In cornparison, studies on CO-stimulatorysignaiing are still at their beginning and are still rather poorly understood 17 10, 7 1 11. One criticai Tqll receptor that has been shown to have CO-stimulatory properties is the CD28 molecule (see section 1-3.2). The physiologicd ligands for CD28,87- 1, and B7-2, have been identifiai as cell-surface molecules expressed by APC [107, 108, 1 19, 7 12, 7131. B7-1 through binding to CD28 can increase cellular tyrosine phosphorylation and activate phosphatidylinositol 3-kinase (PI3 K) [ 154, 7 141. Other early signal transduction events observed after CD28 ligation include activation of the Itk kinase [ 146- 1481, activation of

PLCyi [715], raf-1 kinase [716], p21m [156], and increase of ca2+ and inositol(1.4, 5) P,

[715, 7171 (see section 1.3.3). Studies have shown that CD28 itself becornes phosphorylated at Tryl91, which is essentiai for the interaction with the p85 subunit of

PI3K [ 154, 7 141. As well this site can interact with the Grb2 adaptor protein [ISZ1541. It has been proposed that at least two signaling pathways are coupled to CD28 depending on the degree of CD28 phosphorylation and the state of cellular activation (7 101. This mode1 reconciles previously noted discriepancies of CD28 signal transfer reactions observed with resting T-cells versus Teil blasts or transformed T-cells. It remains to be showa, however, which of these CD28 signaling pathways functions as a CO-stimulatorysignal for the TCR compex.

Co-stimulatory signals are required for expression of the high affmity IL-2 receptor, IL-2 production, and a proMerative T£eU response (see section 1.3) 188, 1 14, 697, 718, 7 191. These events are greatly diminished in T-ceiis of mice lacking CD28 ceil surface expression subsequent to the targeted disruption of the CD28 gene [113]. It has been shown that TCR and CD28 stimulation together lead to the activation of the nuclear factor-kB (NF-), which has been directly impiicated in the transcription of the IL-2 and the IL-2 receptor gene 1114,720-7221. It has also been previously reported that the TNF- dependent activation of NFKB is, at least partly, mediated via an acidic sphingomyelinase

(A-SMase) 1368, 7233. This enzyme catalyzes the breakdown of sphingomyelin to phosphorylcholine and ceramide that eventualiy triggers the proteolytic degradation of 1-KB required for the subsequent nuclear translocation of NFKB (see section 1.5.7.3) [636,

723 1. These observations prompted us to investigate whether the A-SMase is also involveci in the CD28 signal pathway of NFKB activation.

Here we show that CD28 tnggers A-SMase activation in resting as well as in activated T-cells. In contrast, A-SMase is not activated by CD3 Iigation. Overexpression of recombinant A-SMase is shown to substitute for CD28 in tbe NFKB activation pathway.

Evidence is provided that A-SMase triggering by CD28 may represent an important co- stimulatory signal for T-ceIl activation. 2.2.1 Ce11 culture and reagents The basic culture media consisted of a mixture of Click'dRPMI 1640 (50%/50% voYvol) supplernented with 5% FCS, 2 mM glutamine, 0.1 m.P-mercaptoethanol, and 50 pg/mi each of penicillin and streptomycin. The human acute leukemic T-ceU heJurkat

(obtained from the Amencan Type Cuiture Coliection, Rockville, MD) was maintained in culture medium in a humidified incubator containing 5% CO,.

Hamster mAb IgG anti-murine CD28 clone 37.51 was obtained from PharMingen

(San Diego, CA), and hamster mAb anti-murine CD3 (hybridoma 145-2Cl1) was kindly provided by Dr. J. Bluestone (Ben May hstitute, University of Chicago, Chicago, IL). Murine IgGl mAb anti-hurnan CD2 (clone 6Fl0.3), mwine IgG2a mAb anti-human CD3 (clone X35), murine mAb IgGl anti-human CD28 clone 28.2, rabbit IgG anti-hamster F(ab), fragment cross-linking Ab, and goat IgG (H+L) anti-murine cross-linking Ab were obtained from Dianova (Hamburg, Gennany).

Highly purifiai recombinant TNFa was kindly provided by Dr. G. Adolf,

Boehringer Research Xnstitute, Viema, Austria. Monensin, chloroquine,

~g~~cerophosphate,p-nimphenyl phosphate, leupeptïn, pepstatin, and trypsin- chymotrypsin inhibitor were obtained from Sigma Chernical Co. (St. Louis, MO). N- methyl-14C-sphingomyelùifrom bovine origin was obtained from Amersham International (Braunschweig, Germany).

Inbred BALB/c mice were obtained from Bornholtgard (Rye, Denmark). Mice of both sexes ranging in age from 6 to 10 wk old were used. 2.2.2 Preparation of peripheral T-cells Single ceil suspensions of spleens and lymph nodes from BALB/c mice were prepared and 1 x 108 celldml werr diluted in PBS containing 2.58 FCS and 3 mM EDTA For Tde~chment negative selecting columns (MTCC-1000; R&D Systems, Minneapolis, MN) were used following the instmctions of the manufacturer. Human PBL were obtained fiom normal donors by leukapheresis and purifed on Ficoll-Hypaque gradients. For depletion of adherent cells, human PBL were subjected to plastic adherence

cultureci for >10 d in the presence of recombinant IL-2 at 10 U/ml. Greater than 98% of these celis expressed CD3'ICW or CD3+/CD8+ phenotypes as detected by indirect imrnunofluorescence anaiysis (data not shown).

2.2.3 Proliferation assay

50,000 Balbk splenocytes were stimuiated in 200 pl of culhue media with 0.5 pg/ml anti-CD3 or 1 pghl anti-CD28 Wb. At time O and 9 h, ceUs were pulsed with dialyzed acidic SMase fiom human placenta (Sigma Chernical Co.). A-SMase was diaiyzed for 18 hr at 4°C against PBS. Thereafter ttK enzyme activity was quantifmi against standard, nondialyzed SMase activity. At day 3, celis were pulsed for 8 hr with 'H- thymidine (1 pCi/weii). Ceils were hawested and the amount of 'H-thymidine incorporated was measured using a p-counter.

2.2.4 Sphingornyelinase assays Acidic and neutral sphingomyelinase were assayed as recently described (3841.

Briefly ,cells were semm starved for 2 hr in culture medium supplemented with 2% BSA. Aliquots of 5 x ld cells were aeaied at 37°C for the indicateù tims with mAb antiCD28, anti-CD2, or anti-CD3 premixed with cross-linking Ab by incubation for 15 min on ice.

The final concentration each of anti-CI>Z8, anti-CD2, or anti-3 was 2 pg/ml and Utof cross-linking Abs was 8 pg/ml. Stimulation was stopped by immersion of samples in methanovdry ice (- 70°C) for 10 s foiiowed by a 15-s centrifugation in a microfuge. Cells were lysed in two distinct buffer systems as descnbed [384]. The protein content of the supematants was measured using a bicinchoninic acid assay (Pierce Chemicai Co., Harnburg, Gemany) with BSA as standard. Equal amounts of protein (between 15 and 50 pg) from celiular lysates were added to 52.25 pl of reaction buffer containing 250 mM sodium acetate, 1 rnM EDTA (pH 5 .O) for A-SMase measurements. For N-SMase assays, the buffer contained 20 mM Hepes (pH 7.4), 10 mM MgCI, 250 pM ATP. N-methyl-I4C- sphingomyelin (56 mCi/mmol) was added ( 1.1 @hl fina! concentration) and the reaction mixtures were incubated at 37°C for 2 h. The reactions were stoppeci by addition of 800 pl

CHCl,/rnethanol (2:l) and 250 pl H,O. Finally, they were vortexed and microfuged at

14,000 rprn for 2 min. To quanti@ the sphingomyelinase activity, 200 pl of aqueous phase, containing the '4C-phosphorylcholine released by the enzyme, was counted using a

B-counter (Canberra-Packard, Downers Grove, IL) Altematively, the arnount of I4C- phosphorylcholine produced was analyzed by TLC using a solvent system CH,OH/O.S% NaCV NH,OH (50:50:1). Phosphorylcholine, glycerophosphorylchoIine, acetylcholine, and choline were used as standards. The amount of '4C-phosphoiylcholine produced was visualized by autoradiography. 2.2.5 Mass measurements of ceramide and sphingomyelin The neutrai Lipid cleavage product of SMases, ceramide, was measured by chamng densitometry of TLC plates as previously described [368]. Briefly, phospholipids were extracted according to the method descnbed by Biigh and Dyer 17241. Neutral iipids were separated from phospholipids in a two-phase methanol-hexane system. For examination of ceramide, neutrai Lipids were separated by TLX3 using a solvent system of CH3CVCH,0W7N NH,OH/H,O (85: 15:OS :OS). After chromatographie separation of lipids, TLC plates were dried and cooled down to room temperature. Plates were exposed for 15 s to a solution of 10% copper sulphate in 8% aqueous phosphoric acid. The Lipids were charred by heating the TLC plate for 10 min at 175°C. Densitometry of charred bands was performed by two-dimensionai laser scanning (Personal Densitometer with ImageQuant 3.22; Molecular Dynamics, Krefeld, Germany). Calculations of total masses were perfonned by use of an exogenous ceramide standard (Sigma Chernical Co.). Sphingomyeiin was extracted with the phospholipid fraction and analyzed by TLC using the solvent system CHC1,/CH,0H/CH3COOH/H20(100:60:20:5).Sphingomyelin, phosphatidylcholine,,phosphatidic acid, phosphoinositol, phosphoethanol, and lysophosphatidylcholine (Sigma Chernical Co.) were used as standards and visualized by charring densitometry. For quantification of sphingomyelin, charred TU3 plates were scanned by two-dimensional laser,densitometry.

2.2.6 Electrophoretic mobility shift assay . Nuclear extracts were prepared as recently descnbed [723].The protein concentration was rneasured using a bicinchoninic assay (Pierce Chernical Co.) with BSA as standard. The NFKB specific oligonuclwtide, containing the two tandemly arranged

NF- binding sites of the HIV-1 long terminal repeat enhancer (5'- ATCAGGGACïTïCCGCîGGGGACïITCCG-3') and its cornplementary oligonucleotide was synthesized on a DNA synthesizer 38 1A (Applied Biosysterns,

Weitentadt, Germany). The oligo was end labeled with Y-~~P-ATP(Amersham

International) using T4 polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany) and annealeci to a lû-fold excess of its complementary strand to obtain an end- labeled double-sttanded otigonucleotide probe. Electrophoretic mobility shifi assays were performed by incubating 6 pg of nuclear extract with 4 pg of poty(d1-dC) (Pharmacia,

Freiburg, Germany) in a binding buffer (5 mM Hepes pH 7.8,s mM MgCl,, 50 mM KCl,

0.5 rnM dithiothreitol, and 10% glycerol) to a final volume of 20 pl for 20 min at room temperature. The end-labeled double-stranded oligonucleotide probe (1 x 10' to 5 X IO' cpm) was then added, and the ceaction mixture was incubated for 7 min at room temperature. The samples were separateci by native polyacrylamide gel electrophoresis in low ionic strength buffer (0.25~Tns-borate-EDTA).

2.2.7 Plasmids, transfections, and CAT assays

A 4X KB HIVng-CAT reporter plasmid containing four tandernly arranged NFKB binding sites of the WAgenhancer linked to the CAT gene [725] was kindly provided by

Dr. R. Schmid (Ulm, Germany). The reporter plasmid was used to measure functional

NFKB activation in Jurkat celis cotransfected with a human acidic sphingomyelinase expression plasmid pHA-SMase. To generate pHA-SMase, the full-1engt.h CDNA for human A-SMase (kindly provided by Dr. K. Sandhoff) 17261 was cloned in both orientations into the high level mdanexpression phsrnid PEF-BOS (kindly provided

Dr. S. Nagata) [727]. Jurkat cells were transfected by using DEAEdextran with 2.5 pg of the 4X KB HIV/Ig+Adorarnphenicol acetyltransferase (CAT) plasmid and increasing arnounts of A-SMase expression plasmid. The native PEF-BOS plasmid was used to keep a total arnount of 5 pg DNA transfeçted. After transfection, ceils were grown for 2 hr at

37'C in culture medium. Ceiis were then left either untreated or treated with PMA (20 ng/ml) for 4 h. The actuai CAT activities were measured in crude cellular extracts using

'%chloramphe~co~(Amersham International) as substrate followed by TLC to separate the native and acetylafed foms, as describeci [728]. For quantification, autoradiographs were analyzed by two-dimensioaal laser scanning. 2.3.1 Activation of A-SMase by CD28 cross-linking Human resting periphed T-ceiis were stimulated with anti-CD28 antibodies in the presence or absence of secondary cross-linkùlg antibodies and assayed for acidic and neutral SMase activities using exogenous radiolabeled sphingomyelin in a micellar system as recently described [384]. As shown in Figure lA, CD28 cross-linking by anti-mouse IgG antibodies resufted in the rapid induction of acidic SMase activity. Increments of A-

SMase activity were detected as early as within 30 s and peaked at 3 min. In contrast, neither anti-CD28 alone nor secondary anti-mouse IgG enhanced A-SMase activity. Notably, CD28 cross-Linking failed to activate the neutral SMase (Figure 1B). Similar kinetics of A-SMase activation were obtained with CD28cross-linked primary splenic T- ceils derived horn BalblC mice (data not shown). Since PI-3 kinase activation seems to depend on the extent of CD28 phosphorylation and of T-cd activation, we next investigated the effects of CD28 cross- iinking on A-SMase activity in PHA-stimulatecf culturecl human peripheral Tds. As shown in Figure IC, CD28 cross-linking of preactivated human T-ceils stimulated the activation of A-SMase. Notably, the amplitude of A-SMase activation was reproducibly found to be reduced when compared to that obtained with TNF or that observecl with CD28-cross-linked resting T-cells. Unlike TNF, CD28 failed to induce the activation of neutral SMase (Figure 1D). Figure 1. CD28 crosslinking activates acidic SMase in human T-cells. Triplicates of 1x107/ml primary resting T-cells (A and B), 10-d PHA-stimulated T-ceus (C and D), or Jurkat Tcells (E and F) were left either untreated, or were stimulated by CD28

cross-linking (a),or treated with 100 nghl recombinant TNFa (O). (A) Human resting

Tcells were treated with anti-CD28 alone (2 pg/ml) (A) or incubated with the secondary cross-linking Ab (O). Cellular lysates were prepared as described in Materials and

Methods and acidic SMase and neutral SMase were measured usiag radiolabeled I4C- sphingomyelin. The increase in SMase activity is estimated fiom the amount of radioactive phosphorylcholine release (100% = arnount released at time O). Basal levels of I4C- phosphorylcholine production were (mg' protein x h"): 2.5qmol (A), 207 pmol (B), 3 -33 qmol (C), 195 pmol (D), 2.34 qmol (E), and 180 pmol (F). The bars indicate SEM time (min) The Jurkat leukemic T-cell he has been extensively used for studying CD28 signaling. Jurkat Tcek were treaîed with anti-CD28 and acidic as weil as neutral SMase activities were measured. As shown in Figure lE, CD28 cross-linking leads to the activation of the A-SMase. The transient activation of A-SMase by CD28 peaked between 2 and 3 min afier cross-linking, which parallels the kinetics observed with pnmary human T- cells. In addition, both amplinide and kuietics of A-SMase activation were compacabie to the levels induced by TNF. N-SMase activation was not detecteû in CD28-cross-iinked Jurkat Tcelis (Figure IF). These data establisb that CD28 can trigger acidic but not neutral SMase in resting Tells as well as in PHA-activaîed Tcells or leukemic T-ceîis. To confirm A-SMase activation by CD28, water-soluble reaction products of the in vitro miceilar assay system were analyzed by TLC. As shown in Figure 2, CD28 cross- linking elicited an enzymatic activity that exclusively produced "~-~hosphorylcholine identfied by a parailel reaction using exogenous bacterial SMase. '%-choline was not detected, which argues against any phospholipase D activity thai might have contributed to SM hycirolysis in the rnicellar assay system. Furthemore, when mass measurements of ceramide were perfoxmed with anti-CD28-stimulated Jurkat T-celis, a tirnedependent increase of cellular ceramide was detected, which stoichiometrically corresponded to a concomitant breakdown of sphingomyelin (Figure 3). Finaily, Jurkat T-cells were preincubated for 1 hr with the endolysomotropic agents monensin, chloroquine, and NH,CI. These agents have been shown to be inhibitory to the activation of the A-SMase following TNF stimulation [384], leaving the N-SMase unaffected. As shown in Figure 4, each of the endolysomotropic agents cornpletely abolished the activation of A-SMase by CD28 cross-linking. These findings therefore strongly support that CD28 cross-linking results in exclusive activation of A-SMase wi thout engagement of the neutral sphingomyetinase pathway . Figure 2. A-SMase activity in CDZS cross-linked Jurkat T-cells analyzed by TLC.

(A) Jurkat T-cells were stimulated in triplicates by CD28 cross-linking. At the indicated times, cellular lysates were prepared and incubated with '4C-sphingomyelin for 2 hr. The aqueous phase was extracted and analyzed by TLC. In a parailel feaction I4C- sphingornyelin was hydrolyzed by bacterial SMase to produce a radioactive phosphorylcholine standard ('%-Pchol, arrow). The '4C-phosphorylcholineproduced was visualized by autoradiography. (B) The bands of (A), representing the arnount of 14C- Pchol produced, were quantitated by two-dimensionai laser densitometer scanning. The bars indicate SEM (n=3).

Figure 3. SMase activity in CD28 cross-linked Jurkat T-ceIls analyzed by mass measurements of ceramide and sphingomyelin. Jurkat T-cells were stimulateci in triplkates by CD28 cross-linking. At the indicated times, neutral lipids and phospholipids were extracted and seperated by TLC. To visualize neutral lipids and phospholipids, TLC plates were charred. For quantification, the charred bands were read by two-dimensional laser densitornetry. CaIculations of total masses were perfomed using exogenous ceramide and sphingomyelin as standards. The bars indicate SEM (n=3). tim (min) Figure 4. CD28-dependent activation of acidic SMase is inhibited by endolysomotropic agents.

Jurkat cells were incubated for 1 hr in the absence (e)or presence of either monensin (10

diminished the basal A-SMase activity by 45%. Cells were then left either untreated or stirnulated by CD28 cross-linking. Acidic SMase activities were measured. Bars indicate SEM (n=3). 1 1 1 I I 1 O 1 2 3 4 5 time (min] One of the most prominent effects of CD28 is to synergize with the TCR to optimally activate Tds.Strikingly, cross-linking of either CD3 or CD2 did not result in

A-SM- activation (Figure 5). Thus, the A-SMase may represent a CO-stimulatory pathway, by which CD28 complements signahg through the TCR cornplex. To examine whether CD28 CO-stimulation can be substituted for by exogenous SMase, mouse

splenocytes were pulsed at O and at 9 hr with increasing concentrations of A-SMase fiom human placent. (Figure 6). Exogenous A-SMase synergized with CD3 cross-linking to

induce a dose-dependent proliferative response, which was comparable to that observed with cells treated with a combination of anti-CD3 and anti-CD28 mAb. Ceiis not exposed to

any mAb and cells stimulateci with anti-CD28 alone remained in a resting state, ruiing out non -speci fic mitogenic effects of the SMase treatment. In addition, kat-inactivateù A-

SMase did not induce ceU proiiferation (data not shown), indicating the requirement for enzymatic activity. At concentrations >7 pg/d exogenous A-SMase appeared to be cytotoxic. Figure 5. CD3 and CD2 fail to activate A-SMase in resting T-celIs.

Human resting Txek were stimulateci in triphtes by cross-linking of either CD28 (O),

CD3 (O),or CD2 (A). At indicated times cellular Iysates were prepared and andyzed for

A-SMase ac tivity. The bars indicate SEM (n=3). time [min] Figure 6. Cellular response to exogenous A-SMase.

Triplicates of mouse spleen ce11 cultures were left mtreated or stimulated for 3 d witti an&

CD3 (0.5 pg/ml) or anti-CD28 (lpgfml). Ceüs were pulsed at tirne O and 9 hr with exogenous A-SMase at the indicated concentrations. The rate of proliferation was rneasured by 'H-thymidine incorporation. The bars represent SEM (n=3). The results are representative for three independent experirnents. u u u -O 2.5 5 7.25

A-SMase (Ulml) 2.3.2 Overexpression of recombinant A-SMase substitutes for CD28 CO-stimulatorysignals On a molecular basis, TCR and CD28 CO-stimulatorysignahg has been shown to converge at the NFKB [722, 7291, which eventudy precipitaies an increase of IL2 production, IL-2 receptor expression, and enhancement of T lymphocyte proliferation. ConfilTning previous reports [722, 7291, incubation of mouse prirnary Tells with either anti-CD3 or anti-CD28 alone did not result in sinnificant NF- activation (Figure 7). In contrast, combined anti-CD3 and anti-CD28 treatment revealed a strong synergism. The specificity of the retarded complexes was demonstrateci by cornpetition analysis with wild- type and mutated HIV-KB oligonucleotides (data not shown). No synergism of CD3 and

CD28 cross-linking was observed in Jurkat Tcells (Figure 7B). Instead, CD28 cross- linking strongly enhanced phorbolester-induced NFKB activation as previously described

[722, 7291. We next tested whether expression of recombinant human A-SMase can substitute for CD28 signaling. Transfection of Jurkat T-cells with a human A-SMase expression plasrnid, pHA-SMase (Figure 8A), resulted in elevated nuciear NFKB activity (Figure 7C).

The kinetics of pHA-SMase-directed A-SMase activities in Jurkat Txells presented in

Figure 8B indicated that recombinant A-SMase was active within the first hour after transfection. Two-dimensional laser scanning revealed a 1.8-, 2-O-, and 2.1-fold increase of NFKB activity at 1 h, 2 h, or 4 hr after transfection, respectively (data not shown). This suggests that A-SMase on its own can hinction as a weak activator of NFKB. A control plasrnid, PHA-SMase,,, containing the A-SMase cDNA in opposite orientation, did not change NFKB activity. Figure 7. Costimulator effects of CD28 and A-SMase on NFKB activation in mouse peripheral T-cells and Jurkat T-cells. Mouse peripheral T-ceUs (A) or Jurkat Tcells (B) were left untreated or treated for 8 hr in the presence of anti-CD28 (1 pg/ml) and/or anti-CD3 (0.5 pg/rnl) or PMA (5 @mi) at

37°C. (C) Jurkat Tdswere transfected for ihe indicated periods of tirne with A-SM- cDNA cloned into pEF-BOS in both orientations (see Fig. 8A). Nuclear proteins were extracted and assayed for NFKB activity.

Figure 8. Expression of human A-SMase cDNA in Jurkat T-cells.

(A) The cDW for human A-SMase was cloned in both orientations irito the expression plasrnid pEF-BOS to generate pHAsMase and pHASMase,, respectively. (B ) Jurkat T- cells were transfected in tripkates with 5 pg of either pHAsMase ot PHAsMase,. At the indicated times, cellular lysates were prepared and analyzed for A-SMase activity. The bars indicate SEM (n=3). 24 ATG TAG 2249 -1 101 1990

2249 GA1 GTA 24 -f99O 101

time (hl To address bctionally the CO-stimulatorypotential of A-SMase, pHA-SMase and an NFKB-CAT reporter plasmid were cotransfected into Jurkat T-cells. As shown in Figure

9, A-SMase dosedependently enhanced transcription from the EUV/I~-KBelement. In addition, A-SMase cooperated with PMA-induced NFKB activation, which corresponds weU with the synergistic effects of CD28 and PMA on nuclear translocation of NFKB

(Figure 7). in contrast, CD28 cross-linking alone did not result in NFKB activation, nor did it synergize with the overexpressed A-SMase. CD28 ligation slightly increased PMA- induced NFB activation, which is likely due to a presumably higher frequency of CD28- cross-linked cells compared to the fraction of ceils successfully transfected with the pH& SMase plasmid. Figure 9. Overexpression of acidic SMase in Jurkat cells leads to activation of NFKB.

Jurkat T-cells were cotransfected with 2.5 pg of WKg-kB-CAT reporter constmct dong with increasing concentrations of pHAsMase. After 2 hr, ceils were left either untreated

(open bars) or stimulated for 4 hr at 37°C with PMA (20 ng/ml, gray bars), CD28 cross- linking (harchd bars), or a combination of PMA and CD28 cross-linking (FIIed bars).

CAT activity of crude cellular extracts was measured using radiolabeled chloramphenicol. The results shown are derived from one representative expriment (n=4). PMA andi-CD28 antKD28 + PMA 2.4 Discussion

CD28 engagement has knshown to have potentialiy ptimpact on the generation of an appropriate adaptive immune response [88, 6971. This cmcial interdependence between the TCR and CD28 may represent one possible mechanism for peripherai immune tolerance. Here we show that CD28 can induce rapid and transient activation of the acidiç sphingomyeiinase pathway in primary T-cells. Because CD3 triggering did not elicit any ceramide production by A-SMase, this lipid messenger system can be viewed as a distinct CO-stimulatorypathway. Lipid second messenger systerns are emerging as important signaling components in a variet of cellular systerns. One possible entry for A-SMase action in Tail activation likely resides in the activation of the nuckar transcription factor NF-. This notion is confmed by our observation that overexpression of recombinant A-SMase mimicked

CD28 effects on NFKB activation in Jurkat T-cells. NF- controls the expression of a number of genes involved in T-ceii activation. Ln particular, NFKB has been shown to regulate the expression of the genes encoding IL-2 and IL-2 receptor-a chah that are instrumental for Tell proliferation. This suggests that A-SMase represents a major mediator of CD28-induced Txeii proliferation. The mitogenic effects of exogenous A-SMase in the presence of anti-CD3 mAb

(Figure 6) appear to confrrm the CO-stimulatory nature of A-SMase activation by CD28 cross-linking. Interestingly, numerous reports suggest that A-SMase activity leads to cellular apoptosis (see section 1.4.6). Such constrast between these reports and Our findings of a role for A-SMase in cellular proliferation is not entirely contradictory since lymphocyte proliferation has often been linked with apoptosis (see section 5.1.1.1). The level of A-SMase activity and of SM hydrolysis may explain such divergances. This notion is illustrated by the cytotoxicity produceci by exogenous A-SMase used at high concentrations (Figure 6). Although, the identification of a secreted isofonn of A-SMase

suggests thaî this may be a physiological mechanims of A-SMase acitivity [358-36û],we

wish to emphasize that this interpretation deserves caution. Sphingomyelin (SM) is

asymmetricaiiy distributed in the plasma membrane: >9S% of SM is found at the outer leaflet of the plasma membrane [730]. Exogenous SMase treatment rnay thus cause extensive SM hydrolysis leading to membrane damage. Furthemore, SMases are also known as bacterial toxins like hemolysins, which destroy eukaryotic membranes [731]. Thus, it cannot be excluded that the mitogenic effects of exogenous A-SMase rnay be secondary, at least in part, to a cellular stress response. Notwithstanding, expression of

recombinant A-SMase cleary activateci transcription fiom a NFKB promoter. This interna1

treatrnent with A-SMase also suggested thaî A-SMase can mediate CD28-induced Tail activation and proliferation through activation of NFKB.

A number of different types of CD28dependent signai transfer reactions have ben described. However, the specific signaling pathways complementing TCR function have not yet been clearly identifieci. Co-stimulation by CD28 rnay be brought about by several pathways: (a) CD28 ligation rnay provide additional, distinct signaling systems like A-

SMase; (b) CD28 rnay enhance the amplitude or duration of a TCR-triggered signal, thereby reaching a threshold to activate Merdownstream signaling cascades; and (c)

CD28 may provide signals similar to the TCR wmplex yet at different tirne points. This rnay result in repetitive stimulation required to maintain a proliferative or functionai activation status. Unlike A-SMase, other signaling pathways ascnbed to CD28 appear to be also engaged by the TCR. For exarnple, PI3K is also coupled to TCR signaling through tyrosine kinases, such as Lck [732]. Further, PLC-y1 and raf-1 kinase are inducible by cross-linking of either CD28 or TCR [7 15,7 161. Given that the TCR and CD28 obviously share quite a number of signaling cascades, synergistic effects rnay involve any of the above mentioned cooperative pathways. The activation of A-SMase rnay be essential to maintain some degree of specificity of tbe CD28 signal. It is important to emphas'i that

CD28 ligation led to A-SMase activation in restbg T lymphocytes as weii as in Tail blasts or transformed Jurkat Tcells. Thus, A-SMase activation occurs independent of the Tell activation status. This is in contrast to the activation of PUK, which requires TCR-induced phosphorylation of the cytoplasmic tail of CD28 at residue Tyr 19 1 [154,7 141. Interestingly, CD28 cross-linking did not result in activation of neutrai sp hingomyelinase. Neutral sphingornyelinase has been implicated in activation of phospholipase A2 and arachidonic acid degradation [384]- Grachidoaic acid metabolites, such as leukotrienes or prostaglandins, have extensively been implicated in cytotoxicity and inflammatory processes. Proinflarnmatory cytokines like TNF and IL-1 activate both acidic and neutral sphingornyelinase [384]. The stimulation of A-SMase in the absence of neutrai sphingomyelinase activation is tàus a property of CD28, which distinguishes this co- stimulatory molecule from ceU surface receptors for proinflammatory cytokines. Results presented in chapter 2 have revealed that CD28, a CO-stimulatory receptor that promotes T-ceU activation and proliferation, can activate the sphingomyelinase pathway, a pathway that has been associated with apoptosis. These seemingiy controversial fmdings may not be so oppsed in view of the research demonstratûig a liak between proliferation and apoptosis, and the fact that CD28 has been associated with

sensitizing T-lymphocytes to AICD (see section 1 -4.3). In fact, the ultimate outcome of

CD28 signaling is probably dependent on the state of the cells. In support of th, IL-2 secretion, which is dependent on CO-stimulatoryactivity, can promote ceil survival in naïve

and recently activated T-ceils, but can also lead to ATCD in prolonged activaîed cells. Hence in ceils where rnany survival pathways are in place, proliferation in response to CD28 rnay occur, whereas in ceiis where there are too few survival factors, a sensitization to AICD may be the final outcome. Sphingomyelinase most likely participates in the CD28 signal by regulating the balance between survival and apoptotic factors and, in doing so, influencing the outcorne of CD28 CO-stimulation.

Interestingly, these differential effects of CD28 are also found among the TNFR superfaxnily members, which are the fmt cellular receptors shown to reguiate sphingomyelinase activity. Arnong these receptors, the CD30 receptor has been shown to promote Bxeii and Hodgkin lyrnphoma cell proliferation, but can also promote Tell

AICD and thymocyte negative selection (see section 1.4.4). Furthermore, CD30, aithough not having CO-stimulatoryproperties on its own, cm potentiate the CD28 CO-stimu1atory signal [733]. In view of this, we hypothesized that CD30's activity might, at least partiy, be similar to A-SMase activation by CD28 in that it coutd modifi the balance between survival versus apoptotic factors and hence the oucome of T-ceIl antigenic and co- stimulation. The study described in the next chapter defines a signaling pathway downstrearn of CD30 that could support such a hypothesis. Chapter 3: Binding sites of cytoplasmic effectors TRAF1,2 and 3 on CD30 and other members of the TNF receptor superfamily4

The rcsults from this chapter were published in Biochemical and Biophysical Reseach Conlmunications 233592-600 (1997). The work generatcd for the figures prcsentcd in this chaptcr werc al1 donc by mysdf. Scientific and technical advicc was given by the other authors. 3.1 Introduction

CD30 is a ceil surface trammembrane protein which was originaliy identified using a monoclonal antibody directed against Hodgkin' s disease Reed-Sternberg cells 1734, 7351. CD30 is also present on cells from other lyrnphomas such as anaplastic large celi lyrnphoma and some non-lymphoid tumors [303, 7361. However, a direct role or mechanism for CD30 in the development or progression of these tumors has never been clearly established. The CD30 protein is stnicnually homologous to the TNF receptor (TNFR) superfarnily of cellular receptors, which includes 0,TNFR pWp75 and Fas 1302, 3031. These receptors are known to have multiple cellular functions including proliferation, differentiation, and apoptosis [103,231,239,737]. Consistent with the pleiotropic roles of receptors within this family, CD30 induces the proliferation of T-ceU type Hodgkindenved ce11 Lines 17381, and stimulates B ceIl differentiation and proliferation 17391, but can trigger the death of anaplastic large cell lymphomaderived ce11 lines 17381, of T-ceii receptor stimulated hybridomas [234], and of thymocytes [240]. The mechanisms by which CD30 mediates these cellular processes are still unclear. Stimulation of T-ceUs with CD30 ligand or anti-CD30 antibodies has shown CD30 signaling regulates p42 MAPK [740] ar.d NF- [741,742]. This activity has been shown for other rnembers of the TNF receptor superfamily. The CD30 receptor belongs to the group of TNFR superfamily rnembers that lack a death domain (se section 1.3.2). It has long been thought that the intracellular tail of these receptors did not share any hornology, however, many of these receptors, including CD40 and TNFRp75, have been shown to interact with the intracellular TNFR associated factors (TRAFs) (see section 1.5.4) 1491- 493, 499, 5071. TRAFs are defined by the presence of a TRAF domain which can be subdivided into a highly conserved C-terminal region and a lesser conserved N-terminal region [491], In addition, they also contain a ring finger domain which mediates the TRAF;! and TRAF5 activation of NF- as has been shown for CD4û [495, 499, 743,

74-41.

Using a yeast two tiybrid approach to idem@ the cellular effectors mediating CD30 signals, we, and other groups, bave isolated TRAF1, TRAM, and TRAF3, as CD30 interacting proteins [234,495,503]. Furthermore, in order to elucidaîe the mechanism of interaction between CD30 and the TRAF signaling effectors, we have used a series of

CD30 derived peptides in TRAF binding experiments. This has dowed us to define the specific residues within the CD30 întraceiiular tail that mediate TRAF interaction. We also report that similar binding sites capable of binding with the sarne specificity to the TRAF proteins are found in the taiis of TNF receptor p75 and CD4û. 3.2.1 Cells, libraries and antibodies The yeast strain LAO, kindly provided by Stan Holienberg, was used for the yeast two hybrid screen. This strain is deficient in histidine, leucine and tryptophan synthesis.

~galactosidaseand HIS3 gene products are under the control of 8 and 4 lexA operons, respectively. Yeast strain SFYS26 was used to confirm interactions in the GALA system

(Clontech, CA, USA). This strain carries the lac2 gene under the control of the GALl promoter. COS7 celis were obtaiaed froxn Dr. J. Woodgett (Ontario Cancer Institute, Toronto, Canada)- The pBTMll6 plasmid, generaîed by Paul Bartel and Stan Fields, conferring tryptophan selection was used as CD3û-LexA DNA biading domain (LexA DB) fusion protein expression vector. The library used for the screen was obtained from Clontech and consisted of a PHA stirnulated human peripheral blood leukocytes cDNA library fused to the GALA activation domain (GALA AD). In the SFY526 yeast strain the pASl vector containing the CD30 taii was used (obtained hmDr. S.J. Eiiedge) to express the CD30- GAFA DNA binding domain (GALA DB) fusion protein. Unrelated proteins p53 (pVA3, Clontech) and laminin (PLAM, Clontech) were expressed in pGBT9 vector (CLONTECH).

X-gal assays to quanti@ f%galactosidase expression were done using the Galacto-

Light Cherniluminescent Reporter Assay Kit (Tropix Inc., Massachusetts, USA). Antibodies against HA (influenza hemagglutinin protein) and FLAG epitopes

(amino acid sequence DYKDDDDK) were obtained from Boeringer Manheim (PQ, Canada) and Kodak (CT, USA), respectively. Rabbit anti-mouse Horse Radish Peroxidase (HRP) (Amersham,ON, Canada) and anti-Gal4DNA Binding Domain PB) (Santa Cruz, CA, USA) were used in this study according to the manufacturers' protocols. Unless othenvise stated, COS7 celis were cultured with Dulbecco's minimai essential mediuni (DMEM) supplemented with 2mM L-glutamine and 10% FCS at 37°C with 5% CO,.

3.2.2 Yeast two hybrid screen The initiai yeast two hybrid screen was perfonned as previously described [745] using the intraceliular tail of CD30 in pBTM116 vector and the Clontech activated TceU library. Bnefly, CD30 cDNA (obtained from Dr. H. Stein) was digested with

BsaWl/BamHI and cloned in frarne hto pBTMl16 and PAS 1 vectors using NcoI-BsaWI linkers. LAO Saccharomyces cerevisiae yeast were sequentially transfod with pBTM116-CD3û/LexA DNA DBD fusion vector and 250 pg of library cDNA using the lithium acetate/DMSO transfection protocol[745]. The transfomants were grown 16 hrs in Yc media lacking L-tryptophan (Trp) and L-leucine (Leu), washed with TE pH 7.5 (10 mM Tris, 1 mM EDTA), and plated on Yc plates lacking L-Trp, L-Leu, and L-histidine (His).

A total of 5x106 transfectants were plated and grown for 2 days, and His+ colonies were then analyzed for expression of P-galactosidase using a fdter assay and cherniluminescent assay. DNA from positive yeast was recovered by lysis with 2% Triton X-100, 1% SDS, 100 rnM NaCI, IO rnM Tris-CL pH8.0, 1 mM EDTA pH 8.0, and acid washed glas beads.

DNA was then electroporated into E. coli HE3 lû 1. Colonies transfected with library DNA were selected for growth on Leu- Mg-agar plates.

cDNA plasrnids isolated by this method were retransfixted in SFY526 yeast, either alone or with the CD30 tail-pAS1 vector, or with unrelated proteins laminin (pLAM) and p53 (pVA3) fused to Ga14 DBD. Clones that produced a positive X-gal assay only in the presence of CD30 were further analyzed by DNA sequencing.

PAS 1 vectors containing either the N-terminal or the C-terminal portion of uie CD30 tail were constmcted by removing half or the full length CD30 tail with either NcoI- SmaI or SmaI-BamHI, respectively, and using a SmaI-BamHI liaker or NcoI-Sd linker to close the vector up while maintilining the reading frame. These vectors were then used to cietennine which half of the CD30 tail bound the isolated clones by measuring f3- galactosidase activity.

3.2.3 Transfections The CD30 expression vector used for COS ceii transfections was constmcted by inserting the CD30 intraceiiular tail (hm the BsaWI digestion site to the end) into the pEBG vector (Wly provided by Dr. James Woodgett, Ontario Cancer Institute), resulting in the production of a GST-fiised fidi length CD30 tail. The CD30 tail halves were constmcted by digesthg the tail in two parts with Smd. The TRAFl full-length clone obtained from the yeast two hybrid screen was inserted into the marnrnalian expression vector pcDNA3 (Invitrogen, CA, USA) engineered with a 5' HA tag. TRAF;! and TRAF3 full-length clones were inserted into a pRK mammalian expression vector with a 5' FLAG tag (obtauied from Dr. Goeddel, Tularik Inc.). For the transfection of COS ceus, 2x10' ceils were plated in 6 well culture dishes and cultured for 15 hrs. For each transfection, 1 pg of each sample DNA was combined with 10 pl of Lipofectamine (Gibco/BRL, MD,

USA) in 1 ml of Opti-MEM medium (Gibco/BRL, MD, USA). After 5 hours incubation at 37 OC with 5% CO,, 1 ml of medium containing 20% FCS was added. After an additionai 16 hrs incubation, the medium was removed and 2 ml DMEM containing 10% FCS were added for another 24 hrs. Cells were lysed in lm1 lysis buffer (1% NP-40, 140 rnM NaCl, 50 rnM Hepes pH7.5, 20 rnM EDTA, 10 mM NaF, 10 mM Na-pyrophosphate, 0.4 mM

Na-vanadate, 1-8 mghl iodoacetamide, and the protease inhibitors leupeptin ( 1pghl), aprotinin (lpg/rnl) and PMSF (100 ughl)) and solubilized for 15 minutes on ice. Celi

Iysates were microcentrifuged at 14,000 rpm for 15 minutes at 4 OC and the supernatants recovered for use in the binding assays. 3.2.4 Peptides used in binding assays Peptides were synthesized by a solid-phase method using the FmdtBu protocol.

Chain assembly was cbed out at the Amgen Institute (Boulder, CO) using a single coupling program on an AB1 43 1 instrument (Applied Biosystems, Foster Cyity, CA). The crude products were purified on a Vydac C4 reverse-phase preparative HPLC column.

Homogeneity of the final products was assessed by analytid HPLC. Characterization was provided by arnino acid analysis and electrospray mass spectrometry.

Three sets of peptides were used. The fmt set of peptides spanned the full-length

CD30 tail; peptides were 20 axnino acids in length, with overlaps of 5 amino acids. The second set of peptides consisted of 3 arnino acid deletions of those peptides of the fmt set that showed positive interaction with the CD30 tail. FinaiIy, a third set of peptides was constructeci containing single alanine substitutions at each amino acid in the smallest peptide from the second set of peptides that stili bound the TRAFs.

3.2.5 Immunoprecipitations and binding assays The supernatants obtained frorn the lysis of the transfected cells were supplemented with 0.5 % SDS and 1% Nadeoxycholate, and transferred to 10 pl of Glutathione- sepharose beads (Pharmacia, Uppsala, Sweden, #17-0756-Ol), or 10 pi of precoated

Streptavidin-beads (Sigma, MO, USA). STREP-beads were pre-coated by incubation for 1 hour at 4 OC with 1 nmol biotinylated peptide in 1 ml lysis buffer, followed by a wash in

500 pl lysis buffer. To ensure that the same amount of TRAF protein was added to the peptides on the STREP-beads, supematants were pooled and redistributed equally. The supernatant-bead rnix was incubated with rnixing at 4°C for 1 hr, followed by 4 washes in 1 ml lysis buffer. Samples were separateci on 10% SDS-PAGE and transferred ont0 nitrocellulose membranes using a semidry transfer system for 1 hr at 0.8 mNcm2. Membranes were blocked overnight with 5% rnilk dissolved in TBS-T (20 mM Tris pH 7.5, 150 rnM NaCl, 1% Tween 20) at 4 OC. Primary antibody was added at 10 pghl for 1 hr at rmm tempemtwe foiiowed by 4 x 15 min washes with TBS-T. The recommended concentration of secondary HRP conjugated antibody was added for a further 1 hour incubation. After five additional washes, blots were developed with ECL reagents (Amersharn, ON, Canada). 3.3.1 Yeast two hybrid approach using the cytoplasmic tail of CD30 as bait In an a~temptto isolate intmcxlluiar signaling effectors downstream of CD30, we, and three other groups [234, 495, 5031, have used a yeast two hybnd approach to determine proteins that could interact with the cytoplasrnic region of CD30 (residues 408- 595). Transfection of LAO yeast containiag CD30-LexA DNA binding dornain (DBD) with the cDNA library fused to the GAL4 activation domain produced 10x106 yeast transfectants. After 16 hrs of growth in selective medium, 5x 106 transfectants from this amplifîed population were plated for histidine-independent growth. From these, 199 colonies were obtained, of which 125 proved positive in a fZlter assay for B-galactosidase activity. A total of 66 clones were sequenced and 42 of these coded for various fragments of TRAF1,TRAF2, and TRAM proteins. (see Figure 1) Positive interactions were reproducible using a CD30-GALA DBD fusion system.

Isolated clones were assayed for their specificity of interaction using unrelated GALA DBD fusion proteins pLAM (Laminin) and pVA3 (p53) as bait. In contrast to CD30, cotransfection of Iaminin and p53 with TRAF 1, 2 and 3 failed to activate expression of f3- galactosidase (see table 1). Figure 1. TRAF clones isolated in the yeast two hybrid screen.

CD30 intracytoplasmic taii was used as bait in a yeast two hybnd system to screen an activated Tell library. Of 125 positive clones that were picked on plates lacking histidine and expressed B-galactosidase, 66 were sequenced. 45/66 clones were fragments of

TRAF 1, 2 and 3 of various sizes. 1sO 282 416 TRAF 1 I I I

Clones Isolated:

TRAF 2

lsolated : C

356 430 568 TRAF 3 I I l

Isolated: C Total: 45/ 66 Table 1: P-galactosidase activity in whole protein extracts from yeast transfected with various baits and various clones of TRAFs 1, 2 and 3.

Yeasts were ~ransfectedwith expression vectors of GALA DNA binding domain fused to various baits and of GALA activation domain fused to the TRAF clones. Proteins were extracted fiom quai numbers of transfectants and p-galactosidase activity measured using a cherniluminescent assay. Growth on plates lackuig histidine correlateci with P-

galactosidase activity (data not shown). (0, background P-galactosidase activity; + and ++ more than 5x and more than 4ûx induction of pgalactosidase above background, respectively). Growth on plates lacking histidine comlated with P-galactosidase activity (data not shown). Bait DNA Target DNA B-Galactosidase activity

œ + ++ + AIthough the TRAF fragments varied in size, they aii contained a region which included rninimally the TRAF homology domain (see Figure 1). Thnee different types of TRAFl clones were isolated. One such clone coasisted of tbe full length TRAF1. The other two types were vuncated versions of TRAFl lacking the N-terminal portion of the protein, one of them encoding almost only the TRAF-c homobgy region. Clones encoding TRAFî apparently contained a splice variant of a previously isoiated protein found in genbank under the name TRAP3, thought to be the human homolog of mouse TRAF2. This TRAF2 variant differed in 23 residues at the Junction of the TRAF-n and TRAF-c homology domain and was more homoiogous to mouse TRAF2 than the TRAP3 sequence.

The TRAF3 clone isolated was identical to the clone found to bind to CD40 (49 1, 492,

5071 and LMP 1 [SM]. To merdefine the region of CD30 that interacts with TRAF 1, 2, and 3, CD30 constructs containhg the N- or C-texminal half of the cytoplasrnic tail were used in the yeast two hybnd system. These deleted CD30 fusion proteins were assayed for their ability to bind to the TRAF 1, TRAF 2, and TRAF 3 clones fused to the Ga14 activation domain. As shown in Table 1, the C-terminal half of CD30 spanning between residues 504 and 595 could still associate with TRAF 1, 2, and 3 however, CO-transfectionof the N- terminal half (res. 408-504) with the TRAF clones faiied to show p-galactosidase induction.

3.3.2 COS ce11 transfections of CD30 with full length TRAF1, 2 and 3 To define the interaction of CD30 with the TRAFs in mammalian cells, COS-7 cells were transiently co-transfected with GST-CD30 and either full-length HA-tagged

TRAFI or full-length FLAG-tagged TRAF;! or 3. As shown in Figure 2, ail three TRAFs were able to CO-precipitatewith CD30. Neither an unrelated HA-tagged protein (GOS8) nor the FLAG tag by itself was able to bind to the GST-CD30 fbsion protein (data not shown). GST by itself also failed to bind either of the three TRAF proteins (Figure2). There is specificity of the TRAF interaction to the C-tenninal haif of the CD30 since oniy clones containhg the C-tenninal half of CD30 CO-precipitatedthe TRAFs (Figure2).

3.3.3 TRAF binding sites on CD30 Gedrich et al. have previously àefmed the approximate TRAF association sites by analyzing tnuicated versions of CD30 [746] and have suggested that the residues 56'PEQE~MSparticipate in TRAF 2 and TRAn binding. They also propose a TM1and TRAF 2 binding site through homology with TNFRp75. in order to define the exact binding sites on CD30 with which the TRAFs interact, we have used biotinylated peptides spanning the CD30 tail immobitized on streptavidin beads to precipitate full-length TRAF proteins expresseci in COS-7 ceiis. As shown in

Figures 3A, 4A and 5A, two regions in the C-tenninal half of the CD30 tdwere able to bind TRAFs. A peptide spanning CD30 residues 553-573 was able to bind strongIy to TRAF2 (Figure 4A) and TRAF3 (Figure SA), but only minimaiiy to TRAFl (Figure 3A). A second peptide (residues 573-595) was able to bind strongly to TRAFl (Figure 3A) and

TRAFï (Figure 4A), but fded to bind TRAF3 (Figure 5A). An unrelated peptide with a scrambled sequence was unable to bind the TRAFs and no non-specific binding was detectable in non-transfected COS-7 cells. Figure 2: TRAF1, 2 and 3 coprecipitates with CD30 in transfected Cos cells and binds to the carboxyl terminal end of the CD30 tail.

GST-CD30, GST-Ntem Idf CD30, and GST-Cterm haif CD30 were immunoprecipitated from Cos-7 ceils CO-transfectedwith various vectors including HA-tagged full length

TRAFl and FLAG-tagged fuii length TRAFu3. The resulting immunopnxipitate was seperated on a SDS-PAGE gel, electroblotted to nitrocellulose, and incubateà with anti- HA/FLAG antibodies. Bound antibdes were visualized using an HRP-labeled anti- mouse antibody and ECL-HRP detection kit.

Figure 3: Deletion and alanine substitutions within the tail of CD30 binding motif defines the exact binding site for TRAFl. A series of biotinylated peptides spanning the intracellular tail of CD30 were used to precipitate qua1 amounts of TRAF 1 expressed in transfected COS ceils. The resulting immunoprecipitates were seperated on a SDS-PAGE gel, electroblotted to nitrocellulose, and incubated with anti-HA antibodies. Bound Abs were visualized using an HRP-labeled anti-muse antibody and ECL detection kit. A) TRAFl binding to peptides spanning the fuii length tail of CD30. The scrambled peptide consisted of a random aminozid synthesized peptide of similar length. B) W1binding to tnincated versions of peptide

12. C) TRAFl binding to single alanine substitution of each amho acid in peptide 12 -12~. pep. 13 -3N -m -9~. -12N -12c -9C -6C -x Figure 4: Deletion and alanine substitutions within the tail of CD30 binding motif defines the exact binding site for TRAF2.

A series of biotinylated peptides spanning the intt-dceilular tail of CD30 were used to precipitate equd amounts of TRAF 2 expressed in transfected COS cells. The resuiting irnmunoprecipitate was seperated on a SDS-PAGE gel, electroblotted to nitroceilulose, and incubated with anti-FLAG antibodies. Bound Abs were visuahxi using an HRP-labeled anti-mouse antibody and ECL detection kit. A) TRAFS binding to peptides spanning the full iength tad of CD30. The scrambled peptide consisted of a random aminoacid synthesized peptide of similar length. B) TRAF;! binding to truncated versions of peptide 10.11 and 12. C) TRAF2 binding to single alanine substitution of each amino acid in peptide 10.1 1 -6c and 12 - 12c. AAAAAAAAAAAA AAAAAA Figure 5: Deletion and alanine substitutions within the tail of CD30 binding motif defines the exact binding site for TRAF3. A series of biotinylated peptides spanning the intrstceliular tail of CD30 were used to precipitate equd amounts of TRAF 3 expressed in transfected COS cells. The resulting irnrnunoprecipitate was seperated on a SDS-PAGE gel, elecfroblotted to nitrocellulose, and incubated with anti-FLAG antibodies. Bound Abs were visualized using an HRP-labeled anti-rnouse antibody and ECL-HRP detection kit. A) TRAF3 binding to peptides spanning the full length tail of CD30. The scrarnbled peptide consisted of a random aminocid synthesized peptide of similar length. B) TRAM binding to tnincated versions of peptide

10.1 1. C) TRAF3 binding to single alanine substitution of each amino acid in peptide

10.1 1 -6~. pp. 11a EADHT P HYPEOET EP ++++++++++AAAAAAAAAA AA++ More precise chamcterization of essential residues in peptide 553-573 was carried out by repeating the binding experiments witn a series of peptides containhg seriai deletions of 3 amino acids from either end of the peptide (see Figures 4B and SB). Tbe

TRAF binding site was pinpointed to a stretch of 12 amho acids between residues 557 and 567. These residues were then individually mutated to alanine to detennine the residues, in this section of CD30, that are responsible for TRAF;! and TRAF3 interaction. As shown in Figures 3C and 4C, for the peptide "HTPHYPEQETE~,mutations of the residues Y'"

, pl,E'~*, Q'~~, E~,T~~~ had the greatest effect on the binding of CD30 to TRAF2 and 3. Although E"' was necessary for TRAE3 binding, the association of TRAFZ with CD30 was affected to a Lesser extent by this mutation. In contrast, E'~was not necessary for TRAF3 binding but required for TRAF;! interaction. Mutations in residues Pss%d Mdss9 had only minor effects on TRAF3 binding. Sirnilar studies on peptide 573-595 (iocated more C-terrninally) restricted the binding site of TRAFl to residues 576-580, and of M to residues 576-583 (Figures 3 and 4). Point mutation studies of the peptide mSDVMLSVEEEG's3 showed that T'AH binding was greatly affected by alanine mutation of any of the residues LS7', SS7' , v~~~or E"' (Figure 3C). Although TRAFZ binding to peptide n6ML~~~~~~"3appeared to be

Iess affecteci by single alanine mutation, and always showed weak residual binding, changes to residues L'", , v"~,E'~ or E'~' resulted in the greatest decreases in binding (Figure 4C). This data identifies a more N-terminal site for TRAFI and TRAF.2 binding than that proposed by Gedrich et al.[746] and is supported in the main by a study by Lee et al [744]. The latter study differs by only minor variations in suggested binding residues, variations that might be due to differences in the techniques emptoyed in each studies. In our opinion, the use of peptides can minimize the problem of improper folding of proteins and therefore bemr reflects the amino acids that can actudy interact in the binding, rather than those necessary for proper protein folding. However, the downside of such peptide studies is that they may not reflect accurately the importance of protein 3D structures on TRAF binding.

3.3.4 Homologous binding sites in other members of the TNF receptor superfamily Some cellular effects of CD30 stimulation are very similar to those of CDN, including the stimulation of B œll activation and differentiation into immunoglobulùi- secreting cells [738], and the enhancement of the secretion of IL-6, TNF and lymphotoxin- a in Reed-Sternberg ceiis 17471. Thus, it would not be surprising if both receptors utilized a similar signaling mechanism. To determine whether there were similar TRAF binding sequences in other members of the TNF teçeptor superfamiiy, peptides whose sequences were homologous to those shown to interact with CD30 were synthesized and andyzed for their ability to bind Ws.

Since CD40 binds to TRAF2 and TRAF3, but not TRAF 1 (see section 1-5 -4.1 ) , we specuiated that CD40 would contain a TRAE.w3-iike sequence but lack a sequence homologous to the CD30 TRAF1/2 binding site. This was in fact the case; the peptide

245~NT~~~~~~~*in the intnceilular tail of CD40 was found to be most homologous to the CD30-wbinding peptide. This CD40 peptide was able to immunoprecipitate both TRAF;! and TRAM but not TRAFI,whereas sirnilar peptides with less homology were unable to do so (Figure 6). However, the tyrosine residue YSa which is critical for TRAF binding to CD30 is not present in the CD40 binding site, suggesting that the surrounding amino acid environment may have an influence on the specificity of the TRAF binding motif. It is also unknown whether this tyrosine residue is phosphorylated in CD30, and how such a phosphorylation would influence CD30 stimulation. Such differences arnong receptors should allow a differentiation of strategies aimed at blocking these interactions. Similady, since TNFRp75 binds only to TRAFl and TRAM, we speculated that we would find a sequence in the intraceilular tail of this receptor homologous to the CD3Q TRAFlmbinding sequence. The common denominator for TRAFl and TRAFZ binding in CD30 is n7~~~~5".Our results showed that peptide "'KDEQVPFSKEECAI? in the TNFRp75 tail exhibited the closest homology to the

CD30-TRAFlllWW2 binding peptide. This TNFRp7S peptide was able to bind both TRAFl and TRAFi! but not TRAF3 (Figure 6). Another peptide denved from TNFRp75 showing less homology was unable to interact with TRAF 1 or 2 (Figure 6). FIGURE 6: Similar binding sequences are found in the intracellular tail of CD30, TNFRp75 and CD40 and define the specificity of TRAF interactions. By homology search using the CD30 TRAF binding sequences and the intracellular taii sequences of CD40 and TNFRp75, biotinylated peptides spanning suc h homolog~us regions were used to precipitate TRAF 1, TRAF;! or TRAF3 expressed in transfected COS cells. The resulting immunoprecipitate was run on a SDS-PAGE gel, electroblotted to nitrocellulose, and incubated with an anti-HA Wl)or anti-FLAG antibody (TWWî and TRAF3). Bound Abs were visualized using an HRP-labeIed anti-mouse antibody and ECL-HRP detection kit. Position of residues used to design the peptides are as indicated. c~30 SDVMLSVEEEG TRAF112 ,r 573-583 :I :I II Binding motif TNFRp75 : KDEQV PFSKEECAF -L 4 17-430 HTPHYPEQETEP TRAF2/3 cD30~s6-567 : Binding motif I III CD40245-256 : SNTAAPVQETLH 3.4 Discussion

The study of intracellular signaliag pathways has developed into a powemil tool to understand the fuoction of cellular receptors as weiï as to define targets that can be used to interfere with these fûnctions. Our study of CD30 signaling has allowed us to identifj TRAF- 1, -2, and -3 as CD30 downstream effectors and to defme the interaction of these proteins with the intraceliular tail of CD30 as well as with other TNFR superfamily members. In addition to TRAF-1, -2, and -3, TRAFS has also been shown to interact with CD30 at a different binding site [490]. At least TRAFS, TRAM and TRAF6 have been shown to mediate the activation of

NF- by CD30 and other members of the TNFR superfamiiy 1490, 495, 499, 504, 743,

7441. However, the mechanism by which TRAFs are regulated through their interaction with cellular receptors is stiii unclear. The use of agonistic anti-CD30 antibodies has reveaied that the activation of NFKB by CD30 stimulation is not due to an increase in

TRAF recruitment [495]. This suggests that activating CD30 probably causes a conformational change of its intraceUuIar tail, leading to the release of TRAFs or the recruitrnent of other signaling molecules, such as ASK- 1 or NIK (see section 1.5.4.2). which, in conjunction with TRAFs, could participate in NFKB activation. However, such signaiing mechanisms remain to be established. Our resu1t.s demonstrate that an interaction is possible between the TRAF-c domain of TRAFs and CD30, which is in accordance with data from other members of the TNFR superfarnily that also interact with this dornain of the * TRAF molecules 17431. Interestingly, while single but different CD30 binding sites were found for TRAFl

(573-595) and TRAF3 (553-573). TRAFî was found to associate with both sequence motifs. This finding suggests various possible signaling schemes. During the finaikation of these studies, Lee et al. reported that the N-terminal portion of TRAM'S TRAF domain binds to the TRAMnaAF3 binding site on CD30, and that its C-terminal portion binds to the TRAF1- binding site [744]. In view of this, it cm be hypothesized that TRAFZ rnay bind both sites on CD30 sidtaneously, thereby causing a conformational change

within the taii of CD30 that would block or open other binding sites. Altematively, the binding of TRAF;? to CD30 may be criticai for CD30's activity, and the presence of two binding sites could reduce the chance rhat TRAF2 will be outcompeted by the binding of other TRAFs. It is also possible that TRAF2's activity is to diplace both TRAFl and

TRAF3 from the CD30 receptor by competing for these binding sites. Hodgkin's lymphoma cells that express a T-ceil-like surface marker phenotype, when stimulated with CD30, undergo proliferation [738]. The fact that TRAFs interact with CD30 might explain such findings. in fact, a proliferative role for TRAFs is implid by the involvement of TRAFl and TRAE.';! in TNFRp75 signaling [493] that can induce cellular proliferation [737], of TRAF-2,-3, -5 and -6 in CD40 signaling [490-492, 499, 5043 that can promote Bell proliferation [103], and of CART 1, another TRAF domain- containing protein, found to be abnonnaiiy expressed in bmt carcinomas [509]- In addition, TRAFl and TRAM have been found to interact with the EBV receptor LMPl and to play a signifiant role in the cellular transformation induced by this receptor [748, 7491. EBV has been postulated to be an important etiologic agent in Hodgkin's disease and other lymphomas, and can lead to cellular imrnortalization and transformation of B cells [750- 7521. Altematively, it has been suggested that TRAFs' activity might be to block cellular apoptosis by binding to the "inhibitor of apoptosis" proteins cIAP1 and cIAP2 (see section

1.5.3) [473, 4741 or by upregulating the activity of the NF- transcription factor (see

section 1 S.7). In view of the interaction of CD30 and TRAFs, it is somewhat surprising that

CD30 has also been shown to play a role in lymphocyte activation-induced ce11 death [234], in thymocyte negative selection 12401, and in the killing of anaplastic large cell lymphomas

[738]. Possible explanations for these findings have corne from studies showing that TRAF2 aiso binds to the TRADD moiecule which Iinks de& receptors, such as the

TNFRp55, directiy to the caspase cascade (see section 1.4.5.7) 13291. It is thought that by binding to TRADD,TRAF;! signais oppose the pro-apoptotic signaling downstream of the death receptor. Such a hypothesis bas corne hmthe generation of TRAF;! lcnockout mice which exhibit an increased sensitivity to TNF-induced apoptosis [514j. Hence CD30 rnight function in ceIl death by either recruking TRADD via TRAFî or by sequestering

TRAF;! from the TNFRp55 and decteasing its pro-survival activity. Support for the latter hypothesis has comme from studies by Ducketî et al., showing that CD30 signaiing caused the degradation of the TRAFS bound to the CD30 receptor, and in so doing, made ceils more sensitive to TNF-induced apoptosis [250]. If such a mechanisrn does occur, one could envision that in the case where the degradation of these factors is inhibited, CD30 could actuaily signal cell survival and cell proliferation, offe~ga possible explanation for the various CD30 effects that have been reported. Our results using tnuications and alanine sans have shown that, aithough the TNF superfamily receptors do not contain a distinct binding domain, they contain conserved amino acid motifs to which the TRAF domains bind. This is reminiscent of the binding of the SH3 and SH2 domains to their respective short amino acid binding sequences found in various cellular receptors and signaling effectors [753] . As fùrther studies progress to determine the exact role of TRAFs, the precise knowledge of these binding sites, contributed by uiis study, could permit the design of strategies aimed at blocking or stimulating TRAF-receptor interactions. Understanding the mechanism of interaction of TRAFs with CD30 will one day be crucial to devising strategies for modifjing such interactions in cells, and hopefùlly ailow control of the homeostasis of abnormal cells such as those found in Hodgkin's lymphomas and in anaplastic large ce11 lymphomas. The findings describecl in Chapter 3 suggests a pathway by which CDU) can influence ceU surival or ceii death. By regulating TRAFs, CD30 couid rnodiw the balance between cellular survival factors and apoptotic factors. In ceUs where many deatb signals are active and the balance between pro-apoptotic and pro-survival factors is compromised, CD30 by itself could actuaily tilt this balance enough to allow death signals to prevaii. This might be the case of anaplastic large ce1 lymphoma-derived celi lines that die in response to

CD30 signaling (see section 3.1). However in ceUs where ceUuiar survival signals are more stable, the CD30 signaiing may simply act by reducing these survival signais, making the celis more sensitive to additional death signals. This would be the case of the CD30 mediated sensitization of T-ceils to Fas-induced AICD (see section 1.4.4). Finally, in ceils where the breakdown of survivai signal is inhibited, CD30 may actuaily promote cellular survival, allowing the cells to proliferate. This would be exernplified in Hodgkin's lymphoma ceUs that proliferate in response to CD30 signaling (see section 3.1).

This hypothesis of the importance of the balance between swival and death factors in reguiating the outcome of T-cell, in response to antigenic stimulation, suggests many mechanism by which receptors can influence this outcome. CD28, in addition to its co- stimulatory activity, rnay influence this balance by promoting the sphingomyelinase pathway and CD30 probably functions by reguiating the levels of pro-survival TRAF factors. However, there is another activity that could regulate this balance: the inhibition of death signals. This activity would promote cdsurvival. One candidate lymphocyte receptor that has been suggested to play such a role is Toso. This gene has been found to inhibit the Fas signal in Jurkat cells. The fourth chapter of this thesis attempts to define the role of Toso in regulating lymphocyte AICD and the balance between survival and death signals. Chapter 4: Toso deficiency does not alter lymphocyte sensitivity to activation-induced apoptosis

The results from this chapter have not yet been published. 1 have generated, with the precious technical help of Drew Wakeharn, al1 the work that has led to the figures pmiented in this chapter, except for the transfection of murine Toso in various ceIl lines (Figure IC), which was donc by Dr. Gary Nolan's group. The injection of blastocysts for the generation of the knockout mice have been done by Drcw Wakeharn, Annick Itie, and Wilson Khoo. 4.1 Introduction

Participation of activated T-lymphocytes in the immune system is dependent on a delicate balance between survivd and de*. Following antigenic stimulation, COgMte T- lymphocytes are aaivated and pmliferate sufficiently to support an immune response. However, uncontrolled T-lymphocyte activation and proliferation can lead to systemic shock as is observed when strong superantigens hyperactivate large numbers of T- lymphocytes [193]. The immune system usually prevents this overstimulation by inducing apoptosis of T-lymphocytes foltowing prolonged activation, a process caiied activation- induced ce11 death 17541. One farnily of cellular receptors that has been shown to be pivotal in maintaining this homeostasis is the TNF-R superfamily. These receptors have been reporteci to play a critical role in promoting survival as well as promothg apoptosis (reviewed in 1755, 7561). Arnong those capable of inducing apoptosis, Fas is a key player in controiiing lymphocyte homeostasis (see section 1.4.4). Mutations in Fas signaLing lead to 1ymphoproliferative and autoimmune diseases in both human and mice 17571, underlining the necessity for a strict regulation of the Fas apoptotic activity. Because of this, rnany researchers have studied the Fas signaling pathway. Upon Fas engagement by its ligand, the Fas receptor clusters and recruits apoptosis inducing proteins into a death inducing signaling complex (DISC) [758]. Formation of the DISC is dependent on protein interactions involving death domains (DD) and death effector domains (DED) (see section 1.4.5.7) [264, 327, 759, 7601. The DDs mediate homophilic interactions benveen the intraceiiular tail of the Fas receptors and the Fas associated protein with a death domain (FADD/MORTl) 1291, 2921. In addition to a DD, the FADD protein contains a DED mediating interaction with the DEI) of caspase-8 (FLICE, MACH, MchS) 133 1, 332,7611. Caspase-8 is a member of the capase family of proteases and is thought to initiate the caspase cascade leading to cellular apoptosis (see section 1.4.5.5) 12681. Although the deof Fas is to induce apoptosis, tbe presence of Fas and Fas Ligand does not aiways imply the activation of the apoptotic program. In fact, a number of studies have shown the existence of Fas' twnor cek that are resistant to Fas-induceci apoptosis [762-7641. It has aiso been shown that following activation of human T-lymphocytes, cells are resistant to Fas-induceci apoptosis for several days despite upreguiated expression

of Fas [765, 7661. Since in most cases, these ceils constitutiveIy express the proteins known to be necessary for the execution of the Fas receptor signaling, other proteins must

be mdulating the Fas signaling pathway. Arnong these, FAP-1, the Fas-associated phosphatase [767, 7681, and FAF-1, the Fas-associated protein factor [769], have been shown to bind to Fas and either inhibit or potentiate, respectively, its activity. However no mechanisms have been defmed to explain how these proteins act upon the Fas signaling pathway. Insight into mec hanisms that regulate Fas apoptosis signaling have recently been provided by the discovery of the viral FLICE-inhibitory proteins (vFLIPs), that are capable of inhibiting caspase-8 activation [46l, 7701. Mammalian cells were found to contain a homologue of vFLIP called cFLIP (CASH, Casper, CLARP, FLAME, 1-FLICE, MRIT, and Usurpin) [771-7781. There are two isoforms of cFLIP. Each contain the two DED domains of caspase-8, but neither have caspase activity (see section 1S.2).

Overexpression of these proteins was shown to block Fas-induced apoptosis in various ce11 lines [77 1, 772, 775, 776, 7781 by competing for binding of procaspase-8 to the FADD proteins located whithin the DISC [779]. Using an ingenious retroviral genetic screen, Hitoshi et al. have recently discovered

a novel cellular receptor of the immunoglobulin (Ig) supergene farnïiy that can inhibit Fas rnediated apoptosis of Jurkat cells [780]. This receptor, calleci Toso, is normaüy expressed in T-, B-, and NK-lymphocytes. The inhibition of Fas mediated apoptosis by Toso was attributed to its capability of upregulating cFLIP expression upon transfection into Jurkat cells. Being a cellular receptor, Toso is amenable to extracellular stimulation, and, because of this, is a promising candidate for therapeutic approaches to modulate Fas apoptosis and homeostasis of the immune system.

In order to dehe the importance of Toso in the physiological regulation of Teil apoptosis, we have generated and studied mice engineered with a nul mutation in the Toso gene. In this report, we characterize a murine homologue of Toso and show that this gene is expressed rnainly on resting B-cells and on activated T-celïs. The study of the mouse containing a nul1 mutation in the Toso gene shows that the kinetics of Fas killing during T- celi activation are not sienificantiy modified by the absence of Tom and thaï Toso is not necessary for FLIP upregulation following Tell activation. Futher, we demonstrate that infection of the pre-Bcell line 7023 with a Toso expressing retrovirus leads to death of these cells. However, the Toso knockout mouse does not have aberrant nwnbers of B- ceiis, nor is the sensitivity of the B-ceUs to Fas-induced apoptosis altered by the absence of Toso. Finally we also show normal NK cell activity in the Toso knockout mouse. 4.2 Materiais and Methods

4.2.1 Cell culture and reagents The 129J/Ola genornic library was obtained from Dr. J. Rossant's laboratory . The cDNA Iibrary was a mouse spleen libraty from Clontech (CA,USA). The pKJl promoter aven Neomycin cassette has been previously reported [78 11. DNA modify ing enzymes were from Boehnnger Mannheim (PQ,CAN). C57B16 mice and ICR mice were obtained from Taconic (NY, USA) and Harlan Sprague Dawley Inc. (IN, USA) respectively. Knockout mice used in these assays were F2 and F3 hybrids of C57B16 and 129J/Ola and were al1 between 6- 12 weeks of age. Experiments were done by comparing littermattes as controi. Anti-CD3e was purifiai from the 145-2C11 hybndoma, a g& of Jefferey

Bluestone. The anti-IgM used is from Jacksons ImmunoResearch Labs (PA,US A).

Purified anti-CD40 and fluorophore tagged antibodies for FACS were purchased fiom Pharrningen (ON, CAN). Fas-L containing media was obtained from a COS cell expressing a recombinant fusion protein of CD8 extracellular domain fused to the Fas- ligand [782], Lipopolysaccharide (LPS), Staphylococcrrs aureus enterotoxin B (SEB), Stapltylococcus aureus enterotoxin A (SEA), Concanavalin A(ConA), phorbol 12-myristate 13-acetate (PMA), Ionomycin, Mitomycin C, and p-nitrophenyl-phosphate were purchased from Sigma-Aldrich (ON,CAN). Geneticin (G4 18) was purchased from Gibco/BRL (NY, USA). Anti-murine antibodies used to measure basal serum antibody levels were from

Southern Biotechnologies Assoc. (AL, USA). The anti-CD3& and anti-Thy 1.2 producing hybridomas ce11 lines KT3 and AT83 respectively were obtained from the American Type Culture Collection (ATCC). Low toxicity-M rabbit complement, lympholite-M, and sheep red blood cells were obtained from Cedarlane (ON,CAN). Cells were cultured in Dulbecco's minimal essential media supplemented with 10% FCS, 2mM L-glutamine, 50 pm /3mercaptoethanol, Penicillin+Streptomycin. For embryonic stem (ES) cells, optimal concentrations of leukemia inhibitory factor &IF) coataining recombinant COS celï supernatant (a generous gift from Dr. Brady) was added to the culture media

4.2.2 Mouse genomic and cDNA library screening

Phage murine 129UOla genomic and spleen cDNA iibraries were screend using the fuii length human Toso gene 17801. Library screens were performed by infecting LE392

E-coli with Aphage murine genomic librq or with Aphage murine spleen cDNA library and performing 3 rounds of lifts ont0 nitrocellulose filters. Filters were screened each time with a fidl length 32P-radiolabelledhuman Toso probe. From the cDNA library, 5 pure positive phage clones were isolated. Each contained the same cDNA as defuied by DNA sequencing. The genomic iibrary yielded 6 pure positive clones each of which were probed with both a 32~-radio1abeIledoligomer derÎved from the 5' and 3' region of the isolated mouse Toso cDNA. Two clones were found to label positively with both probes. Those genomic fragments were inserted into the pBS K-plasmid vector (Stratagene, CA, USA) and used to produce the knockout constnict.

4.2.3 Knockout construct and screening of the modified gene Short sequences were obtained by sequencing the Iibrary isolated genomic DNA fragments using a series of oligomers derived from the mouse Toso cDNA sequence. A

6.5 kbp fragment was isolated from the 5' end using BglII restriction digestion enzyme and was used as a long arm for the knockout construct. This fragment isolated was confmed by using a 32~-radiolabelledoligomer derived from the sequence. A 650 bp short arm was produced by polymerase chah miction using oligomers derived from the sequence of the 3' end of the gene (S'GTGAATACGTGAGCITGGGCTACC3' and

S'CAAGTGATGG-GGGAï'TACAGTGAA3').The long and short ann were ligated on either end of a Neomycin resistance cassette under the control of the murine pKJ 1 gene promoter in the same orientation as the Toso translation sense [78 11. The site specific insertion of this knockout conscnict into embryonic stem (ES) d genomic DNA was fmt screened by polyrnerase chah reaction (PCR)using primers designeci in the 3' end of Neo and in the genomic DNA flanking region downstream of the 3' end of the short am. This was confirmed by digesting ES cellular DNA with EcoRl and probing with a 32~- radiolabelled polymerase chah reaction synthesized fiagrnent of genomic DNA (284bp' S'CC~GCCAATTTCAGACAACAAC3' and S'TGTITAATATGATGTGTCA- GGCTG3') located in the 3' flanking region of the short arm. The mice were scrieened by PCR using three primers. A comrnon primer (S'CKAAGGCA'ITCAGAT- AGTCAGGTGA3') was located in the short atm region and the two other prirners were from either the 3' region of Neo (5'AGGGCCAGCïCA?TCCTCCCACKAT3') or the 3' region of DNA that was excised by the knockout constmct (S'AACTCT- GCCCCTGCTCC?TCATlTCC3'). In doing so the band obtained from the rearranged ailele was of 400bp and that of the native gene was of 450 bp.

4.2.4 Generation of gene targeted mice D3 ES cells derived fiom 129J/Ola were cdtured on a mitomycin C treated ernbryonic fibroblast feeder layet and in the presence of optimal concentrations of LE, to prevent differentiation. 5x10~ES cells were electroporated with 5 nM of knockout constnict (0.34kV' 250pF) and grown in the presence of 3ûûpg/ml G4 18. Surviving colonies were isolated and screened individually by PCR for gene rearrangement. Positive

ES clones were confmed by southem blotting using the flanking probe. These ES clones were then injected into E3.5 CS7/BL6 derived blastocysts and reirnplanted into pseudopregnant ICR mice. Chimenc male mice produced were bred with C57lB16 females and agouti colored offsprings were screened for the presence of the rearranged allele. These heterozygote mice were then bred together to obtain homozygous knockout rnice. 4.2.5 Northern blotting The mouse multi-tissue RNA blot was obtained from Clontech (CA,USA). The other blots were done by isolating tissue or cellular RNA using Trizol as described by the manufacturer. (Gibco/BRL, NY, USA) 15-20 pg of RNA was separated on a formaldehyde denaturing agarose gel and ethidium bromide staiaed to look for ribosomal

RNA bands and evaluate the level of RNA degradation. The RNA was then transferred to a nitrocellulose membrane and probed using 32~-radiolabeliedfidl length mouse Toso cDNA or FLIP cDNA.

4.2.6 Jurkat and 7023 infection with retrovirus carrying mouse Toso cDNA These infections, as well as the evduation of Tell killing and cellular survival of infected celis were done by Dr. Nolan's group as previously described [780].

4.2.7 FACS analysis

Cellular surface receptors were chosen to distinguish various ce11 population and the cellular activation status. 1x106 celis were stained using saturating amounts of fluorochrome-tagged antibodies. 1Oûûû viable ceils were analyzed using a FACScalibur flow cytometer and Cell Quest software (Becton Dickinson, CA, USA).

4.2.8 Ce11 isolation, activation and apoptosis induction

Ce11 suspension of splenocytes were obtained by using a 70pm mesh screen.

Splenocyte activation was done by stimulating either T-cells or B-cells. T-cell proliferation and activation was done by putting 7.5~10'splenocytes on a 10 cm tissue culture dish in the presence of 1 pg/d anti-CD3 for 36 hrs. Cells were then transferred into media containing 10U/ml of IL-2. CeUs were split to maintain a confluency between 50% and 80%. At various time points, ceils were placed in the presence of various concentrations of apoptosis inducing agents. In the case of Fas-ligand containing media, udess otherwise

stated, 1/5 the final volume was used. In the case of activaîion-induced ceU de&, following 3 days of incubation in IL-2 containing media, cells were restimulated on a plate

precoated with IO pglml of anti-CD~E.In certain experirnents, ceUs were activated using 1

pg/d ConA, 1pg/rnl SEA, or 10 ng/d PM.+ 500 ng/ml Ionomycin for2theindicated time

points. Bceils were activated fiom splenocytes by continuous incubation with either 50 pg/rnl LPS, lûpglrnl anti-1gM or t pglrnl anti-CD40 in the culture da

In the case of pwified T-cells, lymph nodes were isolated and T-cells purified using a Tcell putifkation column as specified by the manufacturer (R&D systems, MN, USA).

The purity obtained using this method was consistentIy between 80-95%. Tells were activated as above, but the starting number of T-cells used was between 1 and 5 x10'. For purification of B-cells. 2x107/ml of isolated splenocytes were incubated with

0.05~volume of supernatant from hybridomas KT3 and AT83 expressing anti-CD3e and anti-Thyl.2 respectively for lhr at 37°C in the presence of 0.1~volume of rabbit compIement. Dead ceils were then removed by gradient centrifugation using Lympholyte- M. By using this approach, 8595% pure B-cells were isolated.

Thymocytes ce11 suspension was obtained using a 70pmesh screen.

4.2.9 Antibody measurements and sheep red blood ce11 stimulation Basal semm antibodies were measured using an anti-rnouse antibody specific ELISA system as specified by the manufacturer (Southern Biotech Assoc., AL,USA). Serurn was obtained from blood by centrifugation in a rnicrotainer serum separator centrifuge tube (Becton Dickinson, NJ, USA). Eliciting of an anti sheep red blood ceil

(SRBC)antibody reaftion was done as previously described [783]. Briefly, 108 SRBC were injected intraperitoneally into mouse on &y O and 14. At day 1, 7, 14 and 20 blood was obtained from their tails and senun prepared as above. ELISA plaies were preaated with 0.25% glutaraldehyde treated SRBCs and blocked with 1% ovalbumin dissolved in

PBS. Various dilutions of mouse serum was added to the plates and the amount of IgG binding was measured as above for the basal serum antibody titers using aikaiine phosphatase conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 antibodies and p- nitrop heny l-phosphate as the alkaline phosphatase substrate. Phosphatase reaction was followed by measuring optical density at 405nm.

4.2.10 NK killing activity

h4ice were injected with 100 pg poly 1-C (Pharmacia, PQ,CAN) intraperitonealy on two consecutive days. On the third day, splenocytes were isolatecl and Live ceiis purifieci by gradient centrifugation using lympholyte-M. These celis were mixed in 96V- bottom plates with 5000 YAC celis preincubated with radioactive "Cr at 3.5 pCi/ml for 1 hour and washed of excess "Cr. Various amounts of splenocytes were added to the YAC cells to obtain the indicated effectoctarget ratio. Foliowing centrifugation of the cells, the supernatant was collecteci and the amount of ''~rrelease was measured using a y-counter

(Canberra Packard) .

4.2.11 In vivo T-ce11 response to SEB

Experiments were done as previously described 17841. Briefly, 100 pg of SEB were injected into the knockout mice. Injections were done by dissolving the SEB in PBS and injecting into the peritoneum of the mice. At various time points, tail bluod was obtained, erythrocytes were lysed in a solution of ammonium chloride, and the remaining ceils were stained for CD4 or CDS and T-ii receptor (TCR) vp8. The œiis were then passed in a FACS machine to determine the percentage of ~$8'ceris present.

4.2.12 Cellular proliferation assays Cells were isolated as abve and 1OOûûû ceUs were plated in a 96 flat bottom weli plate in media containing the indid growth stimuli. At the indicated time points, the ceiis were pulsed for 18hrs with 1pCi of 'H-thymidine and then harvested and washed using a 96 weli Canberra-Paçkard ceIl harvester. The iaçorporated thymidine was counted using a Canberra-Packard B-counter and used as a measure of proliferation. 4,3,1 Mouse Toso cDNA isolation

A cDNA was isolated fiom a mouse splenic library using the full length human Toso cDNA as a probe. Five individual phage clones were obtained and ali contained a sequence from the sarne cDNA. The longest clone contained a full length open reading frame for a gene 68% similar and 61% identical to human Toso (Figure 1A). The leader peptide, the extraceliular Ig-like domain, the hydrophobic transmembrane domain, and the hydrophobic signal sequence found in the human Toso gene were equaliy found in the rnouse gene [780].

To try to confinn that this gene is the mouse homolog of human Toso, we have looked at its expression pattern. The mouse Toso, similas to human Toso, was found at high levels in the lymphocytic compartments and at low levels in the Iungs (Figure 1B). Northerns washed at high stringency showed a major product of approximately 1.5 kb with the presence of two weaker transcripts of higher size. These altemate transcripts are also found in the human Toso RNA blots [780]. This similar expression pattern suggest that the mouse Toso we have isolated is the correct homolog of the published human Toso gene. However, it does not absolutely deout the existence of another homolog. in an attempt to show a functional similarity between both the human and the murine Toso genes, the mouse Toso gene was inserted into a retroviral vector and used to infect the human Jurkat T-ce11 line and the murine 7023 pre-B cell line. Similar to human Toso, upon overexpression, the mouse homolog induced death of the 7023 œll Iine (Table

1A). However, the apoptosis inhibiting activity that the human Toso has in Jurkat cell lines was not conserved with the murine Toso gene (Table 1B). Since this difference could be explained by the artSicial insertion of a murine gene into a human cell line, it was not taken as proof that the mouse Toso lacked Fas inhibiting activity. Figure 1. Isolation, characterization, and expression pattern of murine Toso.

(A) A murine spleen cDNA library was screened using the human Toso gene as probe. Five clones giving stmng signals were isolated. Al1 these clones contained the same cDNA of varying Length. One clone had a fidl open reading frame of 1.2 kbp thaî encoded for a protein that was 68% similar and 61% identical to the human Toso protein. Characteristics of the human Toso protein were also found in the murine Toso, including an Ig-like extracellular domain, a trammembrane domain and an Ïntraceiiular tail.

(B) A commercial addtlembryo tissue northern blot was hybridized using the murine Toso cDNA as probe. For the lymphoid specific tissue blot, the indicated lymphoid organs were isolated from a wild type mouse and the RNA was extracteci. 20 ug of total RNA was separated using a denaturing agarose gel and transferred to a nitroceilulose membrane- The membrane was hybridized using the full-length murine Toso cDNA as probe. Toso expression (indicated by the arrow) was found to be strong in the spleen, lymph nodes, and bone rnarrow. Weak expression of Toso was also identified in the thymus and lung. A 1.2 Kbp

similarity to human TOSO: 68% identity to human TOSO: 61 % Table 1. Effect of Murine Toso overexpression in 7023 and Jurkat ce11 lines.

(A) 2x106 7023 preB cells were cultwed with the vinises indicated above at 32 'C for 12 hours. Cells were incubakd at 37 "Cfor 3 days to aUow integration and expression of each retrovirus. After 3 days without treatment, the number of viable celis was counted with trypan blue. For pBabeMN-Lac2 infected cells, we stained cells with X-gal and calculated the persentages of virus-infected ceîis under microscopic observation. The percentage of Lac2 positive celis was 89 8.Data are expressed as the mean of tnplicate cultures.

(B) Jurkat Tcells were infected with retrovinises carrying pBabeMN-lad, pBabeMN- h4.8 (human Toso), pBabeMN-m4.8-1 to 4 (rnouse Toso). The persentages of lac2 positive cells were counted under microscopic observation of X-gal staining. The percentage of Lac2 positive cells was 38 %. Cells were cultureci with 10 nghl of anti- human Fas rnAb for 24 hours and apoptotic cells were counted with acridine orange and ethidium brornide. The percentage of apoptotic cells are expressed as the mean f SD of tripkate cultures. 7023 cell line. infected virus Viable cells (x1 O-=) Day O Dav 3

Jurkat T-cell fine infected virus % apoptotic cells 4.3.2 Generation of Toso knockout mice Genomic DNA fragments containing the Toso gene were isolated fiom a murine genomic DNA iïbrary using the mouse Toso cDNA as a probe. Sequencing of this DNA revealed that the genomic sequence contains 7 introns within the coding region of the gene.

The knockout construct was assembleci using a 6.5 kbp fragment found whithin an intron located in the 5' leader sequence of the gene and a 0.65 kbp fragment that was synthesized by polymerase chah reaction downstream of the last methionine of the gene. Both fragments were inserted on either side of a Neomycin (Neo) expression cassette. Using this construct, genomic riearrangement causes the Neo expression cassette to replace almost the entirety of the coding region of the gene (Figure 2A). An artificial EcoRI restriction enzyme site was also added which reduces the normal 6kbp EcoRI fiagrnent to a 2.3 kbp size when probed on a southem blot with a flanking probe designed downstream of the knockout construct (Figure 2A). By electroporating this constmct in ES ceils we obtained a homologous recombination rate of 1/125. Two celi lines were isolated that showed rearrangement by PCR and southem: A4D and B2C (Figure 2B). Although the B2C celi line, showed a second integration site when probed with the Neo gene (Figure 2B), the A4D cell luie contained only one single integration site. Both celi lines were injecteci into blastocysts and implanted into pseudopregnant mice. The chimeras produced were bred until germ line transmission occurred in the progeny. These mice were then analyzed for heterozygosity of the rearranged allele. Heterozygous miœ were bred together to obtain homozygosity of the rearranged allele. Screening of the mice were done by PCR (Figure

3A) and confirrned by southern (Figure 3B). Northem blot analysis of various tissues of the knockout mouse showed the absence of any transcript for the Toso gene (Figure 3C). Figure 2. Generation of ES cells that have rearranged with the Toso knockout construct.

(A) The murine cDNA was used to isolate fragments of genomic DNA containing the Toso gene, from a phage library. One isolated fragment contauied the full coding sequeuce of Toso, which was divided into 8 exons. The fmt intron, at the beginning of the gene, was within the leader sequence and contained a 6.5 kpb fragment that was used as a long ami in the knockout constnict. The short amwas generated by a PCR reaction downstream of the last methionine in the coding sequence of Toso. A neomycin selection cassette was inserted between these anns, which generated an extra EcoRI digestion site that reduced the genomic EcoRI digest fragment from 6 kbp, in the wild type allele, to 2.3 kbp, in the rearranged aliele, when probed with the flanking probe downsmof the short arm sequence. In doing so, ali of the fiinctional Toso gene sequence is effectively removed.

(B) ES ceils were electroporated with the Toso knockout constnict and selected in media containing G418. Clones that were positiv~by PCR for the rmanged dele were grown and their genomic DNA was extracted. 15 pg of genomic DNA was digested with

EcoRI or XbaI, seperated on an agarose gel, and transferred to a nitroceiiulose membrane.

These membranes were probed either with the flanking probe or a Neomycin probe to look for the rearranged aiiele (2.3 kbp) and the possibility of a Neomycin random integration event, respectively. Clones A4D and B2C were found to have a correctly rearranged allele, and clone A4D contained a single Neomycin integration site. Genomic: U I- sequenc TM domain Last Met \

1 \ I 6.Skbp t #O a Construct: NE0

TOSO NE0 probe flanking probe (XbalCut) (EcoRI cut) Figure 3. Generation and identification of Toso knockout mice.

(A) Mouse canying the Toso knockout mgeddele can be screened by PCR using a cornmon oligomer in combination with oIigorners thaî are either specific for the Neomycin cassette (400 bp product) or for the deleted endogenous gene (450 bp product). (B) Further confirmation is accomplished by doing a southern DNA blot from isolated tail genomic DNA which is probed using the flanking probe (see legend for Figure 2B). (C) Confirmation of the absence of Toso expression in the knockout mouse was done by isolating total RNA from various tissues and probing them with the fuil Iength Toso cDNA (see legend for Figure 1B). Previous to the transfer of the RNA gel to a membrane for probing, the gels were stained with ethidium brornide and ribosomal RNA bands were visualized to assess possible RNA degradation and to confm that sirnilar amounts of RNA were loaded. e 6 kbp

+2.3 kbp

'pleen LymphNode Thymus Lung Liver

EtBr stain 4.3.3 General status of the Toso knockout mice TIK TOSOknOckout mice were bom in the correct MendeLian ratios and looked healthy (data not shown). The thymus and spleen cellularity were unchanged (data not shown). Thymus, spleen, inguinal and axillaty lymph nodes, mesenterie lymph nodes, and bone marrow were isolateci, stained far the presence of various lymphocytic populations, and compareci to wild type mice. The T£eU populations were unchanged as measured by staining for Thy 1.2, CD3, CM,CDS, CD25, and CD69 (&ta not shown). The Bcell numbers and populations were equally not changed as measured by stainùig for

IgD, IgM, B220 and CD23 (data not shown). Pre Bceli populations were also normal as measured by staining with anti-CD43 and anti-BPI (data not shown). In addition, the presence of NKl. 1 positive ceils was comparable to wild type rnice (data not shown). This data suggests that the Toso knockout mice do not have a defect in the production of the various lymphocytic cells, nor in the maintenance of these cells.

4.3.4 Activation of the T-lymphocytes in the Toso knockout mouse Since Fas kiliing occurs in activated lymphocytes, we first assessed the capability of activating Toso knockout lymphocytes. Titrations of soluble and plate bound anti-CD3& were added to purified T-cells and their cellular proliferation was measured by 'H- thymidine incorporation. Figure 4A shows that an optimal cellular proliferation was obtained at approx. 0.7 pghl soluble anti-CD3& for both wild type and Toso knockout rnice. At this concentration, and at lower concentrations, no significant differences in proliferation was seen between the wild type and the knockout mouse. We did see a difference at higher concentrations of soluble antiCD3e treatrnent in which the ~oso-'-T- cells appeared to proliferate more (Figure 4A). However, this difference seen at higher doses was very suspect because it was not found at the lower concentrations that activated cellular proliferation to a similar dem. In addition, the dinerence did not reproduce itself when the ceiîs were stimuiated with plate-bound CD3e. In fact, in the latter case, an opposite trend was observed (Figure 4%). Furthemore, the trend was inconsistent when foiiowed over various days of anti-CD3e stimulation (Figure 4C). Because of this, we are not convinced that Toso-/- T-celis have a hue significant ciifference in proliferation following anti-CD3e treatment. We cannot, however, mie out the possibility that treaîment with soluble anti-CD3e at high doses can influence cell activation differently than can plate bound anti-CD~Eand that this difference only manifests itseif at a specific time point.

Various Tceli proliferation inducing agents were also used to stimulate cellular proliferation arnong a mixed lymphocytic population. in these cases neither of the stimuli showed a signifiant difference in stimulation ben~eenwild type and Toso knockout splenocytes (Figure 4D). Figure 4. In vitro ~oso"T-ce11 proliferation is normal

T-ceils were isolated from splenocytes and lymph nodes by negative selection on a column that retained B-ceils and macrophages. 100000 T-celis were plated in media containing the indicated growth stimuli. At the indicated tirne points, the amount of DNA synthesis was evaluated by pulsing the cells with 'H-thymidine, washing them, and harvesting them onto fiberglass friters. The level of radioactivity retained on the filters was counted and corresponds to the amount of 'H-thymidine that was incorporated into the cell's DNA.

(A) T-celis were plated for 3 days in media containing the indicated amount of soluble anti-CD~E,after which they were pulsed with 'H-thymidine. The bars indicate SEM (n=6).

(B) Tells were plated for 3 days in media containing the indicated amount of plate bound anti-CD~E,after which they were pulsed with '~-th~midine.The bars indicate SEM

(n=6). (C)

(C) T-ceiis were incubated in 1.25 pghl anti-CD3~for the indicated days, afier which they were pulsed with 'H-thymidine. The bars indicate SEM (n=6).

(D) T-cells were stimulated for 3 days with the indicated stimuli (ConA- 1 pg/rnl, SEA-1 pg/ml, PMA -10 ng/rnl, Ionomycin -500 ng/ml, soluble anti-CD~E-0.7 pg/ml, IL-2 -10

U/d), after which they were pulsed with 'H-thymidine. The ban indicate SEM (n=3).

4.3.5 Activation of B-cells and NK-cells in the Toso knockout mouse B£ells were purifiai fiom splenocytes by Tell depletion and gradient centrifugation. TosoJ- BeUs did not show a strong statistical ciifference in proliferation when cornpared to Toso4- B-ceUs following stimulation with LPS, anti-CWO, or or anti-

IgM over a 3 day pend (Figure 5 A,B,C). This suggests that the Toso knockout mouse has B-ceiis that are functional in their response to various growth stimuli. To look at basai

B-celi fknction in vivo, serum concentrations of various antibody isotypes were measured.

Variability was large between mice, but we did not see a significant defect in producing any of the major antibody isotypes by the Toso knockout mouse eigure 6A). In an attempt to look at the efficiency of Tcell-Bceil iiiteractions in mounting an immune response, sheep red bIood cells (SRBCs) were injected intraperitonealy into mice. The antibody response one sees to this antigen is T-celldependent and involves a primaty IgM production quickly foliowed by class switching and a strong secondary anti-SRBC IgG response [785]. The Toso knockout mouse mounted a similar IgM response to the wild type mice (data not shown) followed by a comparable vigorous IgG response to the SRBCs (Figure 6B). Since Toso had also been shown to be expressed in NK celis (Dr. G.P.Nolan, personai communication), we also checked the activity status of NK ceils in the Toso knockout. To do this, NK celis were preactivated by injecting poly 1-C intraperitonealy into the mice. Splenocytes were then purified and incubated with ''~rlabelld MHCr YAC cells [786]. These ceUs are NK specific targets and the release of "~rfrom thern is a measure of the activity of NK cells. This expriment shows no defect in NK cell activation and lysis activity in the Toso knockout mouse (Figure 7). Figure 5. In vitro ~oso"B-ceIl proliferation is normal

(A) Belis were purifleci by depleting the Tell popdation from splenocytes of Toso wild type and knockout mice. 1OOOOO celis were stimuiated with media containing 50 pg/rnl LPS (A), 1 ug/ml anti-CD40 @), or 10 ug/d anti-IgM (C) for the indicated days.

Foiiowing this, they were pdsed with 'H-thymidine obtain a measure of cellular proliferation. The bars indicate SEM (n=3).

Figure 6. The Toso knockout mouse can generate a T-helper-dependent B-ce11 humoral immune response.

(A) Basa1 serum Ig levels were measuced on two wild type (+/+), two hetorozygote (+/-), and two homozygote (4)Toso knockout mice from isolateci blood serum. The

results are shown as an O.D. vdue at 405 nm obtained using a alkaline phosphatase based anti-mouse antibody ELISA system specific for the indicated antibody isotypes. (B) A T-helperdependent Bxeii humoral immune response was elicited in Toso knockout rnice using sheep red blocxi ceiis (SRBCs). 10' SRBCs, or PBS (as indicaied), were injectecl I.P., at &y O and 15, into wild type (+/+), hetorozygote (+/-), or homozygote (4)Toso knockout mice. At day 1, 7, 4 and 21, tail blood was removed to isolate serum. The anti-SRBC IgG antibodies contained whithin the serum was measured with an ELISA assay using SRBCs as target and an anti-mouse IgG antibody-alkaline phosphatase detection system. Results are shown as the mean O.D. at 405 qm obtained from the ELISA readings. The bars indicate SEM (n=6). +/+ SRBC

+/- SRBC

-/-SRBC

+/+ PBS - +/- PBS -/-PBS

1 st SRBC inj. ~o&t Figure 7. The Toso knockout mouse has normal NK killing activity. Mice were injected I.P. with 100 ug of poly IC for two consecutive days to activate the NK cells. On the third day, splenocytes were isolateci and mixed with 5000

"~rpulsed YAC cells, as NK targets, at the indicated ratios. The amount of "~rrelease was analyzed as a measure of the YAC ceil lysing activity of the NK cells. Spontaneous "Cr release was rneasured from YAC cells incubated in the absence of splenocytes. The maximal "Cr release was obtained by chernical lysis of the YAC cells. Spontaneous release was less than 10% of the maximal release. Results are shown as 46 specific "~r release=(*'~rrelease - Spontaneous ''~rrelease) / maximal release. oogg9,0,n%zs Splenocyte:YAC cell ratio 4.3.6 Status of various apoptosis stimuli in the Toso knockout T-lymphocytes

Since the defect in Tell apoptosis was the major fuiding in the original report on the characterization of the huiction of the human Toso molecule, various approaches were used to look at Tcell apoptosis. We fmt looked at the kinetics of Toso expression in lymphocytes. Although Toso is highly expressed in splenocytes, this expression is almost entirely due to the presence of resting B-ceUs (Figure 14A). Resting Telis did not express Toso (data not shown). Splenocyte Toso RNA levels drop following 36 hours of stimulation with anti€D3&, presumably due to a decrease in the percentage of Boelis

(from 55% of the population at day 1 to 20% of the population at day 2). Foliowing this, incubation of the splenocytes in IL-2 containing media for 1 day, which stimulates Tells to proliferate, was associated with a strong increase in Toso RNA levels reaching a maximum at day 2. Finaily this upregulation of Toso RNA in the activated Tcells decreases and disappears by the fourth day in IL-2 (F3gure 8). This short kinetics of Toso upregulation following Tceli activation was also seen in the original characterization of human Toso [780]. During our activation protocol, CD4' cells became sensitive to the presence of Fas on their surface and died when Fas-ligand was added exogenously (Figure

8). However, activated CD8' cells were unaffécted even though Fas was expressed on their surface. This is in contrast to freshly isolated and non-activated lymphocytes which become rapidly sensitive to Fas (data not shown). In view of this finding, the defect in

Fas-induced killing that we see in activated CD8' T-cells could represent the lag period that has ken suggested to happen in human peripherai blood lymphocytes stirnulated with PHA

(7661. Although we did see a trend of increased Fas killing sensitivity of CD4' ~oso*'- cells at early time points following activation, we were unable to show that this difference was statistically significant (Figure 8). Wetherefore conclude that the absence of Toso did not signifïcantly increase the sensitivity of either activated CD4' or CD8+ ceils to Fas stimulation (Figure 8).

The Fas killing activity was also assessed by activating lymphocytes with anti-

CD~E,incubatïng them in IL-2,and at various times, restimuiating them with anti-CD3&

This second restimdation induces a Fasdependent Tcell apoptosis [198-200, 2321. Our data shows no difference in death of either ceii types between the wild type mouse and the

Toso knockout mouse (Figure 9A). Ceiis stimuiated with continuous anti-CD3~aIso become sensitive to Fas killing and we did not see a defect in either CD4' or CD8' killing following this continuous stimulation (Figure 9B). In an attempt to look at other stimuli that can activate T-cells to undergo apoptosis, Fas-ligand was added to splenocytes activated with various Tdactivation agents for only one day, a time where Toso should be expressed. No ciifference was seen in the ~oso"

T-cells stimdated with either anti-CD3~anti€D3&+IL-2, PHA+IL-2, or PMA+Ionomycin

(Figure 10). Of these stimuli PMA+Ionomycin had been shown to strongly upregulate the transcription of Toso RNA [780]. The effect of Fas killing on these activated Jurkats was decreased by this trieatment and it was suggested that this might occur via Toso's upregulation of FLIP RNA levek However, we did not see any difference in apoptosis between CM' ceils of the Toso knockout mouse and wild type mouse following their stimulation by these agents (Figure 11A). As well, Tdsshowed a strong induction of

FLLP RNA foliowing PMA+Ionomycin treatment even in the absence of Toso (Figure 1 1B). Figure 8. The Fas-àependent AICD of ~oso"-T-cells is not significantly differeat than that of wild type cells.

Splenocytes were isolated from wiid type (+/+), heterozygote (+/-), or homozygote

(4)Toso knockout mice and cultured, at day 0, in media containing 1 pglmi anti-CD3~ antibody. After 36 hrs they were transferred in media containing 10 U/ml IL-2. At the indicated times, the ceiis were harvested and either incubated with Fas-L containing supernatant for 24 hr (A), stained for the expression of Fas (B), or, in the case of wild type cells, used to purify RNA and do a northern using full length Toso cDNA as probe (see legend for Figure 1B) (C). In the case of Fas-L treatment, following the incubation with Fas-L, the cells were stained for CD4 CD8 and with 7-AAD.Results shown represent the mean % decrease of live (7-AAD-) cells, as compared to unûeated controls, arnong the

CWand CD8'population (as indicated). Multiple concentrations of Fas-L were tested in this assay but only the results obtained from an optimal concentration are shown here (see

Materials and Methods). The bars indicate SEM (n=6). +/+ CD4

+/- CD4

-/- CD4

+/+ CDS

+/- CD8

-1- CD8 s

FAS expression: Figure 9. Various protocols of AICD fail to demonstrate a difference in ceIl death between ~oso"'and wild type T-cells.

(A) CeUs from wild type (+/+), heterozygote (+/-), md homozygote (-/-) Toso knockout mice were prepared as in Figure 8, but instead of using Fas-L to induce apoptosis, the ceiis were reincubateci, at the indicated times, with 10 ughl plate-bound anti-CD~Efor 24 hr. Following this incubation, the cells were stained for CD4, CD8 and with 7-AAD. Resuits shown represent the mean % decrease of iive (7-AAD-) cells, as compared to untreated controls, arnong the CD4' and CDS' population (as indicated).

(E!) Isolated splenocytes were treated with continuous soluble anti-CD3e antibody

(1 pg/ml). At the indicated times, they were incubatecl with Fas-L containing supernatant for 24 hrs. Following this incubation the cells were stained for CD4, CD8 and with 7-

AAD. Results shown represent the mean % decrease of live (7-AAD-) cells, as compared to untreated controls, among the CMand CDS' population (as indicated).

Figure 10. Fas killiag of ~oso" T-cells Collowing vanous activation stimuli is normal.

Splenocytes were isolated from wild type (+/+), heterozygote (+/-), or homozygote

(4)Toso knockout rnice and incubated with the indicated stimuli (soluble anti-3~ -1 pg/ml, IL-2 -10 U/d, PHA - 1 pg/rnl, PMA -10 ng/ml, Ionomycin -500 ng/ml). After 24 hr, Fas-L containhg supernatant was added to the cultures for 24 hrs foilowing which the celis were stained for CD4 CD8 and with 7-AAD. ResuIts shown represent the mean % decrease of iive (7-AAD-) CD4' cells, as compared to untreated controls.

Figure 11. PMA+Ionomycin stimulates normal FLIP expression in the

TOSO"- lymphocytes and sensitizes them equivatently to Fas killing.

(A) Lymph node cells were isolated from wild type (+/+), heterozygote (+/-), or homozygote (4)Toso knockout mice and incubated with PMA (10 ng/mi) + Ionomycin (500 ng/mi). At the indicated times Fas-Lcontaining supernatant was added to the cultures foliowing which the ceiis were stained for CD4, CD8 and with 7-AAD. Results shown represent the mean % decrease of Live (7-AAD-) cells, as compared to untreated controls, among the CWand total cell population (as indicated). The bars indicate SEM (n=6).

(B) Total RNA was isolated from lyrnph nodes, purified lyrnph node T-cells, or ceil treated as abve (section A) for 24 hrs from wild type (+/+) or homozygote (-/-) Toso knockout rnice (as indicated). The RNA was used to do a northern using fùil length FLP cDNA as a probe (see Iegend for Figure 3C). &Y 1 day 2 day 2

-1- To assess the Fas pathway in vivo, SEB was injected into mice and the deletion of

TCR 48' celis in the blood was followed over a period of 16 days. SEB binds

specificaiiy the MHCII molecule to the 438, especially vp8.1/8.2/8.3, as weii as to the vp3

[787]. This interaction with tbe TCR is independent of the antigenic specificity of this receptor [788]. nie injection of SEB in mice induces an initial proliferation of the TCR

vp8' population as the cells are activated, followed by gradua1 deletion of the ceiis that is at

least partiy mediated by Fas-Fas ligand interactions [787, 789-79 11. In our expriment the

deletion can be easily seen by looking at the CWpopulation (Figure 12A) and the initial proliferation can be best seen in the CD8+population (Figure 12B). In either case we coufd not see a reproducible difference between T-cells from the Toso knockout mouse and the

wild type mouse. The 46' population was unaffécted by the treatment (data not shown).

These results suggest that Toso knockout mice have neither a defect in penpheral TdlFas

killing in vitro nor in vivo as measured be these various assays.

4.3.7 Thymocyte apoptosis in the absence of Toso Apoptosis is an important rnechanism of negative selection in thy mocytes. Cellular staining with CD3, CD4, CDS, CD25, and CD44 did not show any difference in the

thymocyte populations between the wild type mouse and the Toso knockout mouse (data

not shown). However, the presence of Toso RNA in thymocyte led us to study various apoptotic stimuli in thymocytes. The CD4TD8' population of thymocytes has previously

been shown to be the primary targets of negative selection (reviewed in [246]). In fact

hyperstimulation of the antigen binding complex on thymocytes using anti-CD~Einduces

apoptosis [792].Upon in vitro addition of this antibody to freshly isolated thymocytes, we did not see any difference in apoptosis of the CD4'CDS' population between the Toso knockout and wild type mice (Figure 13). Figure 12. In vivo stimulation of AICD via SEB activation of T-cells is normal in the Toso knockout mouse.

In vivo AICD of activated T-celis was analyzed by injecting I.P. either PBS or 100 pg SEB into wild type (+/+), heterozygote (+/-), or homozygote (-/-) Toso knockout rnice

(as indicated). At the indicated times, tail blood was removed, efythrocytes were lysed, and the remainïng blood cells were stained for CD4 and CD8 and for the TCR vp8.1/8.2 and 436. Results show the Iv$8.1/8.2+ ceils arnong the CM+ and CD8' ceil populations. The $6' populations were unchanged by the treatment (data not shown). ++/+ PBS ++/+ SEB ++/- SEB --1- SEB & -1- SEB

days

+/+ PBS

A +/+ SEB +/- SEB +-1- SEB 4-SEB

days Figure 13. Induction of thymocyte death by CDS&and Fas stimulation i s not aitered in the Toso knockout mice.

Thymocytes were isolated from wild type (+/+) or homozygote (4)Toso knockout rnice. The celis were left either untreated, treated with anti-CD3 (10 pg/rnl), or treated with Fas-L containing supernatant for 24 hrs, following which they were stained for

CD4 CD8 and with 7-AAD. Results shown represent the mean % Live (7-AAD3

CD4+CD8+celJsamong the total ce11 population analyzed. The bars indicate SEM (n=3). FAS-L Although thex is controversy on whether Fas plays a role in thymocyte seleetion, the CM'CD8' double positive population is sensitive to Fas stimulation [793]. Upon addition of Fas-ligand to thymocytes, the Toso knockout mouse cek died as efficiently as the wild type thyrnocytes (Figure 13). A recent report by Yeh et al. has suggested chat

ConA stimulation of thymocytes can block Fas killing via the upregulation of FLIP RNA

[452]. In view of these findings we waated to find whether this biock in Fas kiiling by

ConA was mediated via Toso. However, contrary to their findings, we were unable to see a block in Fas killing by stimulation with ConA of the thymocytes from our mice (data not shown). Our inabililty to reproduce these results might be due to the use of a recombinant Fas-L protein rather than a anti-Fas antibody. It has, in fact, been previously reported thai certain celis are more sensitive to Fas-L than to anti-Fas antibody[794]. In addition, the mice strains that this group used were dso different than ours which might also explain our inability to reproduce their findings. Because of this we were unable to assess the role of Toso in ConA inhibition of Fas killing.

4.3.8 The effect of the absence of Toso on the apoptotic response of activated B-cells to Fas We first attempted to look at the apoptotic response of B-cells in the absence of Toso because of the finding that overexpression of Toso in the pre-B ceil iine 7023 cooid induce them to undergo apoptosis (Figure 1C). RNA expression of Toso was found primarily in resting B-celis purified from the spleen. However following LPS activation of these B-cells, the Toso RNA levels rapidly dwreased and disappeared within 48 hours (FigureI4A). The presence of Fas on these celis appeared at the first day of stimulation and al1 cells were found to express Fas afier 2 days of stimulation with LPS (Figure 14B).

Although this was the case, we did not see a reproducible difference in Fas killing of Tosa-

'-Bcells following either LPS or antiCD4û stimulation (Figure 14B). The activated B- cells from the Toso knockout mouse did not show a difference in sensitivity to Fas-ligand added at various concentrations (Figure 14C). Wewere therefore unable to fmd an in vitro difference in sensitivity to Fas kihgof BceUs lacking Toso. Figure 14. Fas expression aad killing in activated B-cells is not affected by the lack of Toso.

(A) Total RNA was isolated from splenocytes, purified B-cells, or B-ceils activated for the indicated times with LPS (50 p@ml ) from wild type (+/+), heterozygote (+/-), or homozygote (4)Toso knockout mice (as indicated). The RNA was used to do a northern using full length TOSO cDNA as a probe (see legend for Figure 3C).

(B) Splenocytes from wild type (+/+), heterozygote (+/-), or homozygote (-/-) Toso knockout mice were treated with either LPS (50 pg/ml) or anti-CD.QO Ab (10ug/mi) for one or two days. At the indicated days, the celis were either stained for Fas expression (lower panel) or were treated with Fas-L for 24 hrs (bar grayh). Following the reatment with Fas-

L, the cells were stained for B220, as a Be11 marker, and with 7-AAD. Results shown represent the mean 96 decrease of live (7-AAD] cells, as cornpanxi to untreated controls, arnong the £3220' popuiation (as indicated). --

(C) Splenocytes from wild type (+/+), heterozygote (+/-), or homozygote (-/-) Toso knockout rnice were treated with LPS (50 pglmi) for 2 days. After the fmt day, various volumes of Fas-L containing supernatant was added to the culture for anorher 24 hrs (as indicated). Following the reatment with Fas-L the cells were stained for B220, as a B-ceii rnarker, and with 7-AAD. Results shown represent the mean % decrease of live (7-AAD-) cells, as compared to untreated controls, arnong the B220'population (as indicated). Splenocyte LPS activation 8 a +/+ +/- -1- fi$ ha III-

Fas-L day 1 -> 2 I

,+l+ -1- - +/+ -1- ,;+/+ -1- , LPS treatment anti-CD40 treatment

Volume of FAS-L culture 208 4.4 Discussion

Many diseased states can occur due to inappropriate homeostasis of the immune system including the growth of unusual cancers, autoimmune diseases, lymphoproiiferative diseases, and states of imrnunodeficiency. Because this critical balance of the immune system is dependent on, not only the activation and proliferation of lymphocytes, but also their control through apoptosis, many stuclies in the last few years have atîempted to understand how Fas' apoptotic signal is controiled. in a search for proteins that could inhibit the Fas apoptotic signal, Kitoshi et al. isolated the human Toso cDNA encoding for a transmembrane protein of the imrnunoglobulin supergene famiiy. This group showed that the overexpression of Toso not only inhibitecl the Fas killing of Jurkats but also upregulated the expression of the Fas inhibitory protein cFLIP [780]. Furthemore, the expression pattern of Toso suggested bat this protein is only expressed at early time points following activation of Jurkat Talis and that the expression is lost after prolonge. stimulation. Previous snidies had show that Tcells isolated from human blood, rapidly upreguiated Fas expression on their surface, but were resistant to Fas-induced apoptosis during the first few days following activation by phytohernagglutinin (PHA) or IL-2 [765, 7661. Susceptibility to Fas was only restored after incubation for several days in IL-2. Since this resistance to Fas correlated with high expression of Toso, it was suggested that Toso might be responsible for this block in apoptosis seen in recently activated T-lymphocytes. Attempting to answer this question was the main purpose of Our study of the Toso knockout mouse. If Toso is criticai in blocking Fas kiüing in mntly activated T-lymphocytes, one would expect that the absence of this molecule would make T-lymphocytes more sensitive to Fa-induced apoptosis at early time points foliowing th& activation. Our attempts to look at this were hindered by the fact that, in Our hands, activation of freshly isolated murine T-lymphocytes did not result in a general inhibition of Fas-induced killing.

Although CD8' cells clearly were resistant to Fas-induced apoptosis, the CD4' cells showed a sensitivity to the Fas-ligand that appeared to conelate with the expression of Fas on their surface (Figure 8). This is in accordance with previous studies that have shown that Fas acts primarily on CD4+ cells whereas CD8' celis are more sensitive to TNF- induced apoptosis [201]. The studies showing a defect in Fas-induced apoptosis of early PHA or IL-2 activated human peripheral blood Tdsreported a generalized resistance to Fas-induced apoptosis, however they diâ not indicate whether the cellular population they were analyzing was mostly CD8' or CD4' ceils [765, 766). Our frnding that only murine CD8' activated T-lymphocytes are strongly mistant to Fas-induced apoptosis could be due to either a difference between the human and the murine T-lymphocytes, or because tbe previous studies used isolation and activation protocols that prirnarily targetted CD8+ ceils. It must also be mentioned that although our activation of CD4' ceus did not completely abrogate Fas killing, the fact that non-stimulated resting T-lymphocytes becarne highly susceptible to Fas kiliing, even though their expression of Fas was lower than on activated ceUs (data not shown), suggests that activation does, in fact, temporariiy reduce the sensitivity of Cm' cells to AICD. However, neither the activated CD8' or CM+~oso-'- T- ceiis were significantly more sensitive to Fas stimulation at any point following activation suggesting that Toso is not criticai for this inhibition of Fas-induced apoptosis in T- lymphocytes. This data does not necessarily contradict the human Toso findings since such findings were done by overexpression of the Toso protein. It is possible that Toso is only partly responsible for activation of inhibitors of the Fas pathway, such as cFLIP, and that its rernoval does not completely abrogate the block in Fas killing. This hypothesis is supported by a ment report showing that many activators of the mitogen-activated proiein kinase pathway (MAPK) can block Fas-induced apoptosis of thymocytes and peripheral T- ceils by inducing the expression of FLIP via activation of MKKl [452]. This block was inhibited by the use of a MKKl inhibitor or a dominant negative forrn of MKK1. To rule out the possibility that increased FLIP RNA transcription induced by activation of the MAPK pathway was occurring via upregulation of Toso, we looked at FLIP transcription in the absence of Toso. Our results show that T-lymphocytes lacking Toso, but stimulated with PMA and Ionomycin, can upreguiaie FLIP RNA transcription as efficiently as wild type T-celis (Figure 11). Since PMA and Ionomycin can also activate the MAPK pathway, it is possible that multiple mitogenic stimuli could participate in blocking Fas-induced

apoptosis and that Toso might only be one of them. Fuaher insight would be gained by determinhg whether overexpression of Toso cm aiso lead to MAPK pathway activation. Many studies have recently concentrated on cFLP expression to try to explain various Fas insensitive T-celi phenotypes. IL-2, for example, has been found to be crucial for the passage of recently activaîed T-cek hma Fas insensitive to a Fas sensitive state

12081. A recent report has suggested that this IL-2 activity is the consequence of a

downregulation of cFLP [219]. However, the actud role of cFLIP in lyrnphocytic apoptosis remains very controversial. Although initial reports suggested that riecently activated T-celis were insensitive to Fas apoptosis due to the expression of the cFLIP

proteins, a more recent report lmking at protein rather than RNA levels, reveal that cFLIP is expressed to simiiar levels in the recently activated Fas insensitive lymphocytes as in Fas sensitive lymphocytes that have been activated for prolonged periods of time [77 1, 7791.

The expression of cmprotein was also not affected by the presence of IL2 at various concentrations. Scaffidi et al. further suggested that the level of FLIP expression found in activated and non-activated T-lymphocytes is not high enough to block Fas-induced apoptosis [779]. Finally they report that, instead, the recently activated T-lymphocytes have a more generalized defect in formation of the DISC and that neither FADD, caspase-8, or cFLP are efficiently aggregated into this complex even in the presence of sufficient Fas receptor stimulation. Cells having reduced DISC aggregation appear to necessitate involvment of the mitochondria to elicit efficient Fas-induced apoptosis (see section I .4.7), contrary to strong DISC forming ceiis that cm directly activate the caspase cascade [295]. It remains possible that the inhibition of Fas by Toso overexpressed in Jurkats is not only due to an increase in cFLIP transcription, but also due to the modification of other regdatory factors. In this respect, Scl-M is highly expressed in recently activated Tells and has been shown to block mitochondrial-dependent apoptosis [136, 295, 7951. As well, RNA fiom the Fas inhibitory phosphatase FAPl gene has been found to be downregulated in Tells following IL-2 stimulation, giving another possible explanauon for the cells becorning sensitive to Fas foliowing prolonged incubation in IL-2 17681. Studying these regdators of Fas signaling following Toso stimulation could prove to be use ful in understanding Toso's effect. Our results do not support a signfïcant and non-redundant role for Toso in regulating lymphocyte MD. Furthemore, the results using SRBCs demonstrate that generation of a T-dependent immune reaction is efficient in the Toso knockout mouse, Of course al1 these results only cover a srnall spectrum of various lyrnphocytic functions. It is still unclear whether an appropriate memory response can be elicited, whether specific infectious agents cm be cleared, whether the mouse is more susceptible to various autoimmune disease models, etc. Although our assays show functional B-celis, the finding that Toso cm induce death of the pre-B ceU line 7023 suggests that further studies of B-ceii precursors in the bone marrow and spleen should be accomplished to determine a possible role for Toso in B-ce11 development or maintenance of a B-celi immune response. It is also possible that we simply have not been able to reproduce conditions where Toso's function in inhibition of apoptosis is necessary. To Merour study of the Toso knockout mouse, it will be critical to better understand the exact function of Toso at a cellular level. This should give us the knowledge necessq to better reproduce various in vivo conditions that would require the presence of Toso. Discovery of the Ligand for Toso and production of good antibodies against Toso will be critical in gaining this insfght. Our results do not necessariiy demonstrate a lack of function of Toso, but rather implies that Toso in itself is not essential for the various lymphocytic functions that we have tested. It may however be an important player when it is present on the surface of these cells, but its fuaction can be wmpensated by other mechanisms in its absence. The data obtained from the knockout mouse does not mie out the possibility of targeting Toso stimulation for therapeutic intervention of the immune system but lessens the possible importance of blocking its activity to achieve such a goal. Chapter 5: Discussion 5.1 Summary and discussion of results

As has been detailed in the introduction to this thesis, regulation of lymphocyte homeostasis is dependent on the function of many cellular receptors. The multiple signaling pathways that are used by these receptors converge on various aspects of lymphocyte regulation including activation, proliferation, difterentiation, survival, and apoptosis. Lymphocyte homeostasis is fmtly dependent on lymphocyte activation. Without activation, lymphocytes neither proMerate nor differentiate. Signals downstrearn of the antigenic receptors (TCR/BCR) are pivotal in the activation process. Once activated, the lymphocyte can react to various other stimuli including co-stimulation by CD28. This CO-stimulationpromotes T-lymphocyte proliferation, maînly by upreguiating the expression of IL-2. In addition, CD28 has been associated with the induction of T-lymphocyte differentiation [693, 6941 and has been demonstrated to influence the sensitivity of T- lymphocytes to AICD by promoting either survival [453-4561 or apoptosis [207]. In addition to co-stimulation by CD28, various other receptors can influence the fate of T- cells. Among these receptors, the TNFR superfarnily is critical. Some cellular receptors belonging to this superfamily, including Fas and TNFRpSS, contain death domains and play a role in inducing TaIl apoptosis. Family rnembers not containing this domain, including CD30 and CM,have been associated with proliferation, differentiation, as well as apoptosis. Other receptors, such as Toso, appear to regulate homeostasis by modulaiing the signaling pathways that influence ce11 swival or cell death. Trying to understand how unique receptors can mediate such different activities and why different receptors can mediate the same activity has been a goal of this research. We have approached this goal by analyzing various signaling pathways downstrearn of these receptors.

5.1.1 The activation of acidic sphingomyelinase by CD28 Many signaling pathways downstream of CD28 have ken identifiai, however most of these are also activated by the TCR (see sections I .2.2 & 1.3.3). Similar signaiing pathways for CD28 and tbe TCR have led people to propose that CD28 rnay simply increase the intensity of the TCR signal. In support of this, the CO-stimulatoryactivity of CD28 has kenshown to lower the need for high TCR occupancy to generate the antigenic signal [796-7981.However a simple amplification of the TCR signal does not fully explain some of CD28's activities, such as its influence on lymphocyte AKD. The results presented in Chapter 2 demonstrate that CD28 çan regdate the A-SMase pathway, suggesting a mechanism by which CD28 may influence AICD. 5.1.1.1 Acidic spbingomyelinase activity and its role in proliferation versus apoptosis WhiIe CD28 is thought to be the prototypic CO-stimulatoryreceptor because of its capabiiity of enhancing antigenic-induced T4proliferation and IL-2 expression (see section 1.3), the results from Chapter 2 provide an explanation for the previously describeci link between CD28 and AICD [207]. The fzt that CD28 can activate the A-SMase pathway, which has been extensively associated with various forms of apoptotic signaling, suggests a mechanism by which CD28 could add to the apoptotic signals that detennine ceil fate. The multiple signaling cascades stimulated by CD28, including enhancement of TCR signaling, regulation of IL-2 production, induction of the expression of survival factors such as Bc1-XI (see section 1.5.5), and stimulation of apoptotic pathways such as the sphingomyelinase pathway, could be the reason for the vaxious cellular effects CD28 has ken reported to have. Hence it is not surprising CD28 has been identified as a promoter of lymphocyte activation, proliferation, survival, and apoptosis. Somewhat surpnsingl y, our results showing that exogenous sphingomyelinase can compensate., at least partly, for CD28 in potentiating anti-CD3~induced TeU proliferation, suggest that the A-SMase pathway rnay participate in CD28's mitogenic activity. Interestingly, this is not the fmt the an apoptotic pathway has been associated with proliferation. The FADD knockout mouse and dominant negative transgenic mice have a defect in Fas-induced apoptosis of T-cells, but, in addition, have a defect in Teil proliferation in resgonse to antigenic stimulation [334-3361. It is not unreasonable to believe that proIiferation and apoptotic signahg rnay be inter-linked. In fact, resting ceIls can concentrate more effort in reversing conditions, such as DNA damage, ihat could initiate apoptotic stimuli. In contrast, proliferating cells may generate more inûacellular death signals due to a hasty enüy into the ce11 cycle. For example, a rushed DNA synthesis couid lead to genetic instabiiity and the activation of apoptotic programs via the p53 nunor suppressor gene pathway [450]. However, exactly how these apoptotic pathways iink to proliferation stdl remains a mystery. To support such a link, rnany studies have analyzed this association between proliferation and ceii death. In fact, studies have shown that T-lymphocytes do not undergo AICD when treated with dmgs that block the G1 to S phase progression [799]. In addition, inhibition of specific mediators of the ceil cycle has been shown to block AICD.

Cdk2 activity has been linked to the AICD of negatively selected thymocytes C800, 8011.

Cyclin-B has been associated with anti-CD3einduced T-cell death [802]. Cyclin-D3 is involved in TCR stimulated AICD of T-celi hybridomas [803]. Findly, cyclin A has been shown to participate in apoptosis induced by the c-Myc transcription factor [804]. In addition, various transcription factors involved in œU cycle progression have been associated with the induction of apoptosis. This is the case of c-Myc which has ben shown to &ive ceils into the cdcycle [805] as well as to promote apoptosis [806-8091. Furthemore, the use of antisense oigonucleotides, to inhibit c-Myc expression, can block AICD in T-ceU hybridomas [808]. This apoptotic activity of c-Myc has recently been associated with the induction of Fas-ligand RNA expression [8 101 and, at least in B-cells, the kinetics of activation of c-Myc appear to be important in determining celi fate [8 1 1- 8 131. However, none of these studies have yet been able to develop a clear and deftnitive pathway that could directly link apoptosis to proliferation.

These findings of proteins involved in both ceU cycle progression and apoptosis have led some researchers to propose the "mitotic catastrophe" hypothesis that suggests that if a ceil enters the ceii cycle at an inappropriate tirne, it would undergo apoptosis. This proposal is intriguing and would suggest thaî forcing a dl into the ceIl cycle might be a mechanism by which antigenic and CO-stimulatory signaling could promote AICD. Our fmding that ASMase can be, at least partly, responsible for CD28's proliferative effect provides a further link by which CD28 may promote either proliferation or apoptosis. The fact that high levels of A-SMase activity are toxic to the ceils suggest that either there is a non-specific toxicity associated with exogenous SMase activity, or that the level of SMase activity detemines whether activation of this signal wili promote proliferation or cell death. It seems plausible chat the intensity of a signal could lead to two different outcomes if both outcomes have common intraceliuiar mediators, as proliferation and apoptosis appear to have. Nonetheless, the identification of signaling pathways downstream of A-SMase, which could explain both its mitogenic and apoptotic activity, will be key in understanding how SMases can play a role in regulating lymphocyte homeostasis. 5.1.1.2 RAFTS and sphingomyelin in signaling Interestingly, a recent renewal of interest in the role of lipids in signaling has stemmed from various reports indicating an important function for lipids in TCR signaling. Lateral clusters of cholesterol and sphùigolipid have been shown to aggregate into lipid

"rafts" 18 14,8 151. Simons and Ikonen have proposed a mode1 by which these rafts can act as platforms for various signaling proteins that regdate cellular activity [8 16-8 181. In support of this, it has been shown that the cross-linking of these rafts dlows the signaling molecules to interact with each other and activate signaling pathway [8 16, 8 181. Among these signaling molecules, the Src family of kinases such as Lck and Fyn, srnail G- proteins, PI3 K, LAT and glycosylphosphatidylinositol (GPI) linked proteins have been shown to be raft associated [8 17,8 19-8221. In view of our findings, it is very exciting that sphingomyelin is found to concentrate in these rafts 18231 and that CD28 has been shown to induce the redistribution of the rafts at the TCR-MHC contiw:t site [824]. Anti-CD~Estimulation fails to cause this redistribution suggesting that this is a CD28 specZc effect [824]. The activation of sphingomyelinase thaî we have seen associated with CD28 was also specific to CD28 and did not occur in response to CD3e signaiing. In this context, it is very teqting to try to iink our results with this activity of CD28. Since sphingornyelin concentrates in these rafts, it is possible thai the generation of ceramide by the sphingomyelinase could strengthen the solidity of the rafts and possibly promote kir aggregation. Such a hypothesis seems plausible since cholesterol, which is very hydrophobie, as is ceramide, promotes the organization of these rafts [823]. in addition, these rafts have been associated with the formation ofcaveolae 18231, which are also thought to be the site of action of A- SMase (see section 1A6.2). Hence, the activation of A-SMase by CD28, may provide an expianation for the aggregation of RAFTS and the activation of various signaling pathways downstream of this CO-stimulatorysignal. 5.1.1.3 CD28 and sphingomyelinase: Future Directions A human genetic defect of deficient A-SMase exists dedNeimann Pick's disease. This disease provides a mode1 for the study of CD28 in TeIi homeostasis. However, no research has clearly looked at the immune system in these patients. This is possibly due to the fact that the main defect, which causes problem in this disease, is a lipid storage defect. in fact the Lysosomes of the cells from these people become loaded with sphingomyeiin and this abnormal accumdation ieads to cellular dysfunction. In addition, Neimann Pick diseased cells usually have some residual A-SMase activity. This confuses results obtained from the study of these mIis. The ment generation of an A-SMase deficient mouse fmaliy has allowed the study of the effect of the total absence of A-SMase on the immune system [360]. This mouse has shown that, in fact, ceiis lacking A-SMase are partly defective in various forms of apoptosis, such as those induced by DNA damage due to y-irradiation.

However, very little has been done to study the function of the immune system in these mice. The roles of A-SMase in CD28's co-stimulatory activity and its Muence on AICD have not yet been anaiyzed using this model. Unfortunately, one problem with such studies is that œlls fkom these mice are also loaded with abnormal amounts of sphingomyelin. Because of this it wiil be difficult to determine whether the observation of a defect in the immune system of these mice is due to the absence of A-SMase, or rather due to the abnonnal accumulation of spbingomyelin. If this becornes of concern, the generation of inducible knockout ceU lines or mice could be of help to resoive this problem. Since the accw11ulation of sphingomyelin is the results of a prolonged absence of A-SMase, the study of celis that have just recently Iost their A-SMase activity could prevent this problem.

In addition to tbese studies, work must be done to determine a possible role of A- SMase activity on the regdation of ra.in T-ceU. The addition of exogenous A-SMase should be studied to determine whether reorganization of rafts occurs at concentrations where CO-stimulatoryactivity has been observed. If it does, this might explain A-SMase's CO-stimulatoryactivity. Furthemore, the tight link between A-SMase and activation of apoptosis suggests the possibility that rafts may not only mediate the organization of antigenic signals, but might also play a role in serving as platforms for the death machinery. If this is so, the role of A-SMase in regulating apoptosis might be explained by a modification of raft stability and aggregation. The activation of the death signal is not only dependent on the presence of the apoptotic signahg machinery, but aiso by the organization of this machinery. In ceUs were the pro-apoptotic factors are not abundant, such an organization of the machinery may be especially crucial. In this respect, A-SMase may be playing a role, via rafts, in controlling the organization of the apoptotic signaling cascade. Hence the role of rafts in AICD must be studied in conjunction with the study of

A-SMase ac tivi ty. 5.1.2 Implications of TRAFs in CD30 signaling Some members of the TNFR superf'y have also been shown to activate the sphingomyetinase pathways as weil as other apoptotic patbways. This is especialiy the case of the death receptors Fas and TNFRpSS. However, the CD30 receptor has not yet

been associated with such activity- Ln view of this, the association of CD30 with the activation of AICD [234, 2401 had rernained unexplained. Our results, presented in Chapter 3 suggest that CD30 can interact with the TRAF survivai factors, suggesting various possibilities by which CD30 may influence cellular survival. S,1,2.1 Interaction of CD30 with TRAFs as an explanation for CD30's reported activities The association of TRAFs with CD30 did not offer an intuitive explanation for the AICD promoting activity of CD30. In fact, TRAFs have been shown to be mediators of cellular survival (see section 1.5.4). Hence the interaction of TRAFs with CD30 wouId rather explain the pro-survival activity associated with CD30, such as is seen in T-ceii like lymphoma ceiis from Hodgkin lymphoma patients. In fact in these cells, CD30 has been shown to promote proliferation rather than apoptosis [738]. In view of these findings, two hypotheses can be drawn: either CD30's pro-apoptotic activity is via another pathway that does not involve TRAI=s and has not yet been identified, or the interaction with Wsis responsible for both CD30's proLiferaiive and pro-apoptotic activity. Aithough the latter hypothesis might be explained by the Iink between proliferation and apoptosis, as mentioned previously, a ment reports suggests another mechanism by which the interaction of CD30 with TRAFs could promote apoptosis. This report has shown that the stimulation of CD30 can induce the degradation of TRAF2 [250]. This would decrease the pro-survival activity of TRAF;! and hence reduce the influence of this anti-apoptotic pathway. Because some death receptors, such as TNFRpS5, activate both the caspase apoptotic pathway as well as the TRAF survivai pathway, this ôctivity of CD30 could change the intracellular balance between survival and apoptotic factors so that the signaling

induced by these receptors now favors apoptosis rather than survival. The implications of the previous maiel defining TRAFs as the main mediaior of CD30 signaihg suggest that CD30 does not induce death directly, but rather sensitizes ceh to apoptotic pathways activated by other receptors. This might be part of CD303 activity, however, there is evidence that in some celis, CD30 can induce AICD independent of Fas signaling [234]. It is unlikely that the degradation of TRAF2 via CD30, by itself, could explain sucb fmdings unless these cells are also undergoing a basai resting pro-apoptotic stimuli and that CD30 activity allows to overcome survival factors and induce œli death.

Surely TRAF;! is sufficient to provide some protection hmTNF-induced apoptosis, since

its absence renders ceils more sensitive to this apoptotic stimuli [514], however the importance of its presence in various other pathways, such as in AICD, is still uncertain. Because of this, we cannot rule out the possibility that CD30 might aiso activate a death pathway, independent of TRAFs. If such a pathways exists, it may be only recruited to the CD30 receptor following a pst-tmmslationai modification step since it has not yet been identified using the yeast two hybrid method. In contrast, it is possible that some cells, such as malignant cells, could faii to degrade TRAF;, in response to CD30. If CD30 is critical to TRAF;! degradation, this might lead to an active TRAF pathway downstream of CD30. Such a regdation of pro- survival factors, via CD30, is very appeahng since it couId explain reports of CD303 pro- apoptotic as weil as pro-survival activity depenàing on the celi type that is studied. 5.1.2.2 Other CD30 binding proteins In addition to the TRAF clones that were isolated from the yeast two hybrid procedure using the CD30 intraceilular tail as bait, another protein, Zyxin, was identifrd multiple times. Zyxin is a LIM (lin-1 1 isl- 1 mec-3) domain containing protein and ail the clones isolated at least rninimaUy contained the LIM domains. In addition to clones of

Zyxin, another protein containing LIM domains was also isolated. These results suggest the possibility that CD30 may bind LIM domains, however this remains to be formally demonstrated. If such an intefaction exists, it is probably weak since we failed to immuuoprecipitate these proteins when overexpressed with CD30 in COS cells usuig Triton-XI00 as a detergent. Nonetheless, this fmding rnay be very exciting in view of the fact that Zyxin has been associated with cytoskeletai reorganization at focal adhesion sites [825] and has been found to interact with Vav [826], which is necessary for capping and TCR signaling [SOI. Such an association could offer further explanation about the Link between CD30,TCR signaling, and AICD. 5.1.2.3 Possible TRAF binding proteins

In addition to defining TRAFs as downstream effectors of CD30 signaling, our data has characterized the TRAF binding motifs. It is exciting that these binding motifs are quite smali in size, reminiscent of SH2SH3 binding motifs [753]. The characterization of SH2 and SH3 binding motifs has ben key in defining signaling pathways that use such interactions. in fact, novel proteins are often screened for possible SHWSH3 binding motifs. It is our hope that the characterization of TRAF binding motifs will dso be of help in fmding other proteins to which TRAFs can bind and to develop our understanding of TRAF signaling pathways. As an initial screen of possible TRAF binding proteins, sean=hing databases have revealed rnany proteins containing sequences similar to those found in the TRAF2/3 binding site of TNFR superfamily members. However, few contained high homology with the TRAFll2 binding site. Among those that did, the 14-3-3~protein has the sequence "DVELTVEE' which contains the amino acids that are consewed among the TNFR superfarnily members that bind TRAFlR ("LW-w'). 5.1.1.4 CD30 and TRAFs: Future Directions The hypothesis that CD30 mediates its effects via TRAF degradation suggests that excessive accumulation of TRAFs may be responsible for the defect in negative selection that is seen in the CD30 knockout mouse. Such an inference could be studied by looking at the amounts of TRAF2 protein in the CD30 knockout thymocytes from mice crossed with a

negatively selecting TCR ûansgenic mouse, such as the HY male transgenic mouse. In addition, this hypothesis suggests that negaiive selection in the TRAF'2 knockout mouse should be enhanced. If both models prove this to be the case, than it would be IikeIy that CD30's interaction with TRAF2 is critical for negative selection. In addition, this mode1 of TRAFZCD30 interaction in mediating CD3û-induced apoptosis should be verified in the T- ceii h ybridomas w hich undergoes CD3ûdependent AICD 12341. Studies should ahbe done to try and understand how CD30 induces the degradation of TRAF2. The finding that degradation of TRAFl dso occurs in response to

CD30 stimulation, suggests htthe TRAFlR binding site on CD30 may be important in the degradation of TRAFs in response to CD30 signahg 12501. T-ceIl hybridomas, that respond to CD30 by inducing AICD [234], should be studied for the degradation of TRAFs following transfection with CD30 containhg mutations in the individual TRAF binding sites. Furthemore, studies should be done to identiw the protein, downstrea. of CD30 that targets TRAF2 for degradation.

In addition, the hypothesis chat TEZAF degradation is necessary for CD307s

apoptotic activity suggests that various tumor ceus, such as Hodgkin's cells, that respond to CD30 by proliferating may be defective in TRAF degradation. In these cases, CD30 might actuaiiy promote ceiiular survival and proliferation. Because of this, the statu of CD30-induced TRAF2 degradation should be assessed in such cells. If it is found that

TRAF2 is not degraded, detennining the mutation in these ceils that is responsible for the lack of degradation would provide helpful information as to how CD30 induces TRAF2 degradation. Furthemore, the consensus TRAF binding motif we have identified should be used to look at various known and unknown proteins to determine whether they possess putative TRAF binding motifs. The isolation of novel, or known, proteins that can interact with

TRAFs will be pivotal in fully understanding the various mechanisrns by which TRAFs mediate their anti-apoptotic effect. The fact that the 14-3-3~protein contain a stretch of

amino acids similar to the TRAFl/;!binding motif is very exciting since 14-3-3 proteins

have been shown to bloclc apoptosis by sequestering phosphorylated BAD (see section

1.5.5.3). Hence it will be critical to assess whether TRAFs can immunopmipitate 14-3-3 proteins. If this proves to be me, it would suggest a completely novel meçhanism by which Wsmay reguiate ceil survival.

5.1.3 The rote of Toso in lymphocyte ALCD Our data shows that both CD28 and CD30 could influence AICD by stimularing death pathways or by uihibiting survival pathways respectively. Toso is another receptor that has been suggested to function by modulating the intracellular death pathways. In fact,

this gene was initiaiiy isolated as a protein that couid block Fas-induced apoptosis when overexpressed in Jurkat cells [780]. Our data studying the knockout mouse does not support a critical role for Toso in regulating AICD, however, it does not desuch a function when present on cells. 5.1.3.1 Mechanism of action of Toso The initial characterization of Toso showed that the extracellular portion of Toso is necessary for its anti-apoptotic activity [780]. This suggests that the interaction of Toso with a ligand is necessary. Since this is observed with the Jurkat celi line, both Toso as

well as its ligand must be expressed on these 41s. However, no ligand for Toso has yet been isolated. The fact that the intraceliular domain of Toso is not necessary for its activity, further suggests that Toso probably does not signal directly to a survival pathway but may rather function by stimulating its ligand to activate such a pathway. This is not absolutely certain, especially in view of the finding that the transrnembrane domain of Toso is necessary for its activity. Two hypotheses have arisen from these findings: either Toso signals directly a sur~ivalsignal via its transrnembrane domain or Toso must be located to the membrane for its pro-survival activity. The latter hypothesis has so far been favored, but there is no direct evidence thaî supports either hypothesis. The use of hybrids of Toso with transmembrane domains from other proteins rnay help solve such questions. If the crucial importance of the transmembrane dornaia is due to a requirement of Toso to be membrane bound, it suggests that Toso may not function on its own, but rather with a coreceptor that may also be membrane bound. Alternatively, it is possible that Toso could function as a multimeric complex that requires the transmembrane domain to form. It is also possible that Toso's ligand is difficult of access and can only be efficiently stimulated by membrane bound Toso. Furthemore, if Toso does not have signahg capability of its own, but rather functions to stimulate a ligand's pro-survival activity, this rnight explain the absence of a phenotype in our knockout mouse. In fact, such a mechanisrn could allow for much redundancy if the Toso ligand can be stimdated by other receptors. In this case, the absence of Toso may not be smcient to prevent the pro-survival signal, but overexpression of Toso would be able to promote it. However, ali these possibilities remain to be forrnally demonstrated. 5.1.3.2 Toso and the expression of FLIP Although the initial report of Toso suggested that it could inhibit Fas signaling by upregulating the RNA level of FLIP, a recent report suggests that the role of FLIP in Fas- induced AICD of lymphocyte is controversial. This group has shown that the levels of

FLIP protein expressed in early activated Fas insensitive Tells is too small to account for the block in Fas killing 17791. As well, this level was not decreased when the ceils became sensitive to Fas, foliowing a prolonged incubation in IL-2, in contrast to the RNA levels [219]. Because of this finding, the importance of FLIP upregulation by Toso remains controversial. It is possible that the overexpression of Toso may upregulate enough FLTP to get such an effect, but it seerns unlikely that this occurs as a physiological mechanisrn to protect cells froin AICD early on after activation. Furthemore, many signals that activate the MAPK pathway via MKKl have been linked to the upregulation of FLIP 14521, suggesting that Toso is not unique in mediating such an activity. Hence the dcimate decision to inhibit AICD probably mmes hmcumulative survival signals, of which Toso is only one. Because of this, the absence of Toso may be only of limited consequenœ and may influence only ceils that have very few other survival signals and are precociously tilting towards ceil death. It is possible that we have not been able to see an effeçt of Toso on AICD because the conditions we have used failed to generate a large enough number of these ceils to have a measurable defect in the Toso knockout.

Alternatively, it is possible that too few cells express high Ievels of Toso for us to rneasure a difference in the Toso knockout mouse. We have tried to bypass such a problem by generating a knockout mouse in which the green fibrillary protein (GFP) has been inserted in frime with the start codon of the Toso gene (data not shown). These mice should express GFP, instead of Toso, but under the control of the Toso promater. By sorting for ceils expressing the GFP protein, it was our hope that these miœ would allow us to pur@ celis that express Toso, from heterozygote GFP:Toso knockin mice, and ceils that should express Toso but no longer do, from homozygote GFP:Toso knockin mice. This would allow us to do experiments on highly puriflecl populations of cells that normally express high levels of Toso, Unfortunately the levels of GFP expressed in these mice turn out to lx too low for us to see a signal either by FACS sorting or fluorescence microscopy (data not shown). Hence we were unable to use these mice to further our understanding of Toso function. 5.1.3.3 A possible role for Toso in B-cells? The only clear positive finding we have had with the mouse Toso gene is that its overexpression in the 7023 pre-Bsll line induces their death. Our fmding that Toso is highly expressed in resting B-cells further supports a possible role for Toso in these cells. Our data also shows that Toso RNA disappears upon activation of B-ceUs with LPS, suggesting that if Toso does play a role in B-cells, it would probably be in resting cells. In view of these findings, it is tempting to speculate that Toso may function in the elimination of resting or immature B-ceiis. This wuld occur possibly during a negative selecring step in the development of these ceiis, or during "death by negiect" in which the ceUs die due to a failure to receive an antigenic signal. However these hypotheses remain to be fody proven. Our only me Ut vivo &ta on Beil aictivity shows that B-ceU:Ti'~iiinteractions are functionai and that Bcells can appropriately secrete antibodies against a foreign antigen such as sheep red blood ceiis. This suggests that Bdfunction xnay not be impaired by the lack of Toso. However, it does not mie out a possible Bceii developrnental role for Toso. in this respect, ceiis that would nodydie during àevelopment may now survive in the absence of Toso. If this is the case, the defect is only minimal since we do not see a gross accumulation of B-cells in the peripheral lyrnphoid organs. The use of transgenic B- ce11 rnice models might be necessary to observe such a defect. 5.1.3.4 Toso and FLIP: Future Directions The studies we have done have so far have failed to identify an immune defect in the Toso knockout mouse. However more expenments should be accomplished. If Toso, in fact, protects recentiy activated Tails from apoptosis, one would expect that these mice would fail to mount appropriate immune responses against a pathogen that strongly activates T-ceîls, since these cells would undergo premature AICD. Even though we show that Toso knockout mice cari generate an immune response against sheep red blood ceils, it is possible that this antigen is not strong enough to aggressively promote AICD. The study of immune responses against live pathogens such as viruses and bacteria will be important in clearly defining the importance of Toso in generating an adequate immune response. In addition, the differential expression of Toso on T'hl versus Th2 type of T-ceils should tw: assessed, especially in view of the differences in sensitivity to AICD that is obsewed between these cells. If a difference is found, the elimination of Th specific pathogens should be studied in the Toso knockout mouse. Furthemore, more studies must be done to look at BuAl development in these mice. As mentioned previously, the use of BCR transgenic mice might be necessary to highlight such a defect. The studies by Scaffïdi et al. suggest that the levels of FLIP RNA may not accurately repriesent the amounts of FLIP protein in a ceii [779]. Hence it will be crucial to detennine whether the transfection of Toso does affect FLIP protein, and not only FLIP RNA. In addition. because tbe MAPK pathway has been associated with the upregulation of FLIP RNA [452], it wiU be important to detennine whether Toso can also activate this pathway. Otber pathways that infïuence Fas signaling upstream of caspase-8 should also be studied. Among these the eficiency of generating the DISC [295], the activation of inhibitory pathways such as via FAP [767], and the inhibition of stimulatory pathways such as via FAF [769), should be analyzed in Toso transfected cells. This is especially important in view of the evidence that FLIP may only play a minor role in controllhg lymphocyte AKD. Understanding mechanisms by which FLIP is upregulated will also be crucial in understanding Toso signaling. Since FLIP upregulation has been associated with the MAPK pathway, various transcription factors activated by this pathway should be studied for their possible role in upregulating FLIP transcription. In addition, the study of the FLIP promoter could provide information to further understand FLEP regulation.

It has also not been mled out that the MAPK pathway may signal the upregulation of FLIP expression by upregulating Toso expression. However, this is onlikely since this pathway can upregulate FLIP RNA in fibroblast ceiis that don? normdy express Toso. Nonetheless, the influence of MAPK activation on the expression of Toso should be analyzed.

The major obstacle that has impeded the understanding of Toso function is that ail the information we have on Toso's activity has corne from overexpression studies. One must be very careful in extrapolating conclusions on the physiological function of Toso from such studies. Overexpression of Toso can produce misleading resutts due to falsely elevated Toso expression levels or to the expression of Toso in cells that would not normaily express it. Because of this, the stimulation of the endogenous Toso protein on cells is cnticai to fully undexstand Toso's role. The development of an anti-Toso antibody or the discovery of the Toso ligand wiil be pivotal in doing such studies. 5.2 Concluding remarks

The results presented in this thesis have contributeû to understand signahg rnechanisrns that are activated by receptors that influence lymphocyte homeostasis by modulahg AICD. Cellular homeostasis can be viewed as a balance between survival and apoptotic stimuli. Research has demonstrated that most cells have the rnachinery ready to activate the apoptotic program. However, the activation of this program is &pendent on the generation of signals that either onginate fiom cellular receptors, such as Fas, or intracellular factors, such as p53. Since pro-caspases have been shown to have low, but measurable aut0catalyt.k activity, a resting cell must keep the apoptotic machinery in check, by having a consitutive basai expression of anti-apoptotic factors, if it is to survive. This basal level of pro- survival factors is probably crucial in determining ceii fate. if the levels are just suficient enough to block basal apoptotic activity, then the slightest increase in apoptotic signaling, such as via SMase and CD28, or the slightest decrease in pro-survival activity, such as the degradation of TRAFs by CD30, may be sufficient to initiate a non-reversible apoptotic program. In such ceils, protein that inhibit apoptotic signaling, such as Toso, may be important to stabilize the celis and prevent weak death signals to overcorne the survival factors. In contrast, in ceUs with overabundant pro-survival factors, very strong apoptotic signals would be needed to induce apoptosis. In the case where a receptor's signal wouId not be strong enough to promote apoptosis, other celiular effect of these receptors may be observed. Hence the proliferating/diHerentiatllig capability of CD30 may be observed when the TRAF;, degradation by CD30 is insufficient to induce cddeath, and the CD28 proiiferative activity may be observai when activation of the SMase pathway is insufficient to promote apoptosis. Such a hypothesis would explain the fact these receptors often have different effects depending on the ce11 type studied. This mode1 proposes that the strength of the apoptotic signal needed to induce œil death is dependent on both the basai leveis of anti-apoptotic factors as weli as those thaî might be upreguiated by pro-survival receptors. The regdation of homeostasis ca. be achieved by directly activating apoptotic signals, such as CD28 and SMase, by inhibiting prosurvival factors, such as TRAFs and CD30, by inhibiting death signals, such as Toso and Fas, and by promoting survival factors, such as activators of NFKB and IAPs. AU these signals will participate in defining the bahce between pro-survival and pro-apoptotic signals that WUdetermine how a ceii wiii react to its next stimuli. It is our hope that by understanding pathways that regulate this balance, science will one day be able to influence the homeostasis of the immune system and prevent diseased states that occur when this homeostasis is defective. 1. Lydyard PM: Immune response. Curr Opin Immunol 1988; l(2): 283-391. 2. Roitt IM, Brostoff J, Male DK: Immunology, 2nd ed. New York Philadelphia

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