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Alterations in transcription factor binding in anergized human CD4+T- lymphocytes Heisel, Olaf 2001

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ALTERATIONS IN TRANSCRIPTION FACTOR BINDING IN ANERGIZED HUMAN CD4 + T-LYMPHOCYTES by OLAF HEISEL M.D., The University of the Saarland, Germany, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Medicine Experimental Medicine Program We accept this thesis as confirming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 2001 © Olaf Heisel, 2001 UBC Special Collections - Thesis Authorisation Form Page 1 of 1 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l . f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t . f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head of my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f 1 y v "^ -r—y A \ . — V -The U n i v e r s i t y o f B r i t i s h C olumbia = Vancouver, Canada Date 10/4/01 ABSTRACT Background: The mechanisms responsible for the induction of human clonal anergy are not well understood. We have utilized an in vitro model of human T-cell anergy to explore the perturbations in cell signaling at the level of IL-2 gene transcription, and to define the contribution of other cytokines to this effect. Methods: An in vitro model of clonal anergy was established using peripheral T-lymphocytes from healthy human donors. CD4 + T-cells were anergized by pre-stimulation with an anti-CD3 mAb followed by restimulation 72 hours later with anti-CD3 with or without anti-CD28. Proliferation was measured by [3H]-thymidine incorporation and IL-2 production using ELISA. Results: CD4 + T-cells anergized with OKT3 displayed a marked reduction in proliferation (P=0.0036) and IL-2 production (P<0.0001) compared with controls. Simultaneous treatment with anti-CD28 prevented induction of anergy measured either by proliferation (P=0.0002) or IL-2 production(P<0.0001). Co-incubation with IL-10 reduced cellular proliferation in OKT3/CD28 pretreated cells by 19% (P=n.s.) and reduced IL-2 production by 40% (P=0.0024). Anergized T-cells demonstrated a reduced binding activity of the AP-1 protein complex to the IL-2 gene promoter. Supershift experiments confirmed that the individual binding of c-Fos, JunB and JunD, but not of FosB to the AP-1 region of the IL-2 promoter was reduced in anergized cells when compared to controls. Furthermore, there was a reduced Stat-binding at the SIE region of the c-Fos promoter in anergized cells. Supershift experiments using specific antibodies against Statl and Stat3 showed that binding of Stat l , but not Stat3, to the SIE region of the c-Fos promoter was diminished. Co-incubation of PBMC with OKT3/anti-CD28 and a blocking gp39 (CD40L) mAb resulted in a significantly reduced proliferation rate (P=0.0003) and IL-2 production (P=0.005). Co-incubation with anti-CD40L mAb also led to a marked reduction of AP-1 binding to the IL-2 promoter similar to that observed in B7/CD28 abrogated cells. Conclusions: T-cell anergy induced by OKT3 is characterized by reduced T-cell proliferation and a profound decrease in IL-2 production accompanied by a reduction in AP-1 binding to the IL-2 gene promoter, with selective reduction in binding of the individual AP-1 components c-Fos, JunB and JunD. The deficiency in binding of Statl to the SIE region of the c-Fos promoter highlights an involvement of the Jak-Stat pathways in the events of clonal anergy. Furthermore, blockade of the CD40-CD40L pathway is able to achieve similar anergizing effects as in cells where B7/CD28 costimulation is abrogated. This highlights the importance of the CD40/CD40L pathway as a second costimulatory pathway. It also provides insight into new mechanisms of clonal anergy, in which co-blockade of both B7/CD28 and CD40/CD40L pathways might lead to a more profound anergy induction and better graft survival. TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables ix List of Figures ix List of Abbreviations xi Acknowledgements < xiv Dedication xv CHAPTER ONE: INTRODUCTION 1 1.1 Hypothesis 1 1.2 General background 3 1.3 The signal transduction network 6 1.3.1 Overview 6 1.3.2 T-cell receptor signal transduction 7 i) Phosphorylation of the TCR^ chain by tyrosine 7 kinases from the Src family ii) Regulation and function of tyrosine kinases from 9 the ZAP-70/Syk family iii) Transmembrane adaptors and TCR signaling 9 iv) Cytoplasmic adaptors and TCR signaling 10 v) Regulation of TCR-induced, sustained C a 2 + 11 mobilization by tyrosine kinases from the Tec/Btk family vi) Stat (Signal Tranducer and Activator of Transcription) 12 i v proteins and TCR signaling vii) Negative regulation of TCR signaling 12 1.3.3 Costimulatory signal transduction via CD28/CTLA-4 and 14 B7.1/B7.2 i) Structure of the CD28 receptor 14 ii) Structure of the CTLA-4 receptor 15 iii) B7.1 and B7.2 15 iv) Differences in expression and function between B7.1 16 and B7.2 v) Functional responses to B7-CD28 interactions 16 vi) Differential regulation of immune responses 17 vii) Augmentation of IL-2 production 18 1.3.4 Intracellular signal transduction following CD28 activation 20 i) The PI-3 kinase pathway 20 ii) The p21 r a s pathway 21 iii) The ITK pathway 21 iv) The sphingomyelinase/ceramide pathway 21 1.3.5 Costimulatory signal transduction via CD40/CD40L 22 i) Overview 22 ii) Structure of the CD40L and its counter-receptor 25 iii) CD40 signal transduction 25 1.3.6 Downstream pathways following TCR/CD28 activation 26 1.3.7 Clonal anergy 28 1.3.8 Signal transuduction at the transcriptional level 32 i) The IL-2 promoter region 32 ii) The NFAT binding site 32 iii) The AP-1 binding site 34 iv) The c-Fos promoter region 35 1.3.9 Role of IL-10 in the induction of clonal anergy 36 CHAPTER TWO: METHODS 39 2.1 Solutions 39 2.1.1 Oligonucleotides and EMSA 46 2.1.2 Antibodies 46 2.1.3 lnterleukin-10 (IL-10) 47 2.2 Methods 47 2.2.1 Donor cells 47 i) Collection 47 ii) Separation by Ficoll-Hypaque 47 iii) Lympho Kwik 48 iv) Cell culture 48 2.2.2 Jurkat cell line 48 2.2.3 CTLLtest 49 2.2.4 Induction of clonal anergy 49 2.2.5 Preparation of nuclear extracts 50 2.2.6 Electrophoretic mobility shift assay 50 i) Co-incubation of oligonucleotides with DNA binding 50 proteins ii) Agarose gel electrophoresis 51 iii) Blotting 51 2.2.7 Supershift EMSA 52 2.2.8 Electrophoresis and immunoblotting 52 i) Determination of Protein Concentration 52 ii) SDS-polyacrylamide gel electrophoresis 52 iii) Western blot analysis 53 2.2.9 Proliferation Assays 54 2.2.10 IL-2 ELISA 55 2.2.11 Statistical Analysis 56 CHAPTER THREE: RESULTS 57 3.1. Introduction 57 3.2 Reduced proliferation and abrogated IL-2 production in 57 anergized human CD4 + T-lymphocytes 3.3 Anergy is charactierized by reduced binding of AP-1 to its IL-2 60 promoter region but NFAT-binding remains unaffected 3.4 Supershift experiments show reduced binding of c-Fos, JunB and 67 JunD to the AP-1 site of the IL-2 promoter 3.5 Supershift experiments show reduced binding of Statl to the SIE- 70 binding region of the c-Fos promoter 3.6 CD40L blockade significantly reduces proliferation rates and IL-2 73 production in PBMC 3.7 CD40L-blockade mimics the transcriptional events of clonal anergy 79 3.8 Influence of IL-10 on CD4 +-T-cell proliferation and IL-2 production 82 3.9 Influence of the p38-MAPK inhibitor SB 203580 on T-cell proliferation 84 vii CHAPTER FOUR: DISCUSSION 86 CHAPTER FIVE: SUMMARY 95 CHAPTER SIX: R E F E R E N C E S 97 viii List of Tables No tables. List of Figures Figure 1a. Schematic overview of the signal transduction pathways 19 in T-cell activation relevant for different approaches to induce clonal anergy (model 1-4). Figure 1b. Schematic display of the IL-2 promoter region 33 Figure 2. Induction of clonal anergy in human CD4 + T-lymphocytes. 59 Figure 3a. AP-1 binding to the IL-2 promoter in nuclear and cytosolic 62 extracts after stimulation with medium and OKT3/CD28 Figure 3b. Time course of AP-1 binding 63 Figure 4. AP-1 binding in anergized human CD4 + T-lymphocytes 64 Figure 5. AP-1 binding in anergized Jurkat cells 65 Figure 6. Analogous experiment using a NFAT specific 66 Biotinylated oligonucleotide probe Figure 7. Supershift experiment using antibodies against c-Fos, 68 FosB, JunB and JunD Figure 8. Western blotting using antibodies against c-Fos, JunB, 69 and JunD Figure 9. Binding to the SIE-region of the c-Fos promoter 71 Figure 10. Supershift experiments using antibodies against Statl 72 and Stat3 Figure 11. Cell proliferation measured by [3H]-thymidine uptake 75 in PBMC and CD4 + T-lymphocytes Figure 12. IL-2 production into the supernatant measured by IL-2 77 ELISA in PBMC and CD4 + T-lymphocytes Figure 13. AP-1 binding in human PBMC after CD40L-blockade 80 using an anti-CD40L antibody Figure 14: Co-incubation of CD4 +T-lymphocytes with IL-10 83 Figure 15: Influence of the p38-MAPK inhibitor SB 203580 on 85 proliferation of stimulated human T-lymphocytes x ABBREVIATIONS [Ca 2 +] calcium ion concentration AP-1 activator protein 1 A P C antigen presenting cell A R A M antigen receptor activation motif B7 antigen encoded by the B locus of chromosome 6 Bel B-cell leukemia Btk Bruton's tyrosine kinase bZIP motif basic-leucine zipper motif Cbl Casitas B-lineage lymphoma CD cluster of differentiation CD40L cluster of differentiation 40 ligand Clnk cytokine-dependent hemopoeitic cell linker C R E c-AMP-responsive element CREB-protein cAMP responsive element binding protein CTLA cytotoxic T lymphocyte-associated antigen CTLL cytotoxic T lymphocyte leukemia cell line DAG diacylglycerol ERK extracellular signal-regulated kinase ESRD end stage renal disease Fas folic acid synthesis Fos Finkel and osteogenic sarcoma FRK fos-regulating kinase g gram Gads Grb2-related adaptor downstream of SHC GM-CSF granulocyte/macrophage colony-stimulating factor gp39 glycoprotein39 = CD40L Grap Grb-2 related adaptor protein GRB2 growth factor receptor binding-2 HLA human leukocyte antigen Hr hour IF-y interferon-y igM immunoglobulin M IL interleukin IPs inositol (1,4,5) tris-phosphate ITAM immunoreceptor tyrosine activation motif Itk inducible T-cell kinase/lnterleukin-2 tyrosine kinase Jak janus kinase JNK c-Jun N-termial kinase Jun JU-Nana (avian sarcoma virus 17) kDa kilo Dalton LAT linker for activation of T-cells Lck lymphocyte specific protein tyrosine kinase M molar mA milliampere MAPK mitogen-activated protein kinase MHC major histocompatibility complex Min minute ml milliliter NFAT nuclear factor of activated T-cell NOD mice non-obese diabetic mice PBMC peripheral blood lymphocytes PBS phosphate buffered saline PI-3 kinase phophatidylinositol-3-kinase PKC protein kinase C PLC phospholipase C PTK protein tyrosine kinase RT room temperature SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis SH src-homology SHP src homology 2 domain-containing protein tyrosine phosphatase SIE sis-inducible element SLAP src-like adaptor protein SOCS suppressor of cytokine signal SLP-76 src homology 2 domain-containing leukocyte protein of 76kDa xii Sos son-of-sevenless src sarcoma SRE serum response element Stat-1, Stat-3, Stat-5 signal transducer and activator of transcription-1, -3, -5 TAD transactivation domain TCR T-cell receptor Th-1,-2 T-helper1,2 TNF-a tumor necrosis factor a TRAF TNF-receptor associated factor TRIM T-cell receptor-interacting molecule Tris tris (hydroxylmethyl) methylamine ZAP-70 zeta-associated protein 70 xiii Acknowledgements My deepest gratitude goes to Dr. Paul Keown who has given me the invaluable opportunity to conduct my research in his laboratory. I would also like to thank all the members of my supervisory committee, Dr. Paul Keown, Dr. John Schrader and Dr. Vince Duronio, for sharing their time and expertise. In addition, my thanks goes to Dr. Pelech and his laboratory staff for all the help they have provided to me during our collaboration. So many people have helped me during the course of my research. I would like to thank all the students, technologists and staff members at the Immunology Lab who have given me invaluable advice and support. I would also like to express my gratitude to the Canadian Red Cross. Without their supply of large amounts of blood samples this research would not have been possible. Finally, a big thank you to my parents and friends, who have cheered me up with their never-ending support. A special thanks goes to my wife, Rochelle - you never stopped encouraging me and helped me through all the ups and downs! xiv I 4edjcgte this thesis to ... my parents, Marlies and Gilbert Heisel, who gave me all the love and support to make this possible ... my wife, Rochelle Heisel, who is the sunshine in my life! xv CHAPTER 1: INTRODUCTION 1.1 Hypothesis Lymphocyte activation is initiated and regulated through a signaling cascade that starts with the engagement of the T-cell receptor and costimulatory receptor(s) (e.g. CD28, CD40). These receptors then transmit their signals through the cytoplasm using protein kinases, which in turn activate transcription factors in the nucleus. Clonal anergy results from an incomplete activation sequence, which may be postulated to comprise the following characteristics: 1. ) Incomplete antigen stimulation leads to the activation of the T-cell receptor without providing the necessary costimulatory signal. 2. ) The missing costimulatory signal fails to properly activate cytoplasmatic protein tyrosine kinases (PTK's), leading to a permanent downregulation of transcription factors at the promoter level. 3. ) Failure to activate the transcription of the IL-2 gene causes markedly reduced T-cell proliferation and IL-2 secretion leading to a downregulation of the immune response. The objective of this study was to establish a model of clonal anergy in human CD4 + T-lymphocytes that would permit investigation of the signaling events controlling IL-2 gene transcription. This was achieved by stimulation of isolated CD4 + T-lymphocytes through their T-cell receptor without giving the B7/CD28 or 1 CD40L/CD40 costimulatory signal. Using this model, the specific regulatory changes at the AP-1 and NFAT binding sites of the IL-2 promoter region, as well as the SRE and SIE binding sites of the c-Fos promoter region, were analyzed in the state of clonal anergy. Furthermore, transcriptional changes in this model were compared to transcriptional changes in three other possible models of clonal anergy. The first of these three models involved induction of clonal anergy using a blocking CD40L mAb to create a CD40/CD40L costimulatory blockade. The second model involved the blockade of p38 MAP kinase using the specific p38 inhibitor SB 203580, while the third model was based on IL-10 co-incubation using recombinant human IL-10. The identification of a molecule which is permanently downregulated even if costimulatory activity is restored at a later time might lead to new therapeutic opportunities. Such a model could lead to the design of agents that are able to specifically inhibit transplant rejection without affecting regular immune responses. 2 1.2 General background During the last decade, renal transplantation has become the treatment of choice for patients with end stage renal disease (ESRD) by offering a superior quality of life and lower mortality rates (Evans, R.W. et al. 1985; Berkoben, M. and Schwab, S. 1999). This is due to improvement of immunosuppressive protocols through the use of modern drugs such as cyclosporine neoral, which has significantly reduced the incidence and severity of acute rejection and improved short-term patient and graft survival (Sketris, I. et al. 1995; Hariharan, S. et al. 2000). The negative side of this progress in transplantation is the necessity for lifetime use of a combined immunosuppressive treatment (e.g. cyclosporine neoral, mycophenolate mofetil and steroids) associated with high economic costs and drug side effects. For example, cyclosporine neoral alone costs $4700 US per year (Cogny-Van Weydevelt, F. et al. 1998) and is characterized by a significant risk of nephrotoxicity (Ader, J.L. and Rostaing, L. 1998) and neurotoxicity (Bechstein, W.O. 2000). Thus, it has become a major goal in transplantation research to look for alternative ways to improve graft acceptance without the need for an aggressive immunosuppressive protocol (Morris, P.J. 1997). One possible way this could be achieved is through the induction of clonal anergy towards the donor organ in cells of the recipient's immune system. Anergy is a state in which a lymphocyte is fully viable but fails to display functional responses when optimally stimulated through both its antigen-specific receptor and other receptors that are normally required for full activation (Schwartz, R.H. 1996). This state was first demonstrated with cloned lines of CD4 + helper T-cells, in which antigen receptors were initially engaged without 3 costimulation (Schwartz, R.H. 1990). It is thought that anergy is induced by foreign antigens presented without costimulation, because such antigens do not stimulate inflammation or activate APCs (Matzinger, P. 1994). Induction of anergy by specific deletion of costimulatory signals has been proposed as a potential method to tolerize a transplant patient to his new graft. However, studies have shown that the long-term effectiveness of this approach, for example by blocking the B7/CD28 costimulatory pathway using CTLA4-lg alone, might be limited. For example, Wallace et al. showed in a mouse model that T-cells were still able to be primed for IL-4 mRNA synthesis even if B7/CD28 costimulation was blocked by CTLA4lg (Wallace, P.M. et al. 1995). In contrast, in vivo studies have indicated the possible therapeutic utility of clonal anergy by simultaneously blocking more than one pathway. By blocking both the receptor-ligand pairs CD28-B7 and CD40-gp39, Larsen et al. showed prolonged survival of transplanted skin and vascularized cardiac allografts in the mouse model. This survival occurred in the absence of immunosuppressive treatment (Pearson, T.C. et al. 1994; Larsen, C P . et al. 1996; Pearson, T.C. et al. 1996). A similar prolongation of cardiac graft survival could be shown by blocking CD28 and CD2 (Woodward, J .E. et al. 1996). The promising in vivo results obtained using animal models have prompted the investigation of intracellular signal transduction events leading to the state of clonal anergy. These studies will be addressed in detail below. Unfortunately, a major limitation of these studies is that the T-cell experiments have been done mainly in vitro using cell lines. To what extent these findings may be extrapolated to normal lymphocyte populations still remains unanswered. For example, it is not 4 known how the downregulation of signal transduction events might be affected by mechanisms that permanently induce cells to enter the cell cycle and proliferate. Comparing several hamster cell lines, Brezina et al. found various irregularities in mitotic cycle kinetics, such as absence of anaphase or separation of chromosomes from the aster in metaphase (Brezina, V. 1977). Also, Newbound et al. showed that the amounts of transcriptionally active phosphorylated cAMP responsive element binding (CREB) protein differ between activated PBMC and Jurkat cells. Following stimulation, P-CREB levels remain elevated in PBMC for up to 24 hours whereas CREB is dephosphorylated in Jurkat cells within 4 hours following stimulation (Newbound, G.C. et al. 1999). Due to these proven differences between native cells and cell lines, it is of crucial importance to investigate the signal transduction events leading to clonal anergy in native cells which have not been altered by transformation and immortalization. The interleukins IL-4 and IL-10 have also been shown to have anergizing effects in vitro (Ebert, E.C. and Roberts, A. l . 1996; Groux, H. et al. 1996; Marcelletti, J.F. 1996; Romano, M.F. et al. 1996). When both were administered together, they were able to prolong the survival of pancreatic islet grafts in non-obese diabetic (NOD) mice (Rabinovitch, A. et al. 1995). However, IL-10 also displays stimulatory effects on T and B cell differentiation, which might increase the risk of allograft rejection. For example, Qian et al. showed that post transplant systemic administration of IL-10 using a mouse model can promote vascularized allograft rejection, and that this may reflect stimulation both of B- and T-cell alloimmune responses. (Qian, S. et al. 1996). 5 While a large amount of research has been conducted on the extracellular molecules involved in anergy, much less is known about the intracellular signaling network and the nuclear events leading to anergy. The development of a simple and reproducible human model would facilitate the exploration of these events at the cytosolic and nuclear levels, and accelerate the search for new therapeutic agents, which may achieve long-lasting and selective immunological unresponsiveness 1.3 The signal transduction network 1.3.1 Overview In addition to T-cell receptor (TCR) activation, T-lymphocytes need a second signal to become fully activated - this is also described as the "two signal model" (Weiss, A. et al. 1984). This co-stimulatory signal is usually provided by the B7.1/2 molecule on the antigen-presenting cell, which binds to the CD28 receptor on the T-lymphocyte. Occupancy of the TCR alone in the absence of co-stimulation leads to a state of clonal anergy where the cells fail to proliferate when restimulated with antigen presenting cells (APC) and non-self antigen (Schwartz, R.H. 1996). In mouse models, this anergic state is characterized by a specific downregulation in IL-2 production whereas production of other cytokines (e.g. IL-3, interferon-y or IL-4) is not significantly affected (Mueller, D.L. and Jenkins, M.K. 1995). 6 1.3.2 T cell receptor signal transduction i) Phosphorylation of the TCRE, chain by tyrosine kinases from the Src family Binding of an immunogenic peptide presented by other T-cells, fixed APCs , purified major histocompatibility complex (MHC) molecules, or ligation of anti-CD3 antibodies leads to the stimulation of the T-cell receptor (TCR) (Sloan-Lancaster, J . et al. 1994; Mueller, D.L., Jenkins, M.K. 1995). This stable interaction between the TCR and MHC-peptide has been called "immunological synapse" (Shaw, A.S. and Dustin, M.L. 1997; Dustin, M.L. and Shaw, A.S. 1999). In a first step, unstable TCR-MHC interactions occur in a broad ring surrounded by an area of integrins which have been concentrated by engagement of their corresponding adhesion ligand (e.g. ICAM-1) on the surface of the stimulating cell (Grakoui, A. et al. 1999). In a second step, the arrangement is then stabilized as engaged TCR-MHC complexes become enriched in a tight central cluster, encircled by a ring of integrins. This transport of TCR complexes into a central cluster appears to depend upon optimal TCR-MHC interaction kinetics, which correlates with the half-life (t1 /2) of the TCR-MHC-peptide interaction and is a measure of the functional stability of TCR-MHC interaction (Monks, C R . et al. 1998). The formation of this immunological synapse is then associated with the propagation of a signal to the interior of the cell. Activated T-cell receptors within the synapse transduce their signal by interacting with cytoplasmic protein tyrosine kinases (e.g. ZAP-70, Lck) through a 17-residue sequence motif called the antigen recognition/immunoreceptor tyrosine activation motif (ARAM/ITAM) 7 contained in the TCR£, and CD3 chains (Iwashima, M. et al. 1994; Bu, J.Y. et al. 1995). The consensus sequence for an ITAM is YxxL(x) 6. 8YxxL (single letter code is used for amino acids, with x denoting any amino acid) (van Leeuwen, J .E. and Samelson, L.E. 1999). CD3-y, -8 and -s each contain one ITAM sequence whereas TCR£, contains three ITAM sequences. Recruitment of protein tyrosine kinases to the ITAMs is enhanced by stimulation of the co-receptors CD4 and CD8, respectively. They activate a cytoplasmic protein-tyrosine kinase Lck through a cysteine-containing motif shared by their cytoplasmatic domains (Weiss, A. and Littman, D.R. 1994; Gervais, F.G. and Veillette, A. 1995). Activation of intracellular protein tyrosine kinases is further enhanced by the membrane-bound phosphatase CD45. This dephosphorylates the C-terminal negative regulatory tyrosine of src-family members (Lck in particular), resulting in an increase in activity (Ashwell, J.D. and D' Oro, U. 1999). The activation of Lck leads to the recruitment of a second cytoplasmatic PTK, ZAP-70, to the TCR cluster through both of its Src homology-2 domains and its phosphorylation (Iwashima, M. et al. 1994). TCR signal transduction is thus initiated by the sequential interaction of two PTK's (Lck, ZAP-70) with TCR ITAMs resulting in the phosphorylation of two critical tyrosines and the generation of differentially phosphorylated isoforms, p21 and p23. Evidence has been obtained for a highly ordered sequential phosphorylation of the six tyrosines of TCR£, ITAMs, and full phosphorylation of all six tyrosines is dependent on the strength of TCR occupancy (Irving, B.A. et al. 1993). 8 ii) Regulation and function of tyrosine kinases from the ZAP-70/Syk family ZAP-70 has been described to play two crucial roles in the earliest biochemical events after TCR crosslinking (Qian, D. and Weiss, A. 1997; Chu, D.H. et al. 1998). First, it plays a role in the phosphorylation of P L C y l , SLP-76 and LAT; second, it plays a role in TCR-induced C a 2 + mobilization, activation of the transcription factor NFAT (nuclear factor of activated T cells) and IL-2 production (Williams, B.L. et al. 1998). Dual-phosphorylated ITAMs recruit ZAP-70/Syk family kinases via their tandem SH2 sequences. iii) Transmembrane adaptors and TCR signaling LAT (Linker for Activation of T-cells) has been identified as one of the first members of a new class of molecules in TCR signaling that function as transmembrane adaptor proteins (Schraven, B. et al. 1999). Following triggering of the TCR, LAT becomes rapidly tyrosine phosphorylated (most likely by ZAP-70), and is then able to recruit critical signaling molecules to the membrane; these include P L C y l , GRB2, Grap and p85 phophatidylinositol-3-kinase as well as Grb2-SH3-binding proteins Sos, c-Cbl and the SLP-Vav complex (van Leeuwen, J .E. , Samelson, L.E. 1999). Mice deficient for LAT have been shown to have their thymocyte development arrested at the CD4-CD8- (double negative) stage, due to an inability to progress past the pre-TCR checkpoint (Zhang, W. et al. 1999). Recently, two other transmembrane adaptor proteins (SIT and TRIM) have been described in T cells (Schraven, B. et al. 1999). It appears that TRIM is a positive 9 regulator (Zhang, W. et al. 1999), possibly acting through Grb-2 and PI-3 kinase. SIT may negatively regulate T cell activation, consistent with the presence of an inhibitory motif in its cytoplasmatic tail and its interaction with the phosphatase SHP-2 (Marie-Cardine, A. et al. 1999). iv) Cytoplasmic adaptors and TCR signaling SLP-76 is an adaptor protein with an amino-terminal region that contains critical tyrosine phosphorylation sites, a central proline-rich domain and a carboxy-terminal SH2 sequence (Peterson, E.J. et al. 1998). It is a prominent substrate for PTK's from the ZAP-70/Syk family. Furthermore, SLP-76, through its proline-rich tract, binds to the SH3 domain of Lck (Sanzenbacher, R. et al. 1999). Thus, co-ligation of the CD4 co-receptor and its associated Lck brings SLP-76 into proximity with upstream TCR signaling components. It is complexed with other adapter proteins, mostly Grb2 and Gads, through proline-rich sequences distinct from those binding Lck (Clements, J.L. et al. 1999; Rudd, C.E. 1999). After tyrosine phosphorylation, presumably by ZAP-70, the SLP-76-Grb2 (or -Gads) complex then interacts with SH2 containing proteins. SLP-76 is also thought to act in concert with the Vav Rac/CDC42 exchange factor (Wu, J. et al. 1996; Bubeck Wardenburg, J . et al. 1998). However, Vav and SLP-76 may still target some distinct downstream pathways (Fang, N. and Koretzky, G.A. 1999); thus, mutation of tyrosine 113 or 128 in SLP-76 eliminates its detectable interaction with Vav yet still allows for activation of NFAT-dependent transcription. 10 Another protein that may function like SLP-76 is the adaptor Clnk, which is expressed preferentially in cytokine-stimulated cells (Cao, M.Y. et al. 1999). Clnk contains a SLP-76 related SH2 domain near its carboxyl terminus, as well as several potential tyrosine phosphorylation and SH3-interacting sites. Clnk overexpression activates downstream regulators of TCR activation, including NFAT. Other recently identified adaptors of uncertain function include Shb (Lindholm, C.K. et al. 1999) and CAST (Yamazaki, T. et al. 1999). v) Regulation of TCR-induced, sustained Ca 2* mobilization by tyrosine kinases from the Tec/Btk family TCR engagement results in the activation of three members of the Tec/Btk family of tyrosine kinases: Itk (inducible T cell kinase), Tec and Rlk/Txk (Gibson, S. et al. 1996; Debnath, J . et al. 1999; Yang, W.C. et al. 1999). Itk and Tec are also activated following CD28 stimulation. Itk, Tec and Btk are recruited to the membrane via the action of an amino-terminal pleckstrin-homology domain that binds products of phosphatidylinositol-3-kinase (August, A. et al. 1997; Li, Z. et al. 1997; Scharenberg, A.M. et al. 1998). Itk-deficient mice show mild defects in T-cell development (Liao, X .C. and Liftman, D.R. 1995). In addition, proliferation in Itk-deficient mice is greatly reduced upon TCR stimulation but enhanced upon CD28 co-stimulation when compared to normal mice (Liao, X . C , Liftman, D.R. 1995; Liao, X .C. et al. 1997). 11 vi) Stat (Signal Transducer and Activator of Transcription) proteins and TCR signaling The Stat proteins are important mediators of signaling by cytokine receptors (Liu, K.D. et al. 1998). Recently, it has been suggested that Stat proteins play a role in signaling through the TCR. A transient phosphorylation of Stat5 by the TCR£, chain was observed after TCR crosslinking, as was phosphorylation of TCR£-bound Stat5 by Lck (Welte, T. et al. 1999). Furthermore, Lafont et al. showed that antigen receptor ligation induces delayed but sustained phosphorylation of Statl on Ser727, which is dependant on phosphatidylinositol-3- kinase mediated signals (Lafont, V. et al. 2000). Although the ultimate function of the Stat involvement in T-cell activation remains unclear, it raises the possibility of cross-talk between antigen receptors and signaling components of cytokine receptors. This creates the possibility that anergic cells might also show alterations in the Jak-Stat signal transduction pathway. vii) Negative regulation of TCR signaling Negative regulators play an important role in fine-tuning antigen-receptor signaling and in switching it off after activation (Ibarra-Sanchez, M.J. et al. 2000). To date, most emphasis in this area has been on phosphatases (Chan, A.C. et al. 1994). An important example is the protein tyrosine phosphatase SHP-1 which is activated by (31- integrins and has been shown to dephosphorylate ZAP-70, LCK and the TCR £ chain (Mary, F. et al. 1999). 12 The Cbl protein also plays an important role in downregulating TCR activation. Until recently, Cbl was thought to function solely as an adaptor, possibly by binding tyrosine 292 in ZAP-70 (Meng, W. et al. 1999; Rao, N. et al. 2000). Recent studies, however; have provided evidence that Cbl can function as an ubiquitin-3 ligase in vitro (Joazeiro, C A . et al. 1999) and also functions as a negative regulator of receptor clustering (Krawczyk, C. et al. 2000). Thus, Bachmaier et al. described Cbl as a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity (Bachmaier, K. et al. 2000). Loss of Cbl function has been associated with transformation (van Leeuwen, J.E. et al. 1999; Zhang, Z. et al. 1999). Another negative regulator of TCR function is SLAP (src-like adaptor protein). SLAP contains SH3 and SH2 domains that are highly homologous to those of src kinases, but it contains no kinase function (Sosinowski, T. et al. 2000). It is not clear whether negative regulatory proteins play a role in clonal anergy. However, Matsushita et al. have found that anergy induction by peptide analogs in a murine system was associated with binding of SHP-1 to ZAP-70 and with subsequent lack of ZAP-70 recruitment to the CD3E, chain (Matsushita, S. and Nishimura, Y. 1997). This suggests that dominant-negative TCR-mediated signaling by altered TCR ligands might lead to the induction of clonal anergy through phosphatases by a yet unidentified signal transduction cascade (Sloan-Lancaster, J . et al. 1994; Matsushita, S., Nishimura, Y. 1997). 13 1.3.3 Costimulatory signal transduction via CD28/CTLA-4 and B7.1/B7.2 i) Structure of the CD28 receptor CD28 is a homodimeric cell surface glycoprotein expressed on 95% of CD4 + T-cells (June, C H . et al. 1994). It is composed of two glycosylated 44 kDa chains, which are members of the immunoglobulin superfamily, each containing a single disulfide-linked extracellular Ig variable-like (V) domain. The extracellular domain is linked via a single transmembrane region to a 41-amino-acid cytoplasmatic domain, which is presumed to be responsible for initiating costimulatory signals (Ward, S.G. 1996). The cytoplasmic domain of CD28 lacks any direct enzymatic activity and is therefore presumed to signal via the recruitment of cellular enzymes. Of specific interest is a consensus sequence motif within the cytoplasmatic domain of CD28 [(p)Tyr1 7 3-Met-Asn-Met] that forms a potential binding site for interaction with signaling proteins, including the SH2 domains of the p85 subunit of PI 3-kinase and growth-factor-receptor binding protein (Grb-2), a ubiquitous adaptor protein (Songyang, Z. et al. 1993; Songyang, Z. et al. 1995). CD28 also contains two proline-rich motifs (Pro 1 7 8-Arg-Arg-Arg-Pro and Pro 1 9 0 -Tyr-Ala-Pro) which conform to the Pro-Xaa-Xaa-Pro SH3 binding consensus sequence, and these regions of the CD28 tail may mediate interactions with signaling proteins (Ward, S.G. 1996). 14 ii) Structure of the CTLA-4 receptor CTLA-4 is a disulfide-linked homodimer with a predicted molecular mass of 20 kDa, and with only a single glycosylation site. CD28 and CTLA-4 are expressed on the cell surface as either monomeric of homodimeric forms (Guinan, E.C. et al. 1994). Both CD28 and CTLA-4 can bind to the same physiological ligands, namely the 60 kDa B7.1 and the 70 kDa B7.2 (Linsley, P.S. et al. 1991; Linsley, P.S. et al. 1991). However, CTLA-4 is a high-avidity receptor as compared to CD28 (June, C H . et al. 1994). CTLA-4 contains a single disulfide-linked extracellular Ig V domain, a transmembrane region and a cytoplasmatic domain of 36 amino acids. It has a consensus binding site (Tyr164-Val-Lys-Met) for the SH2 domains of the p85 subunit of the PI 3-kinase (Schneider, H. et al. 1995). iii) B7.1 and B7.2 B7.1 and B7.2 are members of the immunoglobulin superfamily that contain two Ig-like domains (June, C H . et al. 1994). B7.1 is a 60 kDa glycoprotein which consists of two extracellular Ig-like domains, a transmembrane region, and a short 19-amino-acid cytoplasmatic domain. In contrast, while B7.2 is a 70 kDa glycoprotein that consists of two Ig-like domains, it also features an extended cytoplasmatic domain that contains phosphorylation sites for protein kinase C (PKC). These phosphorylation sites potentially indicate a signaling function for antigen presenting cells. B7.1 and B7.2 bind CTLA-4 with 20-100 fold higher affinity than they bind CD28 (Gimmi, C D . et al. 1991; Linsley, P.S. et al. 1991; Azuma, M. et al. 1993; Freeman, G.J. et al. 1993; Freeman, G.J . et al. 1993; Guinan, E.C. et al. 1994; June, C H . et al. 1994), with B7.2 being at most 2-3 fold less active than B7.1 (Linsley, P.S. et al. 1994). 15 iv) Differences in expression and function between B7.1 and B7.2 A number of differences between B7.1 and B7.2 have been reported that suggest differential immune regulation by these molecules. For example, B7.2 is rapidly expressed in B-cells following activation, whereas maximum B7.1 expression appears significantly later (Azuma, M. et al. 1993; Boussiotis, V.A. et al. 1993; Freeman, G.J . et al. 1993; Lenschow, D.J. et al. 1994). Furthermore, in contrast to anti-B7.1 mAbs, anti-B7.2 mAbs are potent inhibitors of T-cell proliferation and cytokine production in vitro (Lenschow, D.J. et al. 1993; Wu, Y. et al. 1993; Chen, C. et al. 1994). v) Functional responses to B7-CD28 interactions Engagement of the CD28 receptor enhances the production of IL-2 mRNA approximately 20-fold (Jenkins, M.K. 1994; Umlauf, S.W. et al. 1995), providing the necessary co-stimulatory signal for maximal T-cell activation. Under conditions of "supraoptimal" TCR occupancy, T-cell activation may occur which is independent of co-stimulation (Bluestone, J.A. 1995). Under physiological conditions, however, absence of the co-stimulatory signal induces a state of clonal anergy (Harding, F.A. et al. 1992; Boussiotis, V.A. et al. 1993; Boussiotis, V.A. et al. 1993; Gimmi, C D . et al. 1993; Tan, P. et al. 1993; Linsley, P.S. et al. 1994). Anergic T-cells can be rescued from this non-functional state by the addition of exogenous IL-2 (Boussiotis, V.A. et al. 1993). This requirement for two stimuli for T-cell activation could have an important role in vivo in establishing peripheral tolerance to antigens not encountered in the 16 thymus (Harding, F.A. et al. 1992; Mondino, A. et al. 1996). CD28 also regulates expression of the intrinsic cell survival factor Bcl-xL, which plays an important role in preventing cells from undergoing programmed cell death (apoptosis) induced by y-irradiation, antibodies to Fas or CD3, or IL-2 withdrawal (Boise, L.H. et al. 1995). Engagement of the CD28 co-receptor leads to the upregulation of a variety of surface antigens such as CTLA-4, the high affinity IL-2 receptor, and the CD40 ligand (CD40L) (Cerdan, C. et al. 1992; de Boer, M. et al. 1993; Cerdan, C. et al. 1995; Ding, L. et al. 1995), each of which is needed for successful progression of T-cell responses (Clark, E.A. and Ledbetter, J.A. 1994; Rudd, C .E . 1996). CD28 mediated costimulation also leads to a strong upregulation of IL-4, II-5, IL-13, y-interferon, tumor necrosis factor a, granulocyte/macrophage colony-stimulating factor (GM-CSF) and the chemokine IL-8 (Thompson, C.B. et al. 1989; de Boer, M. et al. 1993; Minty, A. et al. 1993; Seder, R.A. et al. 1994; Wechsler, A.S . et al. 1994). vi) Differential regulation of immune responses There is a body of evidence that the interactions involving CD28 and B7 family members function to differentially regulate the immune response. For instance, CTLA-4, unlike CD28, is not expressed constitutively and is only expressed maximally 2-3 days after T-cell activation by TCR/CD3 and CD28 ligation (Lindsten, T. et al. 1993). In mutant mice, it has been shown that CTLA-4 cannot replace CD28 in providing costimulation, ruling out functional redundancy 17 (Shahinian, A. et al. 1993). Antibodies to CTLA-4 can enhance T cell proliferation, whereas crosslinking of the antibodies have been shown to inhibit proliferation of native T cells (Linsley, P.S. et al. 1992; Linsley, P.S. et al. 1994; Walunas, T.L. et al. 1994; Krummel, M.F. and Allison, J .P. 1996; Walunas, T.L. et al. 1996). This suggests that blockade of CTLA-4 removes, whereas aggregation of CTLA-4 provides, inhibitory signals that downregulate T cell responses. vii) Augmentation of IL-2 production Activation of the co-stimulatory CD28-B7 pathway augments IL-2 production at least in part by inhibiting the selective degradation of cytokine mRNAs (Lindstein, T. et al. 1989; Umlauf, S.W. et al. 1995). Without co-stimulation this degradation process proceeds rapidly and leads to reduced IL-2 mRNA levels. Engagement of the CD28 receptor by B7 also results in the expression of anti-apoptotic proteins of the Bel family, notable Bcl-x L, in T-cells (Boise, L.H. et al. 1995). It also results in the production of cytokines (Schwartz, R.H. 1992), such as interleukin-2 (see Figure 1). Thus, costimulation promotes the survival of T cells that encounter an antigen, allowing autocrine cytokines to initiate clonal expansion and differentiation. 18 19 1.3.4 Intracellular signal transduction following CD28 activation As mentioned previously, the CD28 receptor transduces its signal to the intracellular compartment via the phosphotyrosine based motif pYMNM, which serves as a binding motif for Src homology 2 (SH2) domains (August, A. and Dupont, B. 1994). Four SH2 containing signaling molecules that bind to this motif have been identified. i) The Pi 3-kinase pathway The lipid kinase PI 3-kinase is a heterodimer, composed of a p85 adapter subunit linked to a p110 catalytic domain. The p110 subunit is able to phosphorylate the D-3 position of the inositol ring generating PI 3-P, PI 3,4-P 2, and PI 3,4,5-P 3, respectively. The p85 subunit has two SH2 domains which are able to bind to a core phosphotyrosine residue and adjacent residues within the pYMNM motif. By binding PI 3-kinase through .its p85 binding motif (Tyr 1 7 3-Xaa-Xaa-Met), CD28 anchors the enzyme to the inner face of the plasma membrane where it can act on its target substrates (August, A., Dupont, B. 1994; June, C H . et al. 1994; Stein, P.H. et al. 1994; Truitt, K.E. et al. 1994). Further downstream, PI 3-kinase is required for activation of the rapamycin-sensitive serine/threonine protein kinase, S6 kinase (p70S6K), thus playing a role in cell proliferation (Monfar, M. et al. 1995). PI 3-kinase is also a key molecule involved in cell survival through prevention of apoptosis (Yao, R. and Cooper, G.M. 1995). Whether failure of PI 3-kinase activation in anergic cells might inhibit cell survival and induce apoptosis is still unclear. 20 ii) The p21 r a s pathway The CD28 pYMNM site binds to the GRB-2-Son of Sevenless (GRB-2-SOS) complex via an SH2 domain (Schneider, H. et al. 1995). Indeed, anti-CD28 mAbs can activate the p21 r a s pathway via the induction of RAS-GTP complexes and via the phosphorylation of Vav and the Grb-2/Sos-associated protein (Nunes, J.A. et al. 1994). SOS converts p21 r a s from an inactive GDP-bound state to an active GTP-bound state. p21 r a s plays a central role in activating the mitogen activated protein kinase (MAPK) pathway. iii) The ITK pathway The CD28 pYMNM motif also binds to the SH2 domain of ITK, a Tec-family protein-tyrosine kinase (August, A. et al. 1994). Given its restricted expression on T-cells, ITK is an attractive candidate for co-signaling (Rudd, C.E. 1996). iv) The sphingomyelinase/ceramide pathway Hydrolysis of the phospholipid sphingomyelin by a specific sphingomyelinase results in ceramide production. Ceramide serves as a second messenger, and may activate downstream targets such as PKC^ (Diaz-Meco, M.T. et al. 1994; Lozano, J . et al. 1994), serine/threonine-specific protein kinases (Mathias, S. et al. 1991) and phosphatases (Dobrowsky, R.T. and Hannun, Y.A. 1992). Since the ceramide pathway has been shown to be activated by Fas (Hannun, Y.A. and Obeid, L.M. 1995; Kolesnick, R. and Fuks, Z. 1995), it is thought to be 21 involved in apoptosis. It has been shown that anti-CD28 mAbs induce the activation of acidic sphingomyelinase (Boucher, L.M. et al. 1995; Chan, G. and Ochi, A. 1995). Moreover, this pathway seems to be required for costimulation since the cell-permeable ceramide analogue C-6-ceramide mimicked the CD28 signal by inducing T cell proliferation and IL-2 gene transcription (Chan, G., Ochi, A. 1995). The ability of CD28 to potentially activate both the PI-3 kinase and sphingomyelinase pathways may be a key distinguishing feature between the signals provided by CD28 and the so-called "death" receptors such as Fas (Cleveland, J.L. and Ihle, J.N. 1995), which lack any known PI-3 kinase consensus binding motif. Thus, there may be synergy between the CD28-activated PI-3 kinase and ceramide-mediated signaling pathways in protecting the cell from apoptosis and promoting cell survival and-or IL-2 production. 1.3.5 Costimulatory signal transduction via CD40/CD40L i) Overview The critical role of the CD40-CD40L pathway was first highlighted by the observation that patients suffering from X-linked Hyper-IgM syndrome were characterized by mutations in their CD40L gene (Banchereau, J . et al. 1994; Foy, T.M. et al. 1996; van Kooten, C. and Banchereau, J . 2000). Further research established the critical importance of the CD40/CD40L pathway in T-cell costimulation (Callard, R.E. et al. 1993; Stout, R.D. and Suttles, J . 1996; Stout, R.D. et al. 1996). CD40L -/- knockout mice have been shown to be severely impaired in their primary T cell responses to protein antigens (Grewal, I.S. et al. 22 1995; Grewal, I.S. and Flavell, R.A. 1996; Grewal, I.S. and Flavell, R.A. 1996). The defective T cell responses of these mice were mapped to an inability of CD40L-deficient T cells to undergo effective clonal expansion in vivo and to enter the cell cycle. CD40 ligation on monocytes and dendritic cells has a multitude of effects including (a) enhanced survival of these cells; (b) secretion of the cytokines IL-1, IL-6, IL-8, IL-10, IL-12, TNF -a; (c) enhanced anti-tumor activity of the monocyte; (d) NO synthesis and (e) upregulation of costimulatory molecules such as B7/CD28 (Foy, T.M. et al. 1996; Grewal, I.S., Flavell, R.A. 1996; Stout, R.D., Suttles, J . 1996). Therefore, the CD40/CD40L pathway has an important impact on T-cell activation through increased IL-12 secretion and B7/CD28 upregulation. It has been speculated that CD40/CD40L interactions can be bi-directional. First, cross-linking CD40L in vivo contributes to the generation of helper function and germinal centers (van Essen, D. et al. 1995). Second, in vitro ligation of CD40L on T cells considerably enhances their cytokine production (Poudrier, J . et al. 1998). However, the signaling mechanism leading to this effect is still poorly understood (van Kooten, C , Banchereau, J . 2000). The CD40/CD40L pathway may play an important role in organ transplantation: CD40/CD40L interactions play a critical role in T cell priming and in the induction of transplantation tolerance. Thus, blocking of the CD40/CD40L pathway has been investigated as a tool to prolong the survival of transplanted organs in experimental models including allografts of heart, skin, aorta, and pancreatic islets (Larsen, C P . and Pearson, T.C. 1997). In this setting, it has been shown to 23 be beneficial to use simultaneous blocking of the B7/CD28 and CD40/CD40L pathways (Elwood, E.T. et al. 1998). Such a co-blockade of both costimulatory systems leads to long-term acceptance of skin and cardiac allografts in a mouse model (Larsen, C P . et al. 1996; Larsen, C P . et al. 1996). / • Furthermore, data by Kishimoto et al. has shown that there is a differential effect of CD28-B7 versus CD40-CD40L blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant (Kishimoto, K. et al. 2000). These studies indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD40-CD40L costimulation pathways in vivo, and may therefore have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans. A system of co-blockade may be applied to clinical studies in the near future. Guinan et al. Have already reported a new method of preventing graft-versus-host-disease (GVHD) via B7/CD28 blockade (de Carvalho Bittencourt, M. et al. 1999; Guinan, E.C. et al. 1999). CTLA-4-lg was added to a mixed culture of irradiated mononuclear cells from the recipient and marrow cells from the donor. After 36 hours, the recipient and donor cells were infused into the patient. The incidence of graft-versus-host disease after transplantation of haploidentical bone marrow (from a donor mismatched with the recipient for one HLA haplotype) was lower than expected. The authors attribute the inhibition of alloreactivity to anergy of the donor T cells, mediated by blockade of the B7/CD28 pathway. This effect of anergy induction may be even more pronounced in future applications using the same model in a setting of B7/CD28 and CD40/CD40L co-blockade. 24 ii) Structure of the CD40L and its counter-receptor CD40L, a member of the tumor necrosis factor (TNF) gene family, is a 33 kDa type II membrane protein, that is preferentially expressed on activated CD4 + T cells and mast cells (Banchereau, J . et al. 1994; Grewal, I.S., Flavell, R.A. 1996). Although CD40L is produced as a type II transmembrane protein, CD40L may be expressed on the cell surface as a heteromultimeric complex (Hsu, Y.M. et al. 1997). The counter-receptor for CD40L is CD40 (40 kDa), a type I transmembrane receptor and a member of the TNF-receptor (TNFR) family. CD40 is found on various APCs , including B cells, dendritic cells, activated macrophages and follicular dendritic cells (Graf, D. et al. 1992; Noelle, R.J. et al. 1992; Armitage, R.J. et al. 1993; Hollenbaugh, D. et al. 1994). iii) CD40 signal transduction Although CD40 has no kinase domain, CD40 ligation to CD40L activates several second messenger systems (van Kooten, C., Banchereau, J . 2000). These include the activation of (a) protein tyrosine kinases (including lyn, syk and Jak3), (b) PI 3-kinase and (c) phospholipase Cy2. The CD40 receptor connects to a new class of receptor-associated proteins known as TRAF (TNF-Receptor associated factor). At the moment, six different 25 members of the TRAF family have been identified. One of the earliest members identified is TRAF-3 (Cheng, G. et al. 1995), a 62 kDa intracellular protein that is expressed in almost all cell types. It contains several domains involved in either signal transduction (e.g. isoleucine zipper domain) or in the formation of homo/heterodimers between CD40 molecules (Xu, Y. et al. 1996). TRAF-2 is thought to ultimately induce N F - K B activation after CD40 cross-linking (Rothe, M. et al. 1995; Rothe, M. et al. 1996). CD40 triggering can also lead to the activation of the transcription factors AP-1 and NFAT (van Kooten, O , Banchereau, J . 2000) as well as the JAK-STAT pathway (Hanissian, S.H. and Geha, R.S. 1997). 1.3.6 Downstream pathways following TCR/CD28 activation One major difficulty in assessing the further downstream events is the partial overlapping of both the TCR/CD3 and CD28 signaling cascades. As mentioned above, p21 r a s can be activated by the CD28 receptor via the GRB-2-SOS complex, but TCR binding can also result in p21 r a s signaling via PKC-dependent and PKC-independent mechanisms (Weiss, A., Littman, D.R. 1994). p21 r a s has several important functions; most importantly, p21 r a s can activate Raf-1. Raf-1, in turn, continues the cascade by activating the MAPKs ERK 1 and 2, and these ERKs then phosphorylate the transcription factor Elk-1 (Marais, R. et al. 1993; Cano, E. and Mahadevan, L.C. 1995). Elk-1 can induce expression of Fos, a critical factor for IL-2 transcription (Mueller, D.L., Jenkins, M.K. 1995; Waskiewicz, A .J . and Cooper, J.A. 1995). Furthermore, the ability of Fos to enhance transcription at the IL-2 promoter is enhanced if it is phosphorylated by 26 Fos-regulating kinase (FRK) (Deng, T. and Karin, M. 1994). p21 r a s-dependent Raf activation is also important for activation of N F K B . Once activated, N F K B binds to the IL-2 promoter (Mueller, D.L., Jenkins, M.K. 1995). Similar to p21 r a s , PI 3-kinase can also be activated by both CD28 (see above) and TCR signaling cascades. TCR activation signals through the SH3 domains of p59 f y n and p56 l c k , leading to the recruitment of PI 3-kinase to the TCR^-CD3 and CD4 receptors (Rudd, C.E. et al. 1994). PI 3-kinase itself has a Bcr homology region which may interact with the small G proteins cdc42 and Rac-1 (Zheng, Y. et al. 1994; Cano, E., Mahadevan, L.C. 1995; Kyriakis, J .M. and Avruch, J . 1996). These molecules can activate the kinases SAPK/JNK and p38/HOG-1, respectively. SAPK/JNK activation correlates well with IL-2 transcription by its ability to phosphorylate the c-Jun transcription factor (Rudd, C.E. 1996). The p38/HOG-1 pathway is also predicted to influence transcription by c-Jun but this has not yet been examined. The overlap phenomenon described above involves stimulation of p21 r a s and PI 3-kinase by both CD28 and TCR signaling. The clue to understanding this phenomenon might lie in the different extent of p21 r a s and PI 3-kinase activation by the TCR and CD28-receptor, respectively. For example, the TCR provides a potent signal for p21 r a s stimulation, whereas CD28 binds PI 3-kinase with a several fold higher affinity than TCR/CD3 (Rudd, C.E. et al. 1994; Rudd, C.E. 1996). Thus, TCR signaling may not reach the threshold necessary for full PI 3-kinase activation. In such a scenario, CD28 signaling may supplement this suboptimal TCR-dependent activation of PI 3-kinase. 27 One pathway which is dependent on TCR activation alone is the generation of IP 3 by a tyrosine-phosphorylated PLOyl (Secrist, J .P. et al. 1991). IP 3 is responsible for a rapid and sustained increase in cytoplasmatic free [Ca 2 +]. This, in turn, leads to activation of calcineurin, a calcium/calmodulin dependent serine phosphatase. The immunosuppressive activity of the classical immunosuppressive drugs cyclosporine A and FK 506 correlates well with their ability to inhibit calcineurin phosphatase activity. One major target of calcineurin is the preexisting cytoplasmatic form of the NFAT transcription factor. Upon activation, NFAT is translocated into the nucleus and binds to one of the two NFAT-binding sites at the IL-2 promoter region (Garrity, P.A. et al. 1994; Rao, A. 1994; Jain, J . et al. 1995). A characteristic feature of NFAT is its cooperative binding with Fos- and Jun-family members to the distal NFAT promoter NFAT site (Jain, J . et al. 1993). This calcium-independent nuclear component of NFAT is thereby also influenced by CD28 dependent activation of Jun, reflecting again the connection between TCR and CD28-receptor signaling. 1.3.7 Clonal anergy Under physiological conditions, clonal anergy in peripheral T-lymphocytes is thought to have two major functions: First, it maintains T-cells unreactive against 28 self-antigens that are not expressed in the thymus and, second, it functions as a back-up mechanism for T-cells that escape intrathymic clonal deletion (Blackman, M.A. et al. 1990; DeSilva, D.R. et al. 1991). There are several ways to induce anergy: Originally, T-cell unresponsiveness was induced in CD4 + helper T-cell clones after stimulation with antigen-MHC complexes in the absence of costimulatory signals provided by antigen-presenting cells (APC) (Mueller, D.L., Jenkins, M.K. 1995; Schwartz, R.H. 1996). This approach was later modified by using antibodies directly against the TCR/CD3 complex (Gajewski, T.F. et al. 1994; Kawaguchi, M. and Eckels, D.D. 1995; Mondino, A. et al. 1996) or immunogenic peptides without co-stimulatory activity (Wotton, D. et al. 1995). Furthermore, cells could be anergized by TCR-activation and blocking the IL-2 proliferation signal using antibodies against IL-2 and the IL-2 receptor (DeSilva, D.R. et al. 1991). Interestingly, clonal anergy can also be induced if CD4 + T-cells are activated by allogenic monocytes in the presence of IL-10 (Groux, H. et al. 1996). Nothing is known yet about the intracellular signaling pathways mediating this IL-10 effect. Even if the basis of most models focuses on changes in the costimulatory pathway, it would be too simplistic to regard the TCR as a simple on/off switch. Instead, multiple discrete pathways may be directly activated upon TCR cross-linking by ligand binding. Thus, optimal TCR-binding may result in optimal signal transduction to the cytoplasm and proliferation, and suboptimal or partial binding 29 in anergy (Sloan-Lancaster, J . et al. 1993; Sloan-Lancaster, J . et al. 1994). Experiments with partially binding peptides show that suboptimal ligand/TCR interaction leads to inefficient recruitment of one or both Src kinases (p56 l c k, p59 f y n) resulting in highly reduced ZAP-70 activity (Sloan-Lancaster, J . et al. 1994). In accordance with these results are findings that antibodies binding to different epitopes on the TCR can cause different IL-2 and IL-4 responsiveness, respectively (Schwinzer, R. et al. 1992). It is not clear whether altered TCR-activation plays a role in anergized cells, as the experimental findings are contradictory. ZAP-70 activation mediates the activation of PLCy-1, generating IP 3and DAG. IP3 leads to the elevation of [Ca 2 +]. Gajewski et al. observed that Th1 cells have a reduced capacity to induce C a 2 + mobilization (Gajewski, T.F. et al. 1994; Andris, F. et al. 1996). On the other hand, Mondino et al. found that C a 2 + mobilization was restored after a 1 day decrease upon the induction of anergy, thus not explaining the long term state of anergy. As the initial inhibition correlated well in time with a reduced TCR/CD3 expression, the observed decrease in Ca 2 +-mobilization may be a consequence of a reduction in TCR/CD3 expression (Mondino, A. et al. 1996). Further downstream, calcineurin, a Ca 2 +/calmodulin dependent phosphatase, mediates the translocation of the cytosolic component of the NFAT-complex (NFATp) to the nucleus where it binds to the IL-2 promoter region. Using a mouse model, some authors found a reduction in NFAT binding in the nucleus of anergized cells (Kang, S.M. et al. 1992; Wotton, D. et al. 1995). It is also noteworthy that the IL-2 promoter contains a proximal and a distal NFAT binding 30 site, and NFAT binds cooperatively with members of the Fos and Jun family to the distal site (Jain, J . et al. 1993; Jain, J . et al. 1995). This makes it difficult to differentiate between a reduced NFAT binding versus reduced Fos and Jun-binding. Using a probe complementary to the proximal site (binding NFAT alone), Mondino et al. could not detect reduced NFAT binding even if cooperative binding with Fos and Jun members at the distal site was reduced (Mondino, A. et al. 1996). The second major function of T-cell receptor binding is the activation of p21 r a s by PKC-dependent and independent mechanisms (Weiss, A., Liftman, D.R. 1994) (see above). Fields et al. demonstrated reduced p21 r a s activation in anergic CD4 + T-cells (Fields, P.E. et al. 1996). Consequently, the downstream mitogen activated kinases ERK-1 and ERK-2, as well as JNK, could be shown to have reduced activities (DeSilva, D.R. et al. 1996; Fields, P.E. et al. 1996; Li, W. et al. 1996). This in turn mediated reduced expression and function of Fos and Jun at the mouse IL-2 promoter. In fact, reduced expression and DNA-binding activity of c-Fos, FosB and JunB could be shown in anergized CD4 + T-cells (Mondino, A. et al. 1996). Thus, a major feature of clonal anergy is defective AP-1 binding at the IL-2 promoter. 31 1.3.8 Signal transduction at the transcriptional level i) The IL-2 promoter region Since production of IL-2 in CD4 + T-lymphocytes is regulated at the transcriptional level (Crabtree, G.R. 1989), recent work has focused on the signaling events at the IL-2 promoter region in anergized T-lymphocytes. The IL-2 enhancer/promoter site consists of several DNA responsive elements including proximal and distal AP-1 and NFAT binding sites, binding sites for octamer proteins and an N F - K B binding site. ii) The NFAT binding site The IL-2 promoter site contains two binding sites for members of the NFAT family of transcription factors (Crabtree, G.R. and Clipstone, N.A. 1994; Nolan, G.P. 1994; Rao, A. 1994). Mutation of both sites is required to eliminate IL-2 promoter function (Nair, A.P . et al. 1994; Zhang, L. and Nabel, G.J . 1994), and in vivo footprinting indicates that both sites are occupied in stimulated cells (Garrity, P.A. et al. 1994). Four members of the NFAT family (NFAT.1, NFATc, NFATx, NFAT3) have been identified so far (Masuda, E.S. et al. 1998). 32 N F A T O C T N F - K B C D 2 8 R E A P - 1 N F A T A P - 1 / O C T El C . A G C A A A A T T T G T T T C A T T C A T T C T A T 43 Figure 1b: Schematic display of the IL-2 promoter region (Jain, J . et al. 1995) After translocation to the nucleus and binding to DNA, NFAT proteins enhance transcription in several ways. First, NFAT proteins recruit or facilitate the binding of AP-1 transcriptions factors to NFAT-AP-1 binding sties (Luo, C. et al. 1996; Masuda, E.S. et al. 1998). The components of the AP-1 transcription factor then mediate the induction of transcription by recruiting co-activators such as CBP (CREB-binding protein), p300 and JAB1 (Jun activation domain binding protein), through their transcriptional activation domains (Karin, M. et al. 1997). These co-activators augment transcriptional activity by recruiting the basal transcription machinery through direct protein-protein interactions and by acetylating histones, which increases accessibility of nucleosomal DNA to transcription factors (Hertel, K.J. et al. 1997). 33 In addition, NFAT proteins also contain their own transactivation domains (TADs). For example, in NFAT1 two different TADs were identified (Luo, C. et al. 1996). One was mapped to the first 100 amino acids in the amino terminus, and the other was shown to be present within the last 200 amino acids in the carboxy terminus. iii) The AP-1 binding site The AP-1 family of proteins includes a number of Fos and Jun proteins (Garrity, P.A. et al. 1994; Jain, J . et al. 1995; Sundstedt, A. et al. 1996). Interestingly, the two families of proteins are downstream targets of both the T-cell receptor and the CD28 co-receptor signaling cascade (Ward, S.G. 1996; Whitmarsh, A .J . and Davis, R.J. 1996). AP-1 DNA binding activity is specific for the palindromic sequence 5' TGAGTCA 3' (Wisdom, R. 1999). DNA-binding by AP-1 complexes is mediated by the basic-leucine zipper (bZIP) motif which is important for dimerization. Jun family proteins contain a single activation domain located amino-terminal to the bZIP motif. In the case of c-Jun, the activation domain is regulated to a large degree by the JNK members of the MAP kinase family (Karin, M. 1995). Fos family proteins contain activation domains both amino-terminal and carboxy-terminal to the bZIP motif, which is centrally located. The Fos C-terminal activation domains are regulated by phosphorylation, although the kinases that regulate this domain remain to be identified (Karin, M. 1995; Skinner, M. et al. 1997). Mutation of the AP-1 site abolishes promoter function in transient transfection assays (Jain, J . et al. 1992). The dimerization affinities of Fos-Jun heterodimers 34 are much higher than those of Jun-Jun homodimers (Ryseck, R.P. and Bravo, R. 1991); thus, Fos-Jun dimers are also more effective at DNA binding and transactivation. Because the AP-1 site of the IL-2 promoter is a relatively low-affinity site (Serfling, E. et al. 1989; Jain, J . et al. 1992; Jain, J . et al. 1995), it may require Fos-Jun dimers for optimal activity; hence, the requirement for protein synthesis for IL-2 gene induction (Shaw, J . et al. 1987) may reflect, in part, a requirement for de novo synthesis of Fos-family proteins (Kovary, K. and Bravo, R. 1991; Jain, J . et al. 1992). It has been determined by genetic studies that AP-1 plays a role in many different signal transduction pathways (Wisdom, R. 1999). Although all possible combinations of Fos-Jun dimers will bind the consensus AP-1 target element, functional assays have revealed some differences in the ability of different dimeric combinations to mediate transcriptional activation (Chiu, R. et al. 1989; Wisdon, R. and Verma, I.M. 1993). For example, using a mouse model Ryseck et al. have shown that, for a given AP-1-containing oligonucleotide, the binding affinities of the different Jun proteins are always c-Jun greater than Juh-D greater than Jun-B (Ryseck, R.P., Bravo, R. 1991). As mentioned previously, AP-1 complexes can also interact with NFAT in a cooperative manner (Jain, J . et al. 1995). iv) The c-Fos promoter region Amongst the Fos and Jun family members, c-Fos forms an important part of the AP-1 complex (Angel, P. and Karin, M. 1991; Karin, M. 1995). The c-Fos promoter is regulated by three major transcriptional elements: the c-AMP-responsive 35 element (CRE), the serum-responsive element (SRE) and the sis-inducible element (SIE). While CRE mostly responds to the second messenger c-AMP and SRE is activated by a variety of stimuli, the SIE region is bound by a complex of the transcription factors STAT1 and STAT3. It has been shown that STAT1 and STAT3 activity is regulated by serine phosphorylation, possibly by ERK MAP kinase (Ihle, J.N. et al. 1994; Wen, Z. et al. 1995; Ihle, J .N. 1996). Furthermore, ERK is downstream of p21 r a s , and can be activated by both T-cell receptor and CD28 ligation (Samelson, L.E. and Klausner, R.D. 1992; Nunes, J.A. et al. 1994; Weiss, A., Littman, D.R. 1994; Schneider, H. et al. 1995; Cantrell, D. 1996; Rudd, C.E. 1996). Using a mouse T-cell clone model, Kang et al. have shown a downregulation of binding at the AP-1 site in anergy (Kang, S.M. et al. 1992), which was further narrowed by Mondino et al. to a specific downregulation of c-Fos, FosB and JunB expression (Mondino, A. et al. 1996). However, whether results obtained from mouse cell lines consisting of identical cells can be extrapolated to normal human lymphocyte populations has remained uncertain (Van Parijs, L. and Abbas, A.K. 1998). 1.3.9 Role of IL-10 in the induction of clonal anergy Another signaling molecule thought to be involved in the induction of clonal anergy is IL-10 (Groux, H. et al. 1996), which has shown tolerizing effects in mice 36 (Rabinovitch, A. et al. 1995; Marcelletti, J.F. 1996). However, IL-10 has also been shown to exacerbate cardiac allograft rejection in mice (Qian, S. et al. 1996). Despite those conflicting results in rodents, Groux et al. were able to induce a long-term antigen-specific anergic state using human CD4+ T-cells in vitro. However, it is not known what signal transduction pathways might be responsible for these anergizing effects. (Groux, H. et al. 1996). In a recent publication, Joss et al. have shown that IL-10 was able to inhibit tyrosine phosphorylation of the CD28 receptor and consequently binding of phosphatidylinositol 3-kinase (PI 3-kinase) (Joss, A. et al. 2000). As described earlier, both events are critical for successful B7/CD28 costimulatory activation. Whether this is the only possible mechanism is not known. It is known that IL-10 activates the JAK-STAT pathway and signals through activation of STAT1 and STAT3 (Finbloom, D.S. and Winestock, K.D. 1995; Wehinger, J . et al. 1996; Liu, K.D. et al. 1998; O. Farrell, A. et al. 1998). However, more recent publications have shown that IL-10 also inhibits expression of both interferon-a and interferon-y induced genes by suppressing tyrosine phosphorylation of Stat-1 (Ito, S. et al. 1999; Shen, X. et al. 2000). For example, Ito et al. could show that injection of IL-10 into mice attenuated interferon-a induced Stat-1 tyrosine phosphorylation in the liver. IL-10 injection also induced expression of Suppressor of Cytokine Signal 2 (SOCS-2) and SOCS-3 . This suggests that Stat-1 inhibition might be due to the induction of SOCS-2 and3 (Shen, X. et al. 2000). This might lead to the speculation of a second mechanism through which IL-10 may cause reduced T-cell responsiveness: IL-10 could lead 37 to reduced Interferon-a and -y induced Stat-1 activation, which could lead to reduced Stat-1 binding to the c-Fos promoter region, which in turn could lead to reduced expression of c-Fos. C-Fos is a crucial component of the AP-1 transcription factor critical for IL-2 synthesis and consequent cell proliferation. This connection between IL-10, Stat-pathway and T-cell activation, if confirmed, could lead to a new perspective on how to induce clonal anergy and could lead to new therapeutic approaches. 38 CHAPTER TWO: METHODS 2.1 Solutions 30% Acrylamide/bis acrylamide/bis (29:1) 30g bottle d H 2 0 73ml - protected the mix from light (covered with aluminum foil) - stored at 4°C -^1.5% Agarose Gel Agarose 1.2g 0.5x TBE 80ml - boiled, stirring constantly - cooled to 50°C and added 2.Out ethidium bromide - poured into a casting form, inserted slot former (1mm) and cooled for 30 minutes - poured in 0.5X TBE to cover gel by 2mm, applied samples and electrophoresed Ammonium Acetate, Saturated 19.2M NH 4 acetate* 740.0g - added dH20 to 500 ml 39 -stored at room temperature - saturated solution - added additional d H 2 0 with a dropper until it went into solution without heating 10% Ammonium Persulfate ammonium persulfate 100mg d H 2 0 1.0ml - made fresh weekly - stored at 4°C Cell culture medium RPMI 1640 medium (Irvine Scientific, Santa Anna, CA) 10% FCS 3% glutamine, 3% penicillin/streptomycin Deionized Formamide - laboratory standard formamide was stirred with mixed bead resin, filtered, aliquoted in small quantities, and kept at -20°C 1% Ethidium Bromide Ethidium Bromide 10.0mg -added d H 2 0 to 1.0ml 40 - stored at 4°C 0.1M HCI concentrated HCI 8.3ml - added 800 dH 2 0 , mixed and adjusted to 1.0L with d H 2 0 5x Loading Buffer Bromophenol Blue 0.10g 50mM Tris Base 0.60g 50mM Na 2 EDTA 1.68g SDS 0.50g Sucrose 40.Og - dissolved Tris and EDTA in 50ml d H 2 0 - adjusted pH to 7.6 - dissolved SDS, Bromophenol Blue, sucrose in Tris-EDTA - adjusted final volume to 100ml with d H 2 0 - stored at room temperature 10 x Loading Buffer 0.25% Bromophenol Blue 0.25g 25.0% Ficoll type 400 25.Og 0.1% SDS (10% SDS stock) 1.0ml - QS to 100ml with d H 2 0 - adjusted pH to 7.5 - stored at room temperature - Bromophenol Blue runs at approximately 400 base pairs 0.5M Na?EDTA Na 2 EDTA 186.1g d H 2 0 800.0ml - mixed until dissolved (sped up by mixing at 50°C for one hour and added 40ml 10N NaOH) - adjusted pH to 8.0 with NaOH - added d H 2 0 to adjust for total volume of 1.0 L and autoclaved or filter sterilized - aliquoted and stored frozen in 40ml quantities 0.1M NaOH NaOH 4.0g - added 1.0L d H 2 0 and dissolved - stored at room temperature Nuclear Lysis Buffer 10.0mM Tris base 0.61g 400.0 mM NaCl 11.7g 2.0mM Na 2 EDTA 0.37g or 2.0 ml of 0.5M Na 2 EDTA stock - added d H 2 0 to 500ml - stored at 4°C 12.5% Polvacrylamide Gel 42 30% Acrylamide/bis (29:1) 4.0ml 5x TBE 2.0ml d H 2 0 3.9ml - mixed together and degassed for at least five minutes - added : 10% ammonium persulfate 70 uJ TEMED 7 pi - mixed by swirling gently - loaded the gel immediately - let set for at least one hour - assembled the gel, added 1xTBE to the chambers, applied samples and electrophoresed Protease K Stock Buffer 2.0mM Na2EDTA 2.0ml (0.5M stock) 1.0% SDS (10% SDS stock) 50.0 ml dH20 448.0ml - Stored at 4°C or stored frozen in 50.0 ml aliquots RBC Lysis Buffer 0.144M NH 4CI 15.2g 0.001 M N a H C 0 3 0.17g 43 - added d H 2 0 to 2.0L - stored at 4°C 10% SDS Stock SDS 50.0/100.Og - added d H 2 0 to 500ml/1L - heated to 65°C to dissolve - stored at 4°C Substrate buffer for IL-2 ELISA 2,2'-Azino-bis-(3-ethybenzthiazoline-6-sulfonic acid) 150mg - added to 500 ml of stock anhydrous citric acid (0.1 M) - adjusted pH to 4.35 - aliquoted 11 ml per vial and stored at -20°C - added d H 2 0 to 400 ml - adjusted pH to 8.0 with 10N NaOH - added d H 2 0 to 500ml - autoclaved or filter sterilized - stored at room temperature 1xTE 4.7 mM Tris HCI 0.37g 1.0mM Na 2 EDTA 1.0ml (0.5M stock) 44 5x TBE Tris base 54.0/108.Og Boric acid 27.0/54.Og Stock Na 2EDTA(0.5M) 20.0/40.0 ml - added d H 2 0 to 1.0/2.0L - adjusted pH to 8.3 - stored at 4°C 10x TBE (Gel Buffer) 0.89M Tris Base 215.6g 0.89M Boric acid 110.0g 0.02 Na 2 EDTA 80.0ml (from 0.5M stock) -added d H 2 0 to 2.0L - stored at 4°C 1M Tris Tris Base 60.9g -added d H 2 0 to 500 ml - adjusted pH to 7.0 2.1.1 Oligonucleotides and EMSA Biotinylated dsDNA oligonucleotides were obtained from University Core DNA Services, University of Calgary, Canada. The following IL-2 derived oligonucleotides were used: AP-1 : 5 ' -TCGAGAAATTCCAGAGAGTCATCAGAAGA-3 ' NFAT: 5 ' -TCGAAAGAGGAAAATTTGTTTCATACAGAAGG-3 ' Non-biotinylated, homologous counterparts were obtained for each oligonucleotide. Both oligos were annealed by mixing them at a ratio of 1:1, heating them up to 70°C and letting them cool down to room temperature slowly. 2.1.2 Antibodies OKT3 anti-CD3 mAb (Ortho Biotech, North York, Ontario) (Chatenoud, L. et al. 1990; Chatenoud, L. et al. 1991; Kung, P. et al. 1979) anti-CD28 anti-CD28 polyclonal antibody (Serotec, Kidlington, UK) anti-CD40L anti-CD40L(gp39) mAb (Calbiochem, San Diego, CA) (Aagaard-Tillery, K.M. and Jelinek, D.F. 1996; Pietravalle, F. et al. 1996) 46 2.1.3 lnterleukin-10 (IL-10) Recombinant human IL-10 (hlL-10, Pharmingen, San Diego, CA) (Howard, M. et al. 1992) 2.2 Methods 2.2.1 Donor Cells i) Collection Blood samples were taken from healthy human donors and stored in EDTA tubes. In some cases, blood bags from healthy donors were obtained from the Red Cross. These blood bags had been discarded for clinical applications because their volume did not reach a minimum standard, but were otherwise free of deficiencies. ii) Separation by Ficoll-Hypaque To separate PBMC from red blood cells, a peripheral blood sample was diluted with PBS (1 part blood to 1 part PBS). A 50 ml tube was filled with 13-15 ml of Ficoll-Hypaque and the diluted blood carefully layered onto the Ficoll. The tube was then centrifuged at 1500 rpm for 30 min and the mononuclear layer removed into another 50 ml tube. The tube was topped with PBS, centrifuged at 1500 rpm for 10 min and decanted. PBMC were transferred into a 14 ml tube and washed 47 twice in PBS (centrifugation at 1000 rpm for 10 min). Finally, PBMC were resuspended in RPMI 1640 medium and used for further experiments or CD4 + T cell isolation using Lympho-Kwik. iii) Isolation of CD4+ T-lymphocytes by Lympho-Kwik CD4 + T-lymphocytes were isolated by negative selection using Lympho-Kwik (One Lambda). In detail, for each 0.1 ml of buffy coat 1 ml of PBS was added and incubated with 0.8 ml of Lympho-Kwik reagent for 20 min at 37°C. Afterwards, 0.2 ml of PBS was layered over the mixture and centrifuged for 2 min at 2000g. The interface and supernatant were removed, and the pellet was resuspended in PBS. Cells were then washed three times in PBS (centrifugation for 1 min at 1000g). The efficacy of purification was estimated by FACScan and the purity was > 95%. iv) Cell culture Cells were maintained in RPMI 1640-medium at 37°C in 5% C 0 2 . In some experiments, cells were co-incubated with IL-10 (PharMingen) at a concentration of 100 U/ml. RPMI 1640-medium was exchanged every second day. 2.2.2 Jurkat cell line Jurkat cells were obtained from Dr. Schrader (Biomedical Research Centre, Vancouver, BC) and maintained in RPMI 1640-medium (10% FCS, 3% glutamine, 3% penicillin/streptomycin added) at 37°C in 5% C 0 2 . The cells were split and the 48 medium was exchanged every second day. Before use, cells were washed in PBS three times at 1500 rpm. 2.2.3 CTLLtest To measure IL-2 secretion into the supernatant, IL-2 dependent CTLL cells were obtained from Dr. Schrader (Biomedical Research Centre, Vancouver, Canada). Cells were washed in PBS (3x) and resuspended in IL-2 free DMEM-medium (10% FCS). 1 x 10" CTLL in 50 ul DMEM were incubated with 50 ul supernatant from stimulated T-cells (under the same conditions as described above) for 24 hours. Then 15 ul/well of MTT (5 mg/ml) were added and the cells were incubated afurther 4 hours. 100 ul of 10% SDS/15% DMF were added per well and the assay was incubated again for a further 8 hours. Extinction was then measured at 550 nm. 2.2.4 Induction of clonal anergy 24-well plates (Nunc) were coated with an anti-CD3 mAb (OKT3) (30 ng/ml) for 3 hours at 37°C. To induce clonal anergy, cells were cultured at a concentration of 2.5 X 10 5 cells/ml at 37°C overnight. Cells were then collected, layered on a Ficoll-Hypaque gradient to remove dead cells, and washed three times in PBS. After a resting period of three days, cells were restimulated at a concentration of 2.5 X 10 5 cells/ml in 24-well plates which had been coated with the anti-CD3 mAb 49 (ranging from 200 ng/ml to 2 ug/ml) and anti-CD28 (2.5 ug/ml) for three hours at 37°C. 2.2.5 Preparation of nuclear extracts After the stimulation period, cells were collected, washed in ice-cold PBS and lysed in cytosolic buffer (10 mM HEPES buffer (pH 7,9), 40 mM KCI, 3 mM MgCI 2, 1 mM DTT, 5% glycerol, 0.2% Nonidet P-40, 1 ug/ml aprotinin, 1 ug/ml leupeptin, and 1 mM PMSF) for 5 to 10 min on ice. Nuclei were removed by centrifugation at 14,000 rpm for 30 s in an Eppendorf microcentrifuge. The nuclei were then washed once with ice-cold cytosolic buffer, pelleted and lysed in hypertonic buffer (20 mM HEPES (pH 7,9), 420 mM KCI, 1.5 mM MgCI 2, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, 1 ug/ml aprotinin, 1 ug/ml leupeptin, and 0.5 mM PMSF) for 30 min on ice. The lysates were then centrifuged for 15 min at 14,000 rpm at 4°C to remove the insoluble fraction. Cytosolic and nuclear extracts were then frozen and stored at -70°C. The protein concentration of each extract was determined by the Bradford assay (Bio-Rad, Hercules, CA). 2.2.6 Electrophoretic mobility shift assay i) Co-incubation of oligonucleotides with DNA binding proteins To perform EMSA, 4 ug of nuclear extracts (4 X 10 6 cell equivalents) were incubated at room temperature for 15 min with 1 ug biotinylated oligonucleotide in 50 the presence of 1 pg poly(dl/dC) and 5 uJ 4-fold binding buffer (20 mM HEPES, pH 7.8, 0.1 mM EDTA, 1 mM DTT, 100 uM PMSF and 10% glycerol) in a total volume of 20 pi. ii) Agarose gel electrophoresis A 1.5%) agarose gel was prepared as described above. 20 pi of the binding mix was mixed with 5 uJ of 5x loading buffer (see above) and applied to the gel. Binding mixtures were then separated on the gel by electrophoresis at 100V in 0.5 TBE buffer. DNA content was checked by illuminating the gel with UV light. Polaroid pictures of the DNA bands which had been made visible by the fluorescent dye ethidium bromide were taken. iii) Blotting After blotting on a Tropilon-Plus™ Nylon Membrane (Tropix), bands were detected using a non-radioactive chemiluminescence method (Southern-Light™, Tropix) according to the protocol provided by the company (Walker, R.G. et al. 1993; Weston, S.A. et al. 1995). In short, the blots were washed 2x in a blocking buffer (0.2%> l-block reagent and 0.5% SDS in PBS) prior to incubation in the blocking buffer for 10 min. They were then incubated in an Avidix-AP conjugate solution (provided in Southern-Light kit) for 20 min at RT prior to subsequent washing in blocking buffer, wash buffer (0.5% SDS in PBS), and assay buffer (provided in Southern-Light kit). After placing on Saran wrap, the blots were incubated in CDP-Sr/arfor 5 min before being exposed to standard x-ray film. 51 2.2.7 Supershift EMSA Supershift experiments were performed by adding 1 ug of antibodies specific for c-Fos, FosB, JunD and JunB (Santa Cruz) to the binding reaction. The rest of the procedure was then performed in a method analogous to normal EMSAs (see above). 2.2.8 Electrophoresis and immunoblotting i) Determination of Protein Concentration Protein concentrations were determined using the method of Bradford (1976). First, a 1.42 mg/ml BSA standard was diluted with dH20 to a total volume of 100 ul/tube in order to create a series of standard tubes ranging from 0 to 30 ug BSA/tube. Second, 5 uJ of each unknown sample was diluted with dH20 to a total volume of 100 ul/tube. To each tube was added 2.5 ml of Bio-Rad Protein Assay Dye Reagent. After gently vortexing to mix and allowing to stand for approximately 5 min, the samples absorbances were read at a wavelength of 595 nM using a spectrophotometer. The concentrations of the samples were then determined using a linear regression plot. ii) SDS-polyacrylamide gel electrophoresis Samples containing proteins were separated using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, U.K. 1970). Proteins were diluted with 30 pi of 5x concentrated SDS-sample buffer (125 mM Tris-Hcl at 52 pH 6.8, 4%SDS, 20% glycerol, 10% p-mercaptoethanol, 0.01% bromophenol blue), boiled for 5 min, and then loaded onto an S D S - P A G E gel. Proteins were subjected to electrophoresis on 1.5 mm thick polyacrylamide gels with 4% stacking gels and 11% separating gels. The gels were electrophoresed for 15 h at 10 mA in running buffer (25mM Tris, 192 mM glycine, 3.5 mM SDS). iii) Western blot analysis After electrophoresis, the separating gel was equilibrated in transfer buffer (20 mM Tris, 12mM glycine, 20% methanol, pH 8.6) for 5-15 min to remove SDS. A nitrocellulose membrane was hydrated in transfer buffer for about 5 min and assembled with the gel into a sandwich between pieces of 3 MM filter paper. The proteins on the gel were electrophoretically transferred to the nitrocellulose membrane for 3 h at 300 mA in a Hoeffer transfer cell system. Transferred proteins were stained using Ponceau S dye by incubating the membrane in the stain for 1 to 5 min in order to visualize the proteins on the nitrocellulose. The excess stain on the nitrocellulose membrane was destained with water. The nitrocellulose membranes were blocked in 5% skim milk in TBS (50 mM Tris-HCl, 150 mM NaCI, pH 7.5) for 1 h. Membranes were rinsed briefly with TTBS (0.05% Tween-20 in TBS) to remove excess blocking solution. The membrane was incubated with the optimized concentration of primary antibody diluted in TTBS containing 0.05% sodium azide for several hours or overnight with agitation at room temperature. After incubation with the primary antibody, the membrane was rinsed in TTBS before incubation with the appropriate secondary antibody (horseradish peroxidase goat anti-mouse or anti-rabbit antibodies, BioRad) for 45 min. The membranes were then washed with TTBS again to 53 remove the excess secondary antibody and a final rinse was performed with TBS to rinse away the detergent Tween-20. The Western blots were incubated with enhanced chemiluminescence (ECL) detecting reagents for 1 min and exposed to film to visualize the immunoreactive protein. If a phosphotyrosine antibody was used, the membranes were blocked overnight at room temperature using low-salt TBS (20 mM Tris, pH 7.5, and 50 mM NaCI) containing 3% BSA. Primary antibody was incubated for 4 h, and alkaline phosphatase conjugated secondary antibody incubated for 3 h. All washes were performed with low-salt TBS containing 0.05% Nonidet P-40 (NP-40). Blots were developed as described above. 2.2.9 Proliferation Assays For proliferation, 5 X 10 5 anergized cells or normal controls were incubated at 37°C in 5% C 0 2 with crosslinked anti-CD3 (200 ng/ml) and anti-CD28 (2.5 ug/ml) in 200 ul RPMI 1640-medium in a 96-well plate (Nunc) for 3 days. On the 4 t h day, cells were pulsed with 1uCi/well [3H]thymidine (DuPont) overnight. Proliferation of cells was expressed as the mean cpm of quadruplicate wells. 54 2.2.10 IL-2 ELISA Sandwich ELISAs were used to detect murine IL-2 in 0.02 ml supernatants removed from cultures after an overnight stimulation period with cross-linked anti-CD3 (200 ng/ml) and anti-CD28 (2.5 ug/ml) in a 96-well plate. At the same time, ELISA plates (Nunc Maxisorb) had been coated with an anti-cytokine capture antibody (PharMingen, San Diego, CA) which was diluted to a concentration of 1-4 p.g/ml in binding buffer (0.1 M N a 2 H P 0 4 , pH 9.0) overnight at 4°C. Plates were then washed at least 4 times with PBS/Tween (0.5 ml Tween-20 to 1 L PBS) and blocked non-specifically by adding 200 ul blocking buffer (10% Fetal Calf Serum in PBS) per well. After incubation at room temperature for 30 min, plates were washed at least 3 times in PBS/Tween. Standards (recombinant human IL-2, PharMingen, San Diego, CA) and samples were diluted in blocking buffer/Tween (0.5 ml Tween-20 and 100 ml Fetal Calf Serum in 900 ml PBS) and 100 ul was added to each well. Plates were then incubated at 4°C overnight. The next day, plates were washed in PBS/Tween at least 4 times. 100 ul/well of biotinylated anti-cytokine detection antibody (PharMingen, San Diego, CA) was added at a concentration of 0.25-2 ug/ml in blocking buffer/Tween. Plates were incubated at room temperature for 1 hour and washed at least 6 times in PBS/Tween. Avidin-Peroxidase (Vector, Burlingame, CA) was then diluted 1:2000 and added at 100 ul/well. Plates were incubated at room temperature for 30 min and washed at least 8 times with PBS/Tween. Substrate buffer (see above) was thawed within 20 min of use, mixed with 10 pi of 30% H 2 0 2 per 11 ml of substrate buffer and immediately dispensed onto the plates at 100 ul/well. The color reaction was 55 stopped by adding 50 u.1 of 1% SDS solution. Plates were then read at 405 nm. IL-2 production was expressed as the mean of triplicate wells (Sander, B. et al. 1993). 2.2.11 Statistical Analysis Data were graphically displayed and statistically analyzed using the statistical analysis program Graphpad Prism, Version 2.01 (Graphpad Software Inc, San Diego, CA). 56 CHAPTER THREE: RESULTS 3.1 Introduction To investigate the induction of clonal anergy, experiments were performed in two phases: In a first step, clonal anergy was induced by stimulation of the T-cell receptor (signal 1) under the abrogation of the B7/CD28 costimulatory signal (signal 2). This was achieved in the absence of antigen-presenting cells (APCs). Cells were then rested for a period between 3-8 days to exclude the initial activation steps from signal transduction events in the state of clonal anergy. In a second phase, cells were then restimulated giving both signals (TCR/MHC and B7/CD28) using specific, activating antibodies at high concentration. This was done to achieve a complete T-cell activation within the model enabling the measurement of differences in signal transduction events between normal cells and anergized cells. 3.2 Reduced proliferation and abrogated IL-2 production in anergized human CD4+ T-lymphocytes To confirm that anergy can be successfully established in the model of anti-CD3-pretreated human CD4 +-T-cells, cell proliferation and IL-2 production into the supernatant were measured and compared to controls (Figure 2). For each assay, cell viability was checked under the microscope and was generally above 90%. 57 Cells were counted using a cell counter and numbers equalized for anergized cells and control cells. When re-stimulated with anti-CD3 and anti-CD28, CD4 + T-cells anergized with anti-CD3 displayed a marked reduction in proliferation (4,824 ± 461 cpm) compared to controls pretreated with medium alone (134,134 ± 36505 cpm, p=0.0036) or to controls pretreated with both anti-CD3 and anti-CD28 (80639 ± 10652 cpm). While medium-pretreated control cells showed a normal production of IL-2 into the supernatant (512 ± 49 pg/ml), anergized cells displayed a complete abrogation of IL-2 secretion (<4 pg/ml, p<0.0001). Cells prestimulated with both signals (anti-CD3/anti-CD28) achieved an IL-2 production similar to medium-pretreated controls (471 ± 22 pg/ml). Medium restimulated controls showed a low background for cell proliferation and an IL-2 production below detection level (<4 pg/ml) in the IL-2 ELISA. 58 A n e r g y Induct ion in C D 4 + T -Ce l l s (1. S tep : ant i -CD3 30 ng /m l ; 2. S t e p : an t i -CD3 2 ng/ml) 175000-1 1 5 0 0 0 0 125000 E 100000 a. B 75000 Prestimulation: • Medium • anti-CD3 • anti-CD3/anti-CD28 I Medium anti-CD3/anti-CD28 Restimulation 600 500 100 0 Medium anti-CD3/anti-CD28 Restimulation Figure 2 : Induction of clonal anergy in human CD4 + T-lymphocytes. Error bars represent three independent experiments. Cells were anergized using a low dose treatment with anit-CD3 (30 ng/ml) overnight. Medium and anti-CD3/anti-CD28 (30 ng/ml and 2.5 pg/ml, respectively) treated cells served as controls. After pretreatment, cells were washed and rested for 3-6 days. They were then restimulated with either high dose anti-CD3/anti-CD28 (2 ug/ml and 2.5 ng/ml, respectively) or medium (control). Cell proliferation measured by [3H]-thymidine uptake was markedly reduced in anergized cells (prestimulation: anti-CD3) compared to controls pre-stimulated with medium. IL-2 production was measured using a standard ELISA system and was almost abrogated in anergized cells. 59 3.3 Anergy is characterized by reduced binding of AP-1 to its IL-2 promoter region but NFAT-binding remains unaffected To investigate the events at the AP-1 region of the IL-2 promoter, nuclear extracts of CD4 +-T-lymphocytes were incubated with an AP-1 specific oligonucleotide in a binding reaction and gel shift experiments were performed. As shown in Figure 3a, the binding of AP-1 was markedly increased in cells stimulated with anti-CD3/anti-CD28 compared to medium-stimulated controls, and was more pronounced in nuclear than in cytosolic extracts, suggesting that activated AP-1 is mostly located in the nucleus. In some experiments, there was a minimal amount of background activity detectable in unstimulated medium-controls. This might be attributed to non-specific stimulatory effects related to the isolation process (Ficoll gradient and Lymphoquick isolation techniques). AP-1 binding peaked after 3 hours, but was still detectable 5 hours and 24 hours later (Figure 3b). In contrast, AP-1 binding was markedly reduced in anti-CD3/anti-CD28 restimulated CD4 +-T-lymphocytes rendered anergic by prior treatment with anti-CD3 alone (Figure 4). Even if this particular experiment presented in Figure 4 shows a 5 hours restimulation time, the same results were observed in experiments with longer restimulation periods of up to 24 hours. Throughout the experiments, medium restimulated control cells often displayed low-level background activation, possibly reflecting some residual activity derived from the 60 original prestimulation event using anti-CD3. As a control, experiments were repeated in the Jurkat cell line to exclude the possibility that costimulation caused by a small fraction of APCs surviving the isolation process might have an impact on the results in native cells. As shown in Figure 5, anergized Jurkat cells anergized by anti-CD3 alone displayed a reduced AP-1 activation after restimulation with anti-CD3/antiCD28. Similar experiments conducted with oligonucleotides specific for the NFAT site did not display any significant differences between anergized cells (pre-incubated with anti-CD3) and normal controls (pre-incubated with medium), suggesting that the inhibition of AP1 does not reflect ubiquitous inhibition of transcription factor binding to the IL-2 gene promoter (Figure 6). 61 Nuclear E . Cytosolic E . I 1 I 1 Figure 3a: AP-1 binding to the IL-2 promoter in nuclear and cytosolic extracts after stimulation with medium (M) and anti-CD3/anti-CD28 (S). This experiment is representative of four independent experiments. Extracts were incubated with AP-1 specific biotinylated oligonucleotides in a binding reaction and gelshift experiments were performed. AP-1 binding is markedly increased after stimulation. 62 Time Oh 3h 5h 24h l 11 11 11 1 M S M S M S M S M= medium S = anti-CD3 +anti-CD28 AP-1 Figure 3b: Time course of AP-1 binding. CD4 + T-lymphocytes were stimulated with medium (M) and anti-CD3/anti-CD28 (S) for Oh, 3h, 5h and 24h. This figure represents one out of four independent experiments. Nuclear extracts were incubated with AP-1 specific biotinylated oligonucleotides. Maximum binding occurs after 3 hours and is sustained over 24 hours under conditions of permanent cell activation. 63 a = medium anti-CD3 + anti-CD28 b = anti-CD3 anti-CD3 + anti-CD28 c = medium medium d = anti-CD3 medium Figure 4: AP-1 binding in anergized human CD4 + T-lymphocytes. One out of six independent experiments is shown. Cells were anergized by incubation with a crosslinked anti-CD3 antibody overnight and then rested. After restimulation with high dose anti-CD3/anti-CD28 for 5 hours nuclear extracts were prepared and binding reactions performed using an AP-1 specific biotinylated oligonucleotide probe. Anergized cells (b) showed significantly reduced AP-1 activation in comparison to medium pretreated controls (a). Lanes c and d represent medium-re-stimulated controls. 64 a = anti-CD3 medium b = medium medium c = medium * anti-CD3 + anti-CD28 d = anti-CD3 * anti-CD3 + anti-CD28 a b e d Figure 5: AP-1 binding in anergized Jurkat cells. Cells of the Jurkat cell line were anergized as outlined in Figure 4. The experiment shown represents one out of three independent experiments. Nuclear extracts were prepared and binding reactions performed using an AP-1 specific biotinylated oligonucleotide probe. As in native CD4 +-T-lymphocytes, anergized cells (d) showed reduced AP-1 activation in comparison to medium pretreated controls (c). Lanes a and b represent medium-re-stimulated controls. 65 a = medium b = anti-CD3 anti-CD3 + anti-CD28 anti-CD3 + anti-CD28 a NFAT Figure 6: Analogous experiment using a NFAT specific biotinylated oligonucleotide probe. One representative experiment out of three independent experiments is shown. There was no difference between anergized cells (b) and medium pretreated controls (a). 66 3.4 Supershift experiments show reduced binding of c-Fos, JunB and JunD to the AP-1 site of the IL-2 promoter To investigate whether individual components of the Fos- and Jun-family members of the AP-1 complex are inhibited in anergy, supershift experiments were performed using antibodies specific for c-Fos, FosB, JunB and JunD (Figure 7). Binding of these antibodies to their respective targets in the AP-1 complex was expressed as a second, heavier band. The binding of c-Fos, JunB and JunD was reduced in anergized cells pre-treated with anti-CD3, while binding of FosB was unaltered. This suggests that c-Fos, JunB and JunD form a critical part of the AP-1 complex by binding to the IL-2 promoter of CD4 + T-lymphocytes whose binding is reduced during the induction of anergy. A Western blot analysis using antibodies against c-Fos, JunB and JunD was performed to determine whether this defect reflected reduced expression or impaired binding of these components to the IL-2 gene promoter. As shown in Figure 8, the expression of c-Fos, JunB and JunD was markedly reduced in anergic cells compared to normal controls. Results for FosB were inconclusive due to specificity problems with the antibody (data not shown). 67 Restimulation with: medium anti-CD3 + anti-CD28 I 11 1 0 0 +c-Fos +FosB +JunB +JunD N A N A N A N A N A N A Supershifted t • 11 * i Zc::r Figure 7 : Supershift experiment using antibodies against c-Fos, FosB, JunB and JunD. One out of three independent experiments is shown. Cells were anergized and restimulated as described above. Nuclear extracts were incubated with an AP-1 specific oligonucleotide probe in the presence of different antibodies. Antibody-labeled AP-1/oligo complexes display an increased molecular mass and form a second band on the gel. Anergized cells (A) were compared with normal controls (N). c-Fos, JunB and JunD demonstrated a second band which was markedly reduced in anergized cells. 68 A N c-Fos JunB JunD -64 kd -38 kd -38 kd Figure 8 : Western blotting of C D 4 + T-lymphocyte extracts using antibodies against c-Fos, JunB and JunD. One out of three independent experiments is shown. Equal protein concentrations were loaded into each well as determined by the Bradford assay. Expression of c-Fos, JunB and JunD was reduced in anergized cells (A) compared to normal cells (N). 69 3.5 Supershift experiments show reduced binding of Statl to the SIE-binding region of the c-Fos promoter Because binding of the important signaling molecule c-Fos was reduced in anergized cells, we investigated the c-Fos promoter itself. This promoter is divided into two major binding regions: SRE and SIE. Gel shift experiments at the SRE locus did not show any significant changes in binding between normal and anergized cells (data not shown). However, using a biotinylated oligonucleotide probe homologous for the SIE region, there was a dramatic decrease in binding at the SIE-region in anergized CD4 + T-lymphocytes when compared to normal controls (Figure 9). Because the SIE region has been reported to be the target of the IL-10 dependent Jak-Stat pathway, supershift assays were conducted using antibodies specific for Statl and Stat3. As shown in Figure 10, binding of the Statl-specific antibody to the SIE-oligonucleotide complex showed a retarded double band in normal controls, which was highly reduced in anergized CD4 + T-lymphocytes. No binding was shown for the Stat3-antibody. The amount of reduction in general SIE binding was variable throughout the experiments possibly reflecting the presence of a yet unknown molecule binding to the SIE promoter. 70 a b e d S I E binding Figure 9: Binding to the SIE-region of the c-Fos promoter. One out of four independent experiments is shown. Human CD4 + T-lymphocytes were anergized as shown in Figure 4. Nuclear extracts were incubated with a biotinylated oligonucleotide probe homologous for the SIE region of the c-Fos promoter. Anergized cells (d) showed a markedly reduced binding to the SIE region compared to normal cells (c). No activation occurred in unstimulated controls for both anergized cells (a) and normal cells (b). 7 1 Restimulation with: medium anti-CD3 + anti-CD28 Figure 10: Supershift experiments using antibodies against Statl and Stat3. One out of three independent experiments is shown. Cells were anergized and re-stimulated as described in Figure 4. Nuclear extracts were incubated with a SIE specific oligonucleotide probe in the presence of different antibodies. Normal cells (N) showed a binding of the Statl-antibody to the SIE-oligonucleotide complex which was highly reduced in anergized cells (A). No binding could be demonstrated for the Stat3-antibody. 72 3.6 CD40L blockade significantly reduces proliferation rates and IL-2 production in PBMC Co-blockade of the CD40-CD40L and the B7-CD28 pathway has been shown to induce long-term cardiac and skin allograft acceptance in mice. To highlight the importance of the CD40-CD40L pathway as a significant costimulatory pathway, we conducted experiments to investigate whether a blockade of the CD40/CD40L pathway in human CD4 + T-lymphocytes could mimic the same downregulation of proliferation, IL-2 production and AP-1 binding to the IL-2 promoter as observed in T-cells where the B7/CD28 costimulatory signal has been abrogated. In proliferation experiments, T cells in the PBMC population were pre-stimulated by anti-CD3 with or without anti-CD28 (Figure 11a). As expected, no anergy was observed in PBMC if the B7/CD28 costimulatory signal was not given, since the APCs could substitute that role (Figure 11a, Lane 3). In contrast, isolated CD4 + T-lymphocytes displayed a highly reduced proliferation rate typical for clonal anergy as described above (Figure 11b, Lane 3). However, co-incubation with a blocking anti-human CD40L mAb significantly reduced the proliferation rate in PBMC (Figure 11a, Lane 3 vs. 4, P=0.0003). Similar differences caused by the anti-CD40L mAb were observed if PBMC were prestimulated with anti-CD3/anti-CD28 (Figure 11a, Lane 5 vs. 6, P=0.0003). In contrast, isolated CD4 + T-lymphocytes did not show significant differences in proliferation through the addition of the anti-CD40L mAb (Figure 11b, Lane 3 vs. 4, P=n.s. and Figure 11b, Lane 5 vs. 6, P=n.s.). This indicates that signaling through the CD40/CD40L pathway can only be blocked in the presence of APCs. 73 Similar results were obtained if IL-2 production into the supernatant was measured by IL-2 ELISA. Co-incubation with anti-CD40L antibody led to a r significant reduction in IL-2 production whether cells were prestimulated by anti-CD3/anti-CD28 (Figure 12a, Lane 5 vs. 6, P=0.0362) or anti-CD3 alone (Figure 12 a, Lane 3 vs. 4, P=0.0005). In isolated CD4 + T-lymphocytes, abrogation of the B7/CD28 costimulatory signal led to a marked reduction in IL-2 production consistent with the state of clonal anergy (Figure 12b, Lane 3). No further reduction could be induced by the addition of the anti-CD40L antibody (Figure 12b, Lane 4). Similarly, anti-CD3/anti-CD28 prestimulated cells did not show a reduction in IL-2 production through anti-CD40L (Figure 12b, Lane 5 vs. 6, P=n.s.). As in the proliferation experiments, this data suggests that blockade of the CD40/CD40L pathway is evident only in the presence of APCs . An additional effect of CD8 + T-lymphocytes might be excluded since they are negatively selected during the isolation procedure. 74 2 0 0 0 0 0 - , 1 5 0 0 0 0 -E g- 1 0 0 0 0 0 -5 0 0 0 0 -(a) PBMC P=0.0003* P=0.0003* Prestimulation with: CZI Medium ^ Medium/anti-CD40L • • a n t i - C D 3 ESS an u-CD3/anti-CD40L mm anti-CD3/anti-CD28 d a anti-CD3/anti-CD28/anti-CD40Li IM1 Control anti-CD3 Medium VZZa Control Medium Medium 2 3 4 5 6 7 8 30000-(b) CD4 + T-Lymphocytes Figure 11: Cell proliferation measured by [3H]-thymidine uptake in PBMC (a) and CD4 + T-lymphocytes (b). Error bars represent three independent experiments. Cells were pre-incubated with anti-CD3, activating anti-CD28 and blocking anti-CD40L antibodies, respectively. After pretreatment, cells were washed and rested for one week. They were then restimulated with either high dose anti-CD3/anti-CD28 (columns 1-6) or medium as a control (control O/M, control M/M). Both 75 untreated PBMC and CD4 + T-cells displayed a strong response if restimulated with anti-CD3/anti-CD28 (column 1). Addition of anti-CD40L did not produce significant differences since the CD40 receptor is mostly unengaged in an environment without T cell activation (column 2). Prestimulation with low-dose anti-CD3 (200 ng/ml) also led to solid proliferation rates in PBMC (above 100 000 cpm), but led to anergy induction and consequent drastic reduction in proliferation rates in CD4+ T-cells lacking the costimulatory signal (column 3) In PBMC, addition of anti-CD40L could reduce the proliferation rate significantly (P=0.0003), but was not able to further reduce proliferation in anergized CD4 + T-cells (column 4). Both CD4+ T-cells and PBMC displayed solid proliferation rates if prestimulated with both anti-CD3 and anti-CD28 (columns 5). PBMC showed a significant reduction (P=0.0003) in proliferation rates in the presence of anti-CD40L (column 6), showing that it is the A P C signaling through the CD40/CD40L pathway. Medium restimulated controls displayed a very low proliferation rate (column 7: anti-CD3 prestimulation/medium restimulation; column 8: medium pretreatment/medium restimulation). 76 S O D -'S) 400-c .2 300-u 3 "g 200-CL «>« 100-(a) PBMC P=0.0005* P=0.0362* Prestimulation with: l l Medium ^3 Medium/anti-CD40L ^ anti-CD3 m$ anti-CD3/anti-CD40L (nnnno anti-CD3/anti-CD28 EZD anti-CD3/anti-CD28/anti-CD40L Control anti-CD3 Medium vrzn Control medium Medium 8 500-t (b) CD4+ T-Lymphocytes g 400-c .2 300-"g 200-Q_ w 100-JL JL o-8 Figure 12: IL-2 production into the supernatant measured by IL-2 ELISA in PBMC (a) and CD4 + T-lymphocytes (b). Error bars represent three independent experiments. IL-2 secretion followed a similar pattern as cell proliferation in Figure 11. IL-2 production in PBMC was significantly reduced by anti-CD40L co-7 7 incubation in anti-CD3 prestimulated cells (column 3 vs. 4: P=0.0005). There was no significant difference in anergized CD4 + T-cells. There was also a significant downregulation in PBMC prestimulated with anti-CD3 and anti-CD28 if anti-CD40L was added (column 5 vs. 6: P=0.0362). However, no significant differences were observed in CD4 + T-cells, thus suggesting that APCs are needed to transmit the CD40/CD40L signal. Column 7 and 8 represent medium restimulated controls analogous to Figure 11. 78 3.7 CD40L-blockade mimics the transcriptional events of clonal anergy PBMC were stimulated with anti-CD3/anti-CD28 and co-incubated with a blocking anti-CD40L antibody. After restimulation with anti-CD3/anti-CD28, PBMC showed a significantly reduced AP-1 binding compared to controls (anti-CD3/anti-CD28 prestimulation without anti-CD40L). This AP-1 pattern was similar to that of anergized cells shown above, suggesting that blockade of the CD40-CD40L pathways induces a pattern similar to blockade of the B7/CD28 pathway. Interestingly, this effect is B7-CD28 independent and occurs even if the CD28 pathway is fully stimulated at the same time. 79 Prestimulation I 1 1 1 1 ' 1 1 M O 0/28 0/28 M M 1 +40 • +40 1 M 1 1 0/28 1 O =an t i -CD3 28 = ant i -CD28 a e Figure 13: AP-1 binding in human PBMC after CD40L-blockade using an anti-CD40L antibody. Data shown represents one out of three independent experiments. Cells were stimulated with anti-CD3/anti-CD28 and co-incubated with a blocking anti-CD40L antibody. Cells were rested and restimulated with anti-CD3/anti-CD28. anti-CD3/anti-CD28 pre-activated cells (c) showed a TCR/CD28 80 induced upregulation of AP-1 binding which was markedly reduced in cells co-incubated with anti-CD40L (d). Pre-incubation with anti-CD40L antibody alone (f) did not lead to a reduction in binding activity compared to controls (e). Lane a+b are medium-restimulated controls. 81 3.8 Influence of IL-10 on CD4+-T-cell proliferation and IL-2 production To examine the effects of IL-10, CD4 +-T-cells were co-incubated with IL-10 and stimulated with anti-CD3/anti-CD28. Viability was assessed at >90% under the microscope and cell numbers were adjusted using a cell counter. Cells displayed a partial (19%) reduction in their proliferation rate compared to anti-CD3/anti-CD28 stimulated controls which was not as marked as that achieved by anti-CD3 alone (Figure 14). Co-incubation of anti-CD3- anergized cells with IL-10 did not reduce their proliferation further suggesting that the abrogation of the B7/CD28 co-stimulatory signal is the principal factor responsible for the induction of anergy in this model. IL-10 co-incubation of anti-CD3/anti-CD28 pre-stimulated cells reduced IL-2 production into the supernatant by 40% (p=0.0024), suggesting that IL-10 has a stronger effect in inhibiting IL-2 production than proliferation (Figure 14). IL-2 secretion in anti-CD3-anergized cells was below the detection level whether cells were co-incubated with IL-10 or not. 82 90000 80000-70000-60000 E 50000 a 40000 30000 20000 10000 0 Co-incubation of CD4 + T-Lymphocytes with IL-10 Prestimulatinn: ' ]anti-CD3/anti-CD28 • anti-CD3/anti-CD28/IL-10 lianti-CD3/IL-10 i lant i -CD3 P=n.s Medium anti-CD3/anti-CD28 Restimulation 500n 400] ! 300 ' 200 100 P=0.0024* Medium anti-CD3/anti-CD28 Restimulation Figure 14: Co-incubation of CD4 + T-lymphocytes with IL-10. Error bars represent three independent experiments. Cells were pretreated with low-dose anti-CD3/anti-CD28 and/or IL-10. After pretreatment, cells were washed and rested for one week. They were then restimulated with either high dose anti-CD3/anti-CD28 or medium (control). IL-10 partially decreased the proliferation (P=n.s.) and significantly reduced the IL-2 secretion (p=0.0024) of anti-CD3/anti-CD28 pretreated cells 83 3.9 Influence of the p38-MAPK inhibitor SB 203580 on T-cell proliferation To investigate the influence of the p38-MAPK inhibitor SB 203580 on proliferation of stimulated human T-lymphocytes, T-cells were incubated with different concentrations of SB 203580 (0, 1, 10 and 25pM). Low (non-toxic) concentrations of SB 203580 were defined between 1 and 10 pM as previously established (Foltz, I.N. et al. 1997; Lee, Y.B. et al. 2000). Increasing concentrations of SB 203580 caused a proportional decrease in T-cell proliferation (Figure 15). The decrease was evident at low concentrations (1 and 10 pM SB 203580, respectively) as well as at high concentrations (25 uM), meaning that decreased T-cell proliferation was not only due to a direct toxic effect of SB 203580. This might indicate that the signaling pathway leading to proliferation of human T-lymphocytes is dependent on p38 MAPK. 84 Figure 15: Influence of the p38-MAPK inhibitor SB 203580 on proliferation of stimulated human T-lymphocytes. Healthy donor T-lymphocytes were purified as described, stimulated with anti-CD3 [2 pg/ml], anti-CD28 [2.5 ug/ml], PMA [30 ng/ml] and ionomycin [1 uM], respectively. Control cells were incubated in media (Med.). SB 203580 was added at concentrations of 0, 1, 10 and 25 uM.. Cells were incubated for 24 h, pulsed with [3H]TdR for 12 h and proliferation was measured. 85 CHAPTER FOUR: DISCUSSION Although short-term and long-term outcomes after organ transplantation have improved considerably (Hariharan, S. et al. 2000), long-term morbidity and mortality still remain substantial problems. Only 50% of cadaveric renal allografts surviving the first year are still functioning after 7.5 to 9.5 years (Cecka, J.M. 1996; Calne, R.Y. 2000). The chronic immunosuppression that organ transplant recipients require for the rest of their lives is associated with severe side effects, including infections, malignancies, nephrotoxicity, and metabolic disorders (Calne, R. et al. 1998; Wekerle, T. and Sykes, M. 2001). The reliable induction of a permanent state of graft-specific immunological tolerance could provide a solution to these pressing problems in the field of allotransplantation. The induction of microchimerism as one method to achieve tolerance normally requires some form of myelosuppression or non-specific T-cell elimination despite the progress in non-myeloablative protocols (Wekerle, T., Sykes, M. 2001). In contrast, clonal anergy as a mechanism of peripheral tolerance can be induced without non-specific T-cell deletion or the use of radiation in both animal and human cells (Sloan-Lancaster, J . et al. 1993; Mueller, D.L. and Jenkins, M.K. 1995; Groux, H. et al. 1996). A number of studies are currently underway to investigate the different ways to induce clonal anergy and its effect on the way immune cells interact. However, only rudimentary research has been done so far to characterize the intracellular 86 changes in signal transduction pathways during the induction and maintenance of clonal anergy. The principal objective of the current studies has been to document the signal transduction events during the state of clonal anergy. Detailed knowledge about changes in signal transduction might be used in future therapies to produce a state similar to clonal anergy in transplant patients. As a first step, a human model of clonal anergy was established. This was achieved by isolating CD4 + T-lymphocytes and stimulating them through their T-cell receptor in the absence of the costimulatory B7/C28 signal. A body of literature has suggested that abrogation of IL-2 production and severe downregulation in T-cell proliferation are key characteristics of the state of clonal anergy (Mueller, D.L., Jenkins, M.K. 1995; Van Gool, S.W. et al. 1999). Mondino et al. have also found severely downregulated mRNA in anergized cells (mondino, A. et al. 1996). Other features of anergized cells have also been described including the lack of cytotoxic activity and downregulation of IL-5, IL-13 and IFN-y production (Van Gool, S.W. et al. 1999). However, since downregulation of IL-2 and T-cell proliferation have been described as being so prominent and consistent in anergized cells, these criteria were taken to demonstrate the validity of the model. Indeed, human CD4 + T-lymphocytes stimulated via the CD3 complex in the absence of the CD-28 co-stimulatory signal, when restimulated after a resting period of 3-6 days, display markedly reduced proliferation and abrogated IL-2 production compared to untreated controls. The viability and histological 87 appearance of anergized cells remained unchanged, making it unlikely that the observed effects could be attributed to cell death or apoptosis. In future experiments apoptosis-specific DNA fragmentation analysis may be performed to also measure ongoing low-level background apoptosis. However, it is unlikely that such a low-level aopoptosis may account for the massive changes in cell proliferation and IL-2 production observed in this model. These results demonstrate for the first time that a state of clonal anergy can be induced in a model of primary human CD4 + T-lymphocytes, as has been previously shown for the mouse (Kang, S.M. et al. 1992; Mondino, A. et al. 1996). Reduced binding of AP-1 to the IL-2 promoter region has been reported to play an important role in the maintenance of clonal anergy using a mouse model (Kang, S.M. et al. 1992; Jain, J . et al. 1995; Karin, M. 1995). As shown in the studies reported here, AP-1 was upregulated after TCR stimulation in both nuclear and cytosolic extracts. This upregulation was detectable after 3 hours, and persisted when cells remained exposed to the stimulus. Anergized cells demonstrated reduced AP-1 binding activity, suggesting that members of the Fos- and Jun family play a crucial role in the signaling events leading to clonal anergy (Jain, J . et al. 1995; Karin, M. 1995). Because these studies were conducted using normal human lymphocytes, it could be argued that APCs surviving the isolation procedure might provide costimulatory signals and thus influence the results. These experiments were therefore repeated in the Jurkat cell line, which is derived from human T-88 lymphoblastoid cells (Dreyfus, D.H. et al. 2000; Radvany, Z. et al. 2000). Downregulation of the AP-1 signal was also shown in the Jurkat cell line, which does not contain any APCs, demonstrating that possible contamination of isolated CD4 + T-lymphocytes with APCs has no measurable influence on the behavior of this model. Supershift experiments showed reduced binding of c-Fos, JunB and JunD to the AP-1 binding region of the IL-2 promoter. In conjunction with the reduced general AP-1 binding in the lower band of the same lane, this provides evidence that c-Fos, JunB and JunD are forming part of the AP-1 complex. Western blotting showed reduced expression of these molecules, which is partially consistent with previously reported observations in mouse derived T-cell clones (Mondino, A. et al. 1996). Mondino et al. could not confirm downregulation of JunD and described a slight downregulation of FosB in their mouse model. In the human model, tb hese findings suggest that not only are the predominantly CD28-dependent Jun family members defective in clonal anergy, but that a predominantly TCR and p21 r a s-dependent Fos family member is also downregulated. This implies that the upstream mechanisms involved in the induction of clonal anergy inhibit both the TCR-dependent and the CD28-dependent pathways. In addition, the CD28-receptor has been shown to both lead to ERK and JNK activation in the Jurkat cell line (Schneider, H. et al. 1995; Ward, S.G. et al. 1995). As of yet, it is impossible to determine whether a single "key signal" is responsible for the induction of clonal anergy or whether an orchestration of multiple events leads to clonal anergy. 89 The implication of c-Fos in reduced AP-1 binding led to examination of the SRE and SIE promoter regions of the c-Fos gene. No abnormalities were observed at the SRE region, but binding to the SIE region was markedly reduced in anergized cells. The SIE region is the binding target of the Jak-Stat pathway (Karin, M. 1995; Wehinger, J . et al. 1996). Supershift experiments using specific antibodies against Stat-1 and Stat-3 demonstrated that Stat-1 was involved in the binding complex, and that Stat-1 binding was substantially reduced in anergized cells. This suggests an involvement of the Jak-Stat pathway in the events leading to anergy. Since it has been shown that IL-10 inhibits expression of both interferon-a and interferon-y induced genes by suppressing tyrosine phosphorylation of Stat-1 (Ito, S. et al. 1999; Shen, X. et al. 2000), it may be speculated that the tolerizing effects induced by either abrogation of the costimulatory signal or IL-10 might partially involve downregulation of Stat-1. Thus, since both clonal anergy and IL-10 seem to use the same downstream transducer to render T-cells anergic it might be suggested that the increased presence of IL-10 and decreased costimulatory activity act synergistically. Furthermore, the observed abrogation in IL-2 production in clonal anergy might also result in incomplete activation of the Jak-Stat pathway, since both the IL-10 dependant Stat-1 and Stat-3-caskades are also activated by IL-2 (Liu, K.D. et al. 1998). 90 Increasing interest has been focussed on the possibility of further enhancing the co-stimulatory blockade and the resulting anergizing effects by blocking several costimulatory pathways simultaneously (Larsen, C P . et al. 1996; Van Gool, S.W. et al. 1999). Prolonged graft survival rates have been observed in several models including allografts of heart, skin, aorta, and pancreatic islets (Larsen, C P . and Pearson, T.C. 1997) using antibodies blocking both the B7/CD28 and the CD40/CD40L pathways. These results led us to investigate proliferation and IL-2 production in both PBMC as well as isolated CD4 + T-lymphocytes which were co-incubated with a blocking gp39 (CD40L).mAb. PBMC prestimulated with anti-CD3/anti-CD28 displayed a significant reduction in proliferation and IL-2 production when co-incubated with gp39 mAb. This confirms the anergizing effects of CD40/CD40L blockade and offers an explanation why co-blockade of B7/CD28 and CD40/CD40L might lead to a more pronounced anergy induction and graft survival. Control experiments using isolated CD4 + T-lymphocytes showed no differences between cells co-incubated with gp39 mAb and untreated controls. This shows that to exercise this downregulation, T-cells need the presence of the A P C to transmit the signal through the CD40/CD40L link. Furthermore, the fact that the B7/CD28 pathway was maximally stimulated in this system through the addition of anti-CD28 antibody shows that changes in proliferation and IL-2 production cannot be attributed to a reduction in B7 expression leading to lower CD28 receptor activation. Therefore, the reduction in 91 proliferation rate and IL-2 secretion may be attributed to the A P C directly stimulating the T-cell through the CD40 ligand. This indicates that costimulation is not only regulated through the activation of the A P C through the CD40 receptor, thus upregulating B7 expression and CD28 costimulation, but also that the A P C may costimulate the T-cell directly through the CD40/CD40L pathway. Such a scenario could suggest a bi-directional link between T-cells and APCs via CD40/CD40L. However, it could also suggest that activation of the A P C through CD40/CD40L interactions might cause the A P C to express an as yet unknown protein. This protein, in turn, could lead to the activation of T-cells. In addition, stimulation of CD4 + T-cells might occur not only through APCs , but also through CD8 + T-cells which are present in the PBMC mix. To investigate whether there are similarities between the type of anergy induced by the abrogation of the B7/CD28 costimulatory signal and anergizing effects observed during the blockade of the CD40/CD40L pathway, gel shift experiments were performed using the AP-1 region of the IL-2 promoter as a probe. PBMC prestimulated with anti-CD3/anti-CD28 showed significantly reduced AP-1 binding to the IL-2 promoter when co-incubated with gp39 mAb. It is also noteworthy that the blocking gp39 antibody exerts these effects in activated T-cells, but has no effect in resting T-lymphocytes. This suggests that the AP-1 binding deficiency is a central event during the induction and maintenance of clonal anergy. Addition of IL-10 throughout the period of initial stimulation produced a limited reduction in both proliferation and IL-2 secretion, suggesting that anergy might be 92 enhanced, but cannot be produced by co-incubation with IL-10. These observations are consistent with results reported in the mouse model of T-cells (Krenger, W. et al. 1994; Weber-Nordt, R.M. et al. 1996; Grunig, G. et al. 1997; Zeller, J .C. et al. 1999). It should be noted that IL-10 appears to exert a more profound inhibitory influence on murine cells than on human cells. In human CD4 + T-cells, abrogation of the B7/CD28 co-stimulatory signal is predominantly responsible for the impaired response. However, the anergy enhancing effect of IL-10 might still play a role in human T-cells under physiological conditions: In recent studies, Van Gool et al. have found that human peripheral blood lymphocytes anergized by co-blockade with anti-CD80, anti-CD86 and anti-CD40 antibodies display a lack of proliferation, cytotoxic activity, and IL-2, IL-5 and IL-13 production, while at the same time showing an enhanced production of IL-10 (Van Gool, S.W. et al. 1999). It may be speculated that this production of high levels of IL-10 in anergized cells could contribute to modulation of antigen-presenting cell activity and to bystander suppression of residually reactive T-cells. The mitogen-activated protein kinase (MAPK) p38 has been implied to have a role in the signal transduction pathway following TCR/CD28 stimulation. Using murine TH1 clones, DeSilva et al. have shown activation of the p38 MAPK pathway upon anti-CD3 plus anti-CD28 crosslinking or PMA plus ionomycin stimulation (DeSilva, D.R. et al. 1997). Furthermore, they have also shown that anergic Th1 cells display decreased p38 activity as well as decreased ERK and JNK activities even though levels of these proteins remained unchanged. To investigate the possible influence of p38 on our human model of isolated CD4 + T-lymphocytes, we used the p38-MAPK inhibitor SB 203580. Interestingly, the inhibitor produced a clear 93 downregulation of proliferation throughout the dosage range employed (Foltz, I.N. et al. 1997). This demonstrates that the observed reduced proliferation rates are not attributable to toxic effects and that T-cell proliferation might be dependent on functional p38 MAPK. Future experiments will have to examine the influence of p38 in greater detail. In conclusion, co-blockade of both the B7/CD28 and the CD40/CD40L costimulatory pathways could be used to induce a long-term state of clonal anergy in transplant patients towards their allograft. The same approach could also be used to induce clonal anergy in bone marrow grafts before transplantation into the host, thereby reducing the risk of severe graft versus host disease. Thus, clinical bone marrow transplantation protocols using blockade of the B7/CD28 pathway alone (de Carvalho Bittencourt, M. et al. 1999; Guinan, E.C. et al. 1999) could be enhanced by the addition of CD40/CD40L blockade. Furthermore, the downstream effects induced during the event of clonal anergy might lead to new therapeutic options in the future. Blockade of AP-1 (or Fos- and Jun members) as well as Stat-1 might prove to be a valuable tool in post-transplant immunosuppression. In future therapeutic approaches, intracellular signal transduction molecules could be targeted using, for example, antisense RNA, retroviral vectors and specific inhibitors. Thus, blocking specific intracellular signal transduction molecules to induce clonal anergy might lead to new therapeutic strategies causing less side-effects than traditional immunosuppression and improve long-term graft survival rates. 94 CHAPTER FIVE: SUMMARY This study was designed to examine the transcriptional changes that occur during the development of clonal anergy. Unlike upstream events at the receptor and cytoplasmic signal transduction level, nuclear transcriptional events have not yet been thoroughly described using a human model. Therefore, a human model of clonal anergy was established in order to investigate transcription factor changes at the nuclear level. Native isolated C D 4 + T-lymphocytes were employed rather than transformed cell lines to ensure that the events observed were due to clonal anergy and not unspecific effects of a transformed cell line. The data presented here are consistent with the hypothesis that clonal anergy is associated with a pronounced reduction of binding of the AP-1 transcription factor to the AP-1 binding region of the IL-2 gene promoter. Further investigation of Fos and Jun members of the AP-1 family showed that this reduction in AP-1 binding is associated with a downregulation of its family members c-Fos, Jun-B and Jun-D. In addition, the expression of c-Fos, one of the major components of the AP-1 complex, is reduced, and there is reduced binding of transcription factors at its SIE region, the target of the Jak-Stat pathway. These findings seem to suggest a contribution of the IL-10-dependent Jak-Stat pathway to the induction and maintenance of clonal anergy, although the exact molecule(s) which form the key initiation steps leading to clonal anergy remains to be determined. 95 This study also investigated the importance of the CD40/CD40L pathway. It determined that blockade of the CD40L signal could induce downregulation in proliferation and IL-2 production and a reduction in AP-1 binding to the IL-2 promoter similar to that observed in the absence of B7/CD28 costimulation. These findings strengthen the hypothesis that both the B7/CD28 pathway and the CD40/CD40L pathway are working in synergy with each other, which might explain why co-blockade of both the B7/CD28 and the CD40/CD40L pathways leads to a more pronounced anergy induction and prolonged graft survival rates. The design of drugs to block the signal transduction molecules described in this study (e.g. AP-1 , c-Fos and Stat-1) during the critical early post-transplantation phase might be able to mimic a state of clonal anergy towards the allograft and lead to higher long-term graft survival rates and graft acceptance without the pronounced site effects of traditional immunosuppressive therapies. 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