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Selective inhibition of signal transduction pathways in human CD4⁺ tlymphocytes Li, Xiaomei 2004

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SELECTIVE INHIBITION OF SIGNAL TRANSDUCTION PATHWAYS IN HUMAN CD4+ T LYMPHOCYTES by Xiaomei Li Bachelor of Medicine, Peking University, China, 1995 Master of Medicine, Peking University, China, 1995 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 Programme of Experimental Medicine We accept thjsJhesis as confirming to thefequlred standard THE UNIVERSITY OF BRITISH COLUMBIA August, 2004 © Xiaomei Li, August, 2004 ABSTRACT We utilized an in vitro T cell activation model to investigate the effect of blocking key signaling pathways downstream of TCR and CD28 molecules by using specific kinase inhibitors. Human peripheral CD4+ T cells were stimulated with anti-CD3 mAb (OKT3) and anti-CD28 mAb, inducing T cell proliferation with maximum IL-2 secretion at day 1 and maximum IL-2 receptor a chain (CD25) expression at day 3 of cell activation. MAP or ERK kinase (MEK) inhibitor U0126, p38 MAP kinase inhibitor SB203580, protein kinase C (PKC) inhibitor Bisindolylmaleimide I and phosphatidylinositol 3 kinase (PI3-kinase) inhibitor LY294002 were added to cell cultures. These inhibitors did not induce cell apoptosis and they markedly reduced T cell proliferation as well as IL-2 production and CD25 expression. This inhibition was time and dose dependent and more profound than the effects of cyclosporine. The suppression of CD25 was largely independent of IL-2. All four inhibitors markedly reduced the binding activity of N F - A T and AP-1 for IL-2 gene transcription. Yet their influence on N F - K B and STAT5 binding activity for CD25 gene transcription was variable. LY294002 markedly reduced N F - K B binding activity, while U0126, SB203580 and LY294002 partially reduced STAT5 binding activity. The combination of the CD28 pathway inhibitor LY294002 with one of the TCR pathway inhibitors further reduced T cell proliferation. The dose relationship study showed that LY294002 worked as an antagonist to the TCR pathway inhibitor at the low end of the scale of inhibition and became more synergistic as T cell inhibition increased. Conclusions: Human CD4+ T cells activated through the TCR and CD28 molecules ii induced cell proliferation with an increase of IL-2 secretion and IL-2 receptor a chain (CD25) expression. Specific inhibitors of the PKC, MEK/ERK, p38 MAP kinase and PI3- kinase pathways caused marked inhibition of T cell proliferation as well as IL-2 and CD25 expression, and indicated that these pathways were important for T cell activation but not essential for IL-2 signaling under TCR/CD28 stimulation. This inhibition occurred at the transcription factor level. These data demonstrated the potential value of these important pathways as future targets for immunosuppressant. iii TABLE OF CONTENTS Abstract Table of Contents List of Tables List of Figures Abbreviations Acknowledgements Dedication CHAPTER ONE: INTRODUCTION 1.1 General Background 1.2 Signal Transduction in T lymphocyte 1.2.1 T cell receptor (TCR) signaling i) T cell receptor, antigen binding and activation of protein tyrosine kinases ii) Adaptor proteins in TCR signaling 1.2.2 CD28/ CTLA-4 costimulatory signaling pathways i) CD28 and CTLA-4 molecules ii) Functions of CD28/ CTLA-4 signaling on T lymphocyte activation 1.2.3 IL-2 and IL-2 receptor a chain gene expression i) Transcriptional control of IL-2 gene 21 ii) Transcriptional control of IL-2 R a chain gene expression 24 iii) IL-2 signaling cascades 25 1.2.4 Activation of signaling pathways downstream 27 of the PTK cascade i) Phospholipase C (PLC) - Ca 2 + - calmodulin pathways 27 ii) Protein kinase C (PKC) pathways 29 iii) p21ras and MAPK pathways 32 iv) PI3 - kinase (PI3K) pathways 35 v) N F - K B activation 36 vi) Stat activation 38 vii) The sphingomyelinase /ceramide pathway 39 1.3 Blocking T lymphocyte activation 42 1.3.1 Blocking T cell activation using cell surface antibodies 43 1.3.2 Src kinase inhibitor 44 1.3.3 Ca2+/calcimeurin pathway inhibitors - Cyclosporine and FK506 44 1.3.4 Protein kinase C (PKC) inhibitors 45 1.3.5 MAP kinase cascade inhibitors 47 1.3.6 Blocking N F - K B signaling cascades 49 1.3.7 Blocking IL-2 induced T cell activation 50 1.3.8 Induction of tolerance 51 1.4 Hypothesis and objective 54 CHAPTER TWO: MATERIALS AND METHODS 2.1 Solutions 2.1.1 Oligonucleotides and EMS A 2.1.2 Antibodies 2.1.3 Inhibitors (calBiochem, USA) 2.2 Methods 2.2.1 Cell culture i) Blood collection ii) Peripheral blood mononuclear cells (PBMC) separation iii) CD4+ T lymphocytes purification iv) Cell activation and proliferation 2.2.2 Blockade of signaling pathways 2.2.3 Detecting IL-2 receptor a chain (CD25) expression using flow cytometer i) Cell surface CD25 molecule labeling ii) Flow cytometer analysis 2.2.4 Cell apoptosis analysis 2.2.5 IL-2 ELISA 2.2.6 Preparation of nuclear extracts 2.2.7 Oligonucleotides and EMSA i) Co-incubation of oligonucleotides with DNA binding proteins ii) Agarose gel electrophoresis iii) Blotting 76 2.2.9 Quantitative Analysis of Dose-Effect 77 Relationships - The Median Effect Equation 2.2.10 Statistical Analysis 81 CHAPTER THREE: RESULTS 82 3.1 Building an in vitro human peripheral 82 CD4+ T lymphocytes activation model 3.2 Inhibitor's toxicity to in vitro CD4+ T lymphocytes culture 86 3.3 Effects of blocking signaling pathways under TCR and/or CD28 87 complex to T lymphocyte activation at levels of T lymphocyte proliferation, IL-2 secretion and IL-2 receptor a chain (CD25) expression 3.4 The suppression of on CD25 expression is partially 96 independent of the reduction of extracellular IL-2 3.5 Role of transcription factors N F - A T and AP-1 in IL-2 gene 100 expression and transcription factors N F - K B and STAT5 in IL-2 R a chain (CD25) gene expression 3.6 Blocking either TCR or CD28 signaling pathways dramatically 104 reduced NF-AT and AP-1 nuclear binding ability 3.7 The effects of blocking TCR or CD28 signaling pathways 109 of the nuclear binding ability transcription factor N F - K B and STAT5 3.8 The quantitative analysis of dose-effect relationships 117 between inhibitors using median effect equation CHAPTER FOUR: DISCUSSION 128 CHAPTER FTVE: SUMMARY 143 CHAPTER SIX: REFERENCES 146 viii LIST OF TABLES Table 3.8.1. UO 126 and LY294002 combination effects 122 on the CD4+ T lymphocyte proliferation Table 3.8.2. SB203580 and LY294002 combination effects 123 on the CD4+ T lymphocyte proliferation Table 3.8.3. Bisindolylmaleimide I and LY294002 combination 125 effects on the CD4+ T lymphocyte proliferation Table 3.8.4. Cyclosporine and LY294002 combination effects 126 on the CD4+ T lymphocyte proliferation LIST OF FIGURES Figure 1.2.1. T cell activation signaling networks 41 Figure 3.1.a. Kinetics of the CD4+ T lymphocyte proliferation 84 Figure 3.1 .b. Kinetics of the IL-2 secretion in activated CD4+ T lymphocyte 84 Figure 3.1 .c. Kinetics of the IL-2 receptor a chain expression on activated 85 CD4+T lymphocyte Figure 3.2. Cell apoptosis study 86 Figure 3.3.1. Effects of the inhibitors on the kinetics of the CD4+ 88 T lymphocyte proliferation Figure 3.3.2. Effects of the inhibitors on the kinetics of the IL-2 secretion 89 IX Figure 3.3.3. a. Effects of inhibitors on the kinetics 91 of the percentage CD25 expression Figure 3.3.3.b. Effects of the inhibitors on the mlfc CD25 expression 92 Figure 3.3.4. Dose -dependent response of the inhibitors on 94 the CD25 expression Figure 3.3.5. Time-dependent response of the inhibitors on 95 the CD25 expression Figure 3.4.1. Effects of extracellular IL-2 on the percentage of 98 CD25 expression Figure 3.4.2. Effects of extracellular IL-2 on the mlfc of CD25 expression 99 Figure 3.5.1. Kinetics of NF-AT and AP-1 binding activity 102 Figure 3.5.2. Kinetics of N F - K B and ST AT5 binding activity 103 Figure 3.6.1. Effects of U0126, SB203580 and Bisindolylmaleimide I 105 on NF-AT binding activity Figure 3.6.2. Effects of LY294002 and Cyclosporine on NF-AT 106 binding activity Figure 3.6.3. Effects of U0126, SB203580 and Bisindolylmaleimide I 107 on AP-1 binding activity Figure 3.6.4. Effects of LY294002 and Cyclosporine on AP-1 108 binding activity Figure 3.7.1. Effects of U0126, SB203580 and Bisindolylmaleimide I 111 on N F - K B binding activity Figure 3.7.2. Effects of LY294002 on N F - K B binding activity 112 Figure 3.7.3. Effects of U0126, SB203580 and Bisindolylmaleimide I 113 on STAT5 binding activity Figure 3.7.4. Effects of LY294002 on STAT5 binding activity 114 Figure 3.7.5. Effects of Cyclosporine on N F - K B and STAT5 binding 115 activity Figure 3.7.6. Summary 116 Figure 3.8.1. The median effect plots and the combination index (CI) 122 of U0126 and LY294002 combination study Figure 3.8.2. The median effect plots and the combination index (CI) 123 of SB203580 and LY294002 combination study Figure 3.8.3. The median effect plots and the combination index (CI) 125 of Bisindolylmaleimide I and LY294002 combination study Figure 3.8.4. The median effect plots and the combination index (CI) 126 of Cyclosporine and LY294002 combination study XI ABBREVIATONS [Ca2+] calcium ion concentration AP-1 activator protein-1 APC antigen presenting cell ARAM antigen receptor activation motif ADAP adhesion degranulation-promoting adaptor protein B7 antigen encoded by the B locus of chromosome 6 Bel B-cell leukemia Bil Bisindolylmaleimide I Btk Bruton's tyrosine kinase Bzip motif basic-leucine zipper motif Cbl casitas B-lineage lymphoma CBP CREB-binding protein CD cluster of differentiation CD28RE CD28 response element CD40L cluster of differentiation 40 ligand Clnk cytokine-dependent hemopoeitic cell linker CRE c-AMP-responsive element CREB-protein c-AMP responsive element binding protein CTLA cytotoxic T lymphocyte associated antigen CTLL cytotoxic T lymphocyte leukemia cell line DAG diacylglycerol DD death domains DED death effector domains DRK fos-regulating kinase DIC death-inducing signaling complex DOK the downstream of protein kinase ELF ETS-related transcription factor ERK extracellular signal-regulated kinase ESRD end stage renal disease Fas folic acid synthesis FADD Fas-associated death domain protein FRAP FKBP-12 -rapamycin-associated protein Fos Finkel and osteogenic sarcoma g gram Gads Grb2-related adaptor downstream of SHC GAPs GTPase activating proteins GEF Guanine nucleotide exchange factor GM-CSF granulocyte/macrophage colony-stimulating factor gp39 glycoproteins 9 Grap Grb-2 related adaptor protein GRB2 growth factor receptor binding-2 HLA human leukocyte antigen HMG high mobility group Hr hour IkB inhibitory kB protein IkK IkB kinase IL interleukin IgM immunoglobulin M INF-y interferon-y IP3 inosital(l, 4, 5) tri-phosphate IP inositolphospholipid ITAM immunoreceptor tyrosine-based activation motif Itk inducible T-cell kinase Jak janus kinase INK N-terminal c-Jun kinase Jun JU-Nana (avian sarcoma virus 17) Kda kilo Dalton Lck lymphocyte specific protein tyrosine kinase LAT Linker for activated T cells LFA-1 Lymphocyte function-associated antigen 1 LY LY294002 m molar mA mlliampere mlfc mean log fluorescent channel MAPK mitogen-activated protein kinase MHC major histocompatibility complex Min minute ml milliliter NFAT Nuclear factor of activated T cells N F - K B Nuclear factor K B PBMC peripheral blood lymphocytes PBS phosphate buffered saline PDK phosphoinositide-dependent protein kinase PI-3 kinase phosphatidyl inositol 3 kinase PI-4,5-P2 phosphatidyl inositol 4,5 biphosphate PKC protein kinase C PLC phospholipase C PTK protein tyrosine kinase PH pleckstrin homology domain PTB phosphotyrosine-binding domain RasGRP Ras guanyl releasing protein Ros reactive oxygen species RT room temperature SB SB203580 SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis SH2/SH3 src homology type 2 or 3 domain SLAP Src-like adaptor protein SMAC supramolecular activation cluster SPAK stress activated protein kinase Xlll SRF serum response factor DIE sis-inducible element SLAP src like adaptor protein SOCS suppressor of cytokine signal SLP-76 src homology 2 domain containing leukocyte protein of 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 TA transactivation TAC transactivation domain TAD transactivation domains TCR T-cell antigen receptor TH-1, -2 T helper 1,2 TNF-cx tumor necrosis factor a TRAP TNF receptor associated factor TRIM T cell receptor interacting molecule Tris tris (hydroxylmethyl) methylamine WASP Wiskott-Aldrich syndrome protein ZAP-70 zeta associated protein kinase of 70 kd xiv ACKNOWLEDGEMENTS I send my deepest gratitude to Dr. Paul Keown for all his direction and guidance to this study. I appreciate this invaluable opportunity of doing transplantation research under his supervision. My sincere thanks go to the committee members - Dr. Vincent Duronio and Dr. Pauline Johnson. They provided me with valuable advice and study chance in their labs and encouraged me through all the ups and downs during this period. I would also like to thank the Canadian Red Cross for their supply of normal human blood samples. To my fellow students, the technologists and staff members at the Immunology Lab, thank you for all the support I obtained for these years. Finally, my deepest love to my family-Mom, Dad, Tao and Wei. You are always the solid foundation of my life. X V I dedicate this thesis to... ...my parents, who always encourage me to work hard and be a better person! x v i CHAPTER ONE: INTRODUCTION 1.1 General Background Transplantation - the replacement of damaged human tissue with that from other people or animals has been one of the humanity's most enduring goals. From 2000 BC till early in the 20th century, transplantation experiments have been performed worldwide. With the improvement of surgical techniques and the thorough understanding of an organ's vascular anatomy, those experiments were finally technically successful but still failed for immunological reasons. Since early in the 20th century, intensive research has been focused on revealing the immunological events in transplantation rejection. Sir Peter Madawar and Maclarfane Burnet first recognized the importance of cellular immunity in graft rejection. It was one of the most important breakthroughs in transplantation immunity and won them the Nobel Prize in 1960. Graft rejection occurs when the recipient's immune system recognizes the allograft as foreign. One of the critical events in this process is T lymphocyte activation. T lymphocyte activation starts with helper T lymphocytes (CD4+ T cells, Th) binding donor antigen in the presence of MHC II molecules and cytotoxic T lymphocytes (CD8+ T cells) binding donor antigen in the presence of MHC I molecules on the surface of antigen presenting cells (APCs) (1, 2). There are many receptors on the T lymphocyte surface, which contribute to the interaction between the APCs presenting the antigenic 1 peptide and the T cell. However, specificity of this interaction is due to the selective binding of the antigen to a discrete TCR. In general, each lymphocyte clone has a single antigen binding specificity that is unique to that clone. In response to a given antigen, only those lymphocytes whose TCRs bind with high affinity will be activated. In depth research continuously evoked the importance of the T cell activation process. The "two signal model" of the T lymphocyte activation process has been used to describe the interaction between T lymphocytes and antigen-presenting cells (APCs). It was originally proposed by Bretscher and Cohn (3) in 1970 in an attempt to account for peripheral self-tolerance. The model assumes that T cells require two signals to be activated by antigen - presenting cells (APCs). The first signal is delivered via T cell receptors (TCR) upon antigen presentation. The second signal, also called "the co-stimulatory signal", is usually provided by the B7.1/2 molecules on APCs, which bind to the CD28 receptor on the T lymphocyte. The second signal has two important features; (i) it has no cognition of the antigen, resulting from receptor counter-receptor interactions not related to antigen specificity, and (ii) it can be produced by a number of distinct molecular interactions that may occur between an APC and a T cell (4). The function of the co-stimulatory signal is to modulate the response threshold of TCR (5).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 thymus (6, 7). CD28 controls a wide range of responses in naive CD4+ T cells, including a decrease of the TCR signaling threshold. It has the ability of regulating specific signaling cascades, 2 as well as promoting a generalized increase in protein tyrosine phosphorylation. In contrast, the major effect of CD28 in memory T cells is to enhance the TCR response, whereas its role in CD8+ T cells is less clearly defined. This is in contrast to its essential role in naive T cells. CD4 and CD8 molecules also contribute to MHC recognition in T helper and cytotoxic T lymphocytes, respectively. Other extracellular proteins such as adhesion molecules CD2 and LFA1 play an enhancing role in T-cell activation. The engagement of TCR and costimulatory molecules with their ligands triggers the activation of a vast and complicated intracellular signaling network. TCR and CD28 signaling commence with an early wave of protein tyrosine kinase (PTK) activity, which is mediated by the Src kinases Lck and Fyn, the 70-kd i\ chain-associated protein kinase (ZAP-70), and members of the Tec kinase family, inducible T-cell kinase (Itk), Tec, and Txk/Rlk. This early wave of protein tyrosine phosphorylation leads to the activation of downstream signaling pathways, including increases in intracellular calcium flux, protein kinase C (PKC), phosphatidylinositol 3 kinase (PI3-kinase), nuclear factor N F - K B , and Ras-mitogen-activated protein kinase (MAPK) activation. These pathways activate transcription factors including NF-AT, AP-1, N F - K B and STAT5 that ultimately lead to the expression of genes that control specific cellular responses such as IL-2 and IL-2 receptor a chain. IL-2 plays a vital role in the growth and clonal expansion of many types of T 3 lymphocytes and in the differentiation of thymocytes, peripheral T and B lymphocytes and other cells of haemopoietic origin. Therefore, IL-2 signaling was referred to as "signal 3" for T lymphocyte activation. The IL-2 required for antigen-driven T lymphocyte proliferation may be derived from the stimulated T lymphocyte itself or from other cells involved in the immune response. Thus the key factor in determining the T cell's ability to mount a proliferate response to IL-2 will be expression of the high-affinity IL-2 receptor on the surface of the antigen activated T cell. The understanding of the molecular and cellular events involved in T lymphocyte activation has provided a rational foundation for immunosuppression. Several immunosuppressive regents had been successfully designed to block key activation signaling molecules. One of the most important was cyclosporine. The clinical use of cyclosporine significantly reduced the incidence and severity of acute rejection and improved the short-term rate of graft survival and patient survival. It has become the core medication for most clinical immunosuppressive protocols. However, many problems still exist. Acute rejection rates remain high. Meanwhile, existing immunosuppressive protocols are very expensive and have serious drug side effects. For example, cyclosporine alone costs $4,700 US per year per patient (8) and is characterized by a significant risk of nephrotoxicity (9) and neurotoxicity (10). Researchers hope to develop drugs that are selective in their action, inducing tolerance of the allograft, and minimizing or negating the morbidity experienced with today's immunosuppressive agents. Fortunately, discoveries in T lymphocyte activation networks provide the knowledge base for new drug design. Many agents have been designed to block key signaling 4 molecules in T cell activation, including antibodies against T cell surface antigens, Src kinase inhibitors, P K C inhibitors and M A P kinase inhibitors. These agents can be classified into different groups based on their targeting the TCR /CD3 complex, costimulatory receptors, or individual signaling components. More investigation is needed to understand the effects of these agents as wel l as other potential immunosuppressants on T cell activation and proliferation, cytokine and cytokine receptor expression, as well as their combined effects on T cell activation. An imal models and clinical trials also need to be set up to check their in vivo and clinical functions in preventing transplantation rejection. 5 1.2 Signal Transduction in T lymphocyte 1.2.1 T cell receptor (TCR) signaling i) T cell receptor, antigen binding and activation of protein tyrosine kinases There are many receptors on the T-cell surface contributing to TCR signaling. However, specificity of TCR signaling is due to the selective binding of the antigen to a discrete T cell receptor. The TCR:CD3 complex comprises six different polypeptide chains thought to be organized into an eight-chain structure (11). These polypeptides include ligand binding TCR subunits a(3 or y8 in a noncovalent association with invariant chains of CD3s, CD3y, CD38 and TCR^. These invariant chains are required for receptor assembly, cell surface expression, and signaling (12). There is a critical region in cytoplasmic domains of the invariant chains called the immunoreceptor tyrosine-based activation motifs (ITAMs). It is also called the antigen recognition activation motif (ARAM). The consensus sequence for an ITAM is YxxL(x)6.8YxxL. When phosphorylated on one or both of its tyrosine residues, ITAM provides binding sites for a number of proteins involved in early TCR signal transduction. There are three ITAMs in each of the TCR<^  chains, and one in each of the CD3 chains (13). Since each TCR can contain a TCR^ dimer and two CD3dimers (s8 and ey), each TCR can contain a total of ten ITAMs (14). Binding of an immunogenic peptide presented by other T-cells, fixed APCs, purified 6 MHC molecules, or ligation of anti-CD3 antibodies leads to stimulation of the T cell receptor (TCR) (15, 16). This stable interaction between the TCR and MHC - peptide has been called "immunological synapse" (17, 18). The mature immunological synapse is defined by a specific pattern of receptor segregation with a central cluster of TCRs surrounded by a ring of integrin family adhesion molecules (19). TCR - MHC first interacts in a broad outer ring and integrins such as LFA-1 concentrate into the central region by engagement of their corresponding adhesion ligand (e.g. ICAM-1) on the surface of the stimulating cell (19). The arrangement is then stabilized as engaged TCR-MHC complexes become enriched in a tight central cluster and integrins molecules are relocated and form a non-overlapping outer ring. This transport of TCR complexes into a central cluster appears to depend upon optimal TCR-MHC interaction kinetics, which correlates with the half-life (tl/2) of the TCR-MHC - peptide interaction (20). Since just a few copies of an antigenic peptide can trigger robust T cell activation, the T cell receptor requires help from other surface molecules to form a stable cell - cell contact, including CD4 or CD8 coreceptors and receptor ligand pairs, such as LFAl/ICAM-1, CD2/CD48, and CD28/CD80 (19). TCR engagement can induce different signals, depending on the precise structure of the peptide MHC complex to which it binds. The dissociation rate of the antigen-MHC complex from the TCR determines the signaling output from the T cell. Antigen-MHC complexes which bind with optimal kinetics of interaction will trigger the engagement of the accessory molecules and the full repertoire of T cell responses (21). Those complexes which do not bind with sufficient affinity will fail to induce the engagement of these accessory 7 molecules, and a partial response wi l l result (22). T cell receptor polypeptides do not possess intrinsic P T K activity. Instead, they associate with cytoplasmic P T K s . Activation of P T K s by the T C R results in a rapid increase in the tyrosine phosphorylation of signaling proteins (23, 24). Tyrosine phosphorylation of these proteins regulates their function and initiates protein-protein interactions that are important for signal transduction (25). Many signaling proteins contain special structural motifs/domains that are able to direct intermolecular interactions. Src homology 2 (SH2) domains are approximately 100 amino acids (26) and recognize short, phosphopeptide motifs composed of phosphotyrosine (pTyr) followed by three to five COOH-terminal residues (27). SH3 or Src homology 3 domains mediate protein - protein interactions, via recognition of regions within proline-rich regions contained the consensus sequence X-Pro-pTyr-X-Pro (28-31). P T B (phosphotyrosine-binding) domains mediate interactions based on phosphotyrosine residues and recognize motifs with the consensus Asn-Pro-X-pTyr (32-35). Interestingly, the specificity for P T B domain binding resides in the amino acids amino terminal to the phosphotyrosine, as opposed to SH2 domains whose binding specificity depends on the carboxyl- terminal amino acids (36). W W domains (named for the two tryptophans (W) located in the protein binding site) are protein regions which bind to other proteins either contain proline-rich regions or phosphorylated serine or threonine residues (37-39). P D Z domains are modules which interact with discrete domains that contain hydrophobic residues carboxyl-terminal of the binding site (40, 41). Pleckstrin homology, or P H 8 domains, direct intermolecular interactions based on associations with phospholipids (42, 43). Increasing evidence indicates that each of these modular regions plays critical roles in localizing effector molecules and creating multimeric-signaling complexes. Four families of PTKs have been shown to be involved in TCR signaling. Lck and Fyn are members of the Src family. They have a unique N-terminal domain with a myristylated glycine at position 2, which is responsible for membrane association. A unique region of approximately 80 amino acids in their N-terminal may direct specific interactions of the PTK with other proteins. They also contain a SH3 domain and a SH2 domain. A conserved tyrosine residue close to their C-terminus acts as a negative regulatory site when phosphorylated (25). Lck and Fyn are critical for initiating TCR signaling and also play important roles in T cell development (44, 45). Syk family PTKs include ZAP-70 and Syk. They are not myristylated and hence are probably not constitutively localized at the plasma membrane. The Syk/ZAP-70 family members have two N-terminal SH2 domains and a C-terminal catalytic domain, but lack an SH3 domain and the C-terminal negative regulatory site of tyrosine phosphorylation, which are characteristic of Src family members (45). Although ZAP-70 is a critical PTK in TCR signaling, Syk is important for B cell antigen receptor and B cell development and is not required for TCR signaling (46). The Tec family PTK preferentially expressed in T cells is Itk. This family of PTKs is characterized by having a N - terminal pleckstrin homology (PH) domain, and a SH2 and SH3 domain, in addition to the C-terminal kinase domain (14). Csk is the fourth family which negatively regulates Src family PTKs. Csk phosphorylates the carboxy-termin tyrosine in Lck and Fyn and by doing so, maintains 9 these proteins in an inactive state (47). The initial step of TCR signaling is the tyrosine phosphorylation of ITAMs. This process is mediated preferentially by Lck and followed by the recruitment of ZAP-70 to the activated receptor, facilitating subsequent tyrosine phosphorylation and activation of ZAP-70 (47). The sequential interaction of Lck and ZAP-70 with TCR ITAMs phosphorylates two critical tyrosines and generate differentially phosphorylated TCR^ isoforms, p21 and p23, which have been observed predominantly in resting and activated T cells (48). 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 (49). Zap-70 has been described to play two crucial roles in the earliest biochemical events after TCR crosslinking (47, 50). First, it plays a role in the phosphorylation of PLCyl, SLP-76 and LAT; second, it plays a role in TCR-induced Ca mobilization, activation of transcription factor NF-AT (nuclear factor of activation T cells) and IL-2 production (57). Dual-phosphorylated ITAMs recruit ZAP-70/ Syk family kinases via their tandem SH2 sequences. 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 (52-54). 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 10 products of phosphatidylinositol-3-kinase (55-57). Itk deficient mice show mild defects in T cell development (58, 59). In addition, proliferation in itk deficient mice is greatly reduced upon TCR stimulation but enhanced upon CD28 co-stimulation when compared to normal mice (59). Recruitment of protein tyrosine kinases to the ITAMs is enhanced by stimulation of the co-receptor CD4 and CD8, respectively. They activate a cytoplasmic protein tyrosine kinase Lck through a cysteine-containing motif shared by their cytoplasmatic domains (45, 60). Activation of intracellular protein tyrosine kinases is further enhanced by the membrane-bound phosphatase CD45. It dephosphorylates the C-terminal negative regulatory tyrosine of Src-family members (Lck in particular), resulting in unfolding Src and an increase in its activity (61). The activation of Lck leads to the recruitment of a second cytoplasmatic PTK, ZAP-70, to the TCR cluster through both of its Src homoloy-2 domains and its phosphorylation (62). ii) Adaptor proteins in TCR signaling Adapter proteins are defined by the lack of enzymatic or transcription activities and the expression of a variety of modular binding domains (e.g. SH2-, SH3-, PTB- or PH domains) or tyrosine based signaling motifs. These domains/motifs enable adapter proteins to mediate constitutive or inducible protein-protein or protein-lipid interactions with other signal transducing components. The main function of adapter proteins is to integrate receptor mediated signals at the intracellular level and to couple signal 11 transducing receptor complexes to intracellular effector systems by organizing the dynamic ensemble of signaling scaffolds (63). LAT (Linker for activation of T cells) is a transmembrane Adaptor protein phosphorylated by ZAP-70/Syk-family PTKs (48). L A T is then able to recruit critical signaling molecules to the membrane including P L C y l , GRB2, Grap and P85 phophatidylinositol-3- kinases (PI-3 kinase), as well as Grb2-SH3- binding proteins Sos, c-Cbl and the SLP-Vav complex (48). LAT is essential in coupling the TCR to PLCy-C a 2 + and Ras signaling pathways. Mutation of SH2 binding sites of L A T inhibited TCR mediated NF-AT/AP-1 activation (64). Mice deficient for L A T has been shown to have their thymocytes development arrested at the CD4" CD8" (double negative) stage, due to an inability to progress past the pre-TCR checkpoint (65). SIT and TRIM have been described in T cells (66). It appears that TRIM is a positive regulator (65), possibly acting through Grb-2 and PI3 kinase. SIT may negatively regulate T cell activation, consistent with the presence of an inhibitory motif in its cytoplasm tail and its interaction with the phosphatase SHP-2 (67). Phosphoprotein associated with glycosphingolipid - enriched micro domains (PAG or Csk binding protein, CBP) emerged as a potential critical negative regulator of Src family kinase activity through its association with Csk (68-70). P A G carries 10 potential tyrosine based signaling motifs among which at least one mediates an association with the Csk. Upon TCR signaling, P A G becomes transiently dephosphorylated by an 12 unknown protein tyrosine phosphatase, resulting dissociation with Csk (77). Csk is known to phosphorylate a carboxyterminal negative regulatory tyrosine of Src-kinases, thereby down regulating their enzymatic activities (63). SLP-76 is an adaptor protein with an amino - terminal region that contains critical tyrosine sites, a central proline -rich domain and a carboxy- terminal SH2 sequence (72). It is a prominent substrate for PTK's from the ZAP-70/ Syk family. SLP-76, through its proline-rich tract, binds to the SH3 domain of Lck (73). Thus, coligation 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 (74, 75). The amino terminal of SLP76 contains three tyrosine phosphorylation sites. Upon TCR engagement, the SLP-76-Grb2 (or Gads) complex then interacts with SH2 containing proteins such as Vav, Rac/CDC42 exchange factor and adaptor protein Nek (76, 77). Evidence demonstrated that SLP-76 is critical for efficient TCR induced tyrosine phosphorylation of P L C x l , inositol phosphate accumulation, C a 2 + mobilization and Ras activation (78). The C-terminal SH2 domain of SLP-76 is associated with adhesion degranulation-promoting adaptor protein (ADAP) (79, 80) and a serine-threonine kinase HPK-1, following TCR stimulation (81). Another protein that might function like SLP-76 is the adaptor CInk, which is 13 expressed preferentially in cytokine stimulated cells (82). CInk contains a SLP-76 related SH2 domain near its carboxyl terminus, as well as several potential tyrosine phosphorylation and SH3- interacting sites. CInk over expression activates downstream regulators of TCR activation, including NF-AT. Other recently identified adaptors of uncertain function include Shb (83) and CAST (84). SLAP-130/Fyb (ADAP) binds SLP-76 (85, 86). It also has been shown to directly bind the Ena/Vasp family protein, E V L , and to be in complexes containing Nek, WASP, Vasp and Arp2/3 (87). Therefore, it links the TCR to the actin cytoskeleton, via the other scaffolds, L A T and SLP-76. The SLAP-130/Fyb knockout mice demonstrated an impairment of T cell proliferation and cytokine production (88, 89). More specifically, the ability of TCR signaling to regulate integrin-mediated adhesion appeared defective. Over expression studies confirmed that SLAP-130 can directly affect integrin adhesion (90) . Determining whether SLAP-130 plays a distinct role in regulating actin cytoskeleton or whether its role in actin cytoskeletal regulation is linked to integrin activation will require further work. Phosphatase SHP-1 is a negative regulator of TCR signaling. It is activated by (31-integrins and has been shown to dephosphorylate ZAP-70, L C K and the TCR% chain (91) . The Cbl protein (Casitas B- lineage lymphoma protein) also plays an important role in downregulating TCR activation. Until recently, Cbl was thought to act solely as an 14 adaptor, possibly by binding tyrosine 292 in ZAP-70 (92, 93). However, studies have provided evidence that Cbl can function as an ubiquitin-3 ligase in vitro (94) and also functions as a negative regulator of receptor clustering (95). Thus, Bachmaier et al. described Cbl as a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity (96). Loss of Cbl function has been associated with transformation (97). Another negative regulator of TCR function is SLAP (Src-like adaptor protein). Its 34 kDa structure contains only an SH3, an SH2 domain and a carboxy-terminal region (98).The molecule co-localizes with endosomes in Jurkat T cells and in Hela cells and appears to co-immunoprecipitate with CD3< ,^ ZAP-70, SLP-76, Vav and L A T . The SLAP knockout mouse strain exhibits higher levels of TCR expression, suggesting that SLAP is involved in TCR endocytosis (99). A homologue, SLAP-2, was recently cloned by screening for negative regulators of B C R signaling and appears to negatively regulate TCR signaling as well (100). The downstream of tyrosine kinases (Dok) family of adaptor molecules share an N -terminal pleckstrin homology domain (PH), a central phosphotyrosine binding domain (PTB) and a C terminus containing motifs. These domains allow for associations with both SH2 and SH3 containing molecules. Dok family members recruit specific multi-molecular complexes containing the inhibitory molecules pl20RasGAP (a negative regulator of Ras), SHIP-1 (a phosphoionsitol phosphatase) and Csk (101-103). 15 SAP is an adaptor protein whose function is controversial. It consists of a single SH2 domain and a short C-terminal tail. The major binding partner of SAP is the receptor S L A M . In a murine model, ligation of S L A M induces tyrosine phosphorylation of SHEP-1, Dok and RasGAP, and leads to the inhibition of IFN-y secretion. These phosphorylation events are SAP-dependent and implicate SAP as a negative regulator of T cell signaling. However, in vitro systems and N K cells, SAP has been implicated as a positive regulator by inhibiting the association of S L A M with negative regulators such as SHP-2 (77). 1.2.2 CD28/ CTLA-4 costimulatory signaling pathways i) CD28 and CTLA-4 molecules CD28 is the primary T cell costimulatory molecule. Upon interaction with ligands B7.1 (CD80) and / or B7.2 (CD86), CD28 transduces a signal which enhances the production of many cytokines important for T cell activation and proliferation, especially IL-2. Conversely, the CD28 homologue cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), (CD152) inhibits T cell responses (104). CD28 molecule is a 44 kDa glycoprotein homodimer belonging to the immunoglobulin supergene family. It is presented on 80% of human T cells and 95% of CD4 + Th cells (705). The CD28 molecule is composed of two glycosylated chains, each containing a single disulfide-linked extracellular Ig variable-like (V) domain. The extracellular 16 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 (106). CTLA-4 is also a disulfide-linked homodimer with only a single glycosylation site. It contains a single disulfide-linked extracellular Ig V domain, a transmembrane region and a cytoplasm domain of 36 amino acids (106). Both CD28 and CTLA-4 can bind to the same physiological ligands, namely the 60 kDa B7.1 (CD80) and the 70 kDa B7.2 (CD86) via the M Y P P Y (in the single letter code for amino acids) motif in the immunoglobulin domain. However, CTLA-4 has a 10-fold higher affinity and a 100-fold higher avidity for B7 ligands compared to CD28 (104). The signaling events of CD28 are just beginning to be understood. CD28 promotes a generalized increase in protein tyrosine phosphorylation (107). Although its cytoplasmic domain lacks any direct enzymatic activity, it includes several binding motifs that may be involved in signal transduction (108, 109). This is initiated by phosphorylation of the Tyrl73-Met-Asn-Met motif within the cytoplasmic domain of CD28 (80). It is followed by recruitment of the signaling proteins, including the SH2 domains of the p85 subunit of PI3-kinase and growth factor receptor binding protein (Grb-2), and T cell-specific protein - tyrosine kinase (ITK) (110, 111). The Tyrl88 is also critical for JNK activation and transcription activation of the IL-2 promoter (109). CD28 also contains two proline-rich motifs (Pro -Arg-Arg-Pro and Pro -Tyr-Ala-17 Pro) which conform to the Pro-Xaa-Xaa-Pro SH3 binding consensus sequence (106). It may be functionally involved in Lck and Tec kinase recruitment and activation (108, 109, 112, 113). How exactly the phosphotyrosine and PR motifs relate to PTK activation and distal signaling cascades is still unclear. We do know, however, that CD28 assists in the assembly of the cortical cytoskeleton and recruitment of lipid rafts to the TCR synapse. Regulation of the cytoskeleton involves the sustained phosphorylation and activation of Vav by CD28. Vav is a GEF for Rac-1, which plays a role in cytoskeleton assembly and possibly also the activation of the JNK cascade. The biochemical signals generated by CTLA-4 are poorly understood. There is evidence suggesting that CTLA-4 might interfere with TCR-associated protein tyrosine kinases such as Lck, Fyn and ZAP-70 (114). The association of CTLA-4 with phosphatase SHP-2 might also explain its inhibitory effects in T cell activation. CTLA-4 has a consensus binding site (Tyrl64-Val-Lys-Met) for the SH2 domains of the p85 subunit of the PI3-kinase (115). The physiological function of this binding still needs to be investigated. Considering that CTLA-4 may induce anergy, it might also be involved in Fyn-Cbl-CrkL-Rap-1 signaling pathway (116). ii) Functions of CD28/ CTLA-4 signaling on T lymphocyte activation The prime function of CD28 is up-regulation of IL-2 production. This results from effects on IL-2 promoter transcription and on mRNA stability. Engagement of the CD28 receptor enhances the production of IL-2 mRNA approximately 20 fold, providing the 18 necessary co-stimulatory signal required for maximal T cell activation. Without co-stimulation, this degradation process proceeds rapidly and leads to reduced IL-2 mRNA levels. Under physiological conditions, absence of the co-stimulatory signal induced a state of clonal anergy (117). Although controversial, increasing evidence supports a role for PI3-kinase in the regulation of CD28 mediated IL-2 transcription. Studies have shown that CD28 costimulation is necessary for the optimal activation of AP-1 and N F - K B , both of which are transcription factors known to be important to the activation of IL-2 promoter. There is a sequence between -164 and -154 of the IL-2 promoter, called the CD28 response element (CD28RE). This sequence is important for CD28 as well as for mitogenic stimulation. Inhibitor studies used CsA and FK506 also showed that CD28 could induce IL-2 production by activating transcription factor NF-AT through calcium - calcineurin pathway via p21 ras (118). CD28 also regulates expression of the intrinsic cell survival factor B c l - X L , 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 (119). It also results in the production of cytokines, such as IL-2. Thus, costimulation promotes that survival of T cells that encounter an antigen, allowing autocrine cytokines at initiate clonal expansion and differentiation. Engagement of the CD28 co-receptor leads to the upregulation of a variety of surface antigens such as CTLA-4, IL-2 receptor a and P, and the CD40 ligand (CD40L), each 19 of which is needed for successful progression of T cell responses {111). CD28 mediated co stimulation also leads to a strong upregulation of IL-4, IL-5, IL-13, y-interferon, tumor necrosis factor a, granulocyte/macrophage colony-stimulating factor (GM-CSF) and the chemokine IL-8 and RANTES (120). The gene promoters of IL-2, GM-CSF, IL-3, INF-y, IL-8 and RANTES have conserved sequenced which are responsive to CD28 signal transduction (106). There is evidence that there are both cyclosporine A-sensitive and -resistant components of the CD28 signaling responses, depending on the activation state of the T cells. The cyclosporine A-sensitive pathway is dominant in activated T cells and tumor cells (121), whereas the cyclosporine A-resistant pathway is dominant in resting T cells (122) CTLA-4 functions differently in regulating the immune response. CTLA-4 is not expressed constitutively and is only expressed maximally 2-3 days after T cell activation by TCR/CD3 and CD28 ligation. Antibodies to CTLA-4 can enhance T cell proliferation, whereas crosslinking of the antibodies have been shown to inhibit proliferation of native T cells (123). This suggests that blockade of CTLA-4 removes, whereas aggregation of CTLA-4 provides, inhibitory signals that downregulate T cell response. B7.1 and B7.2 have also been reported to differentially regulate lymphokine production and the appearance of Thl versus Th2 subset. B7.2 is rapidly expressed in B cells following activation, whereas maximum B7.1 expression appears significantly later (124). In contrast to anti-B7.1 mAbs, anti-B7.2 mAbs are potent inhibitors of T cell proliferation and cytokine production in vitro (125). 20 1.2.3 IL-2 and IL-2 receptor a chain gene expression i) Transcriptional control of IL-2 gene It is generally accepted that triggering of the C a 2 + signal and the induction of p21ras activation through TCR are key events to induce expression of IL-2 and its high-affinity receptor. Regulation of IL-2 production by the TCR requires the coordinate action of multiple transcription factors, which include NF-AT, AP-1, N F - K B and Oct-1 (126). These transcription factors when activated bind to the promoter region upstream of the IL-2 gene to switch on transcription of the gene. Activation of the IL-2 promoter by CD28 costimulation is dependent on the synergistic activation of the I N K and N F - K B cascades (127, 128). The IL-2 promoter contains « 300 bp 5' of the transcription start site. It contains two binding sites for members of the NF-AT family of transcription factors (129). Mutation of both sites is required to eliminate IL-2 promoter function (130), and in vivo footprint indicates that both sites are occupied in stimulated cells (131). Four members of the NF-A T family (NF-AT 1, NF-ATc, NF-ATx, NF-AT3) have been identified so far (132). After translocation to the nucleus and binding to DNA, NF-AT proteins enhance transcription in several ways. First, NF-AT proteins recruit or facilitate the binding of AP-1 transcription factors to NF-AT-AP-1 binding sites (132). The components of the Ap-1 transcription factor then mediate the induction of transcription by recruiting co-21 activators such as CBP (CREB-binding protein), p300 and JAB1 (June activation domain binding protein), through their transcriptional activation domains (133). These coactivators augment transcriptional activity by recruiting the basal transcription machinery through direct protein-protein interactions and by acetylating histones, which increases accessibility of nucleosomal D N A to transcription factors (134). In addition, NF-AT proteins also contain their own transactivation domains (TADs). For example, in NF-AT two different TADs were identified (135). 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. Mutation of the AP-1 binding site abolishes function of the IL-2 promoter in transient transfection assays. In vitro, this site binds dimers of Fos and Jun family proteins (Fos, FosB, Fos-1, Fos-2, Jun B and Jun D) (136). The two families of proteins are downstream targets of both the T cell receptor and the CD28 co-receptor signaling cascade (106). AP-1 binding activity is specific for the palindromic sequence 5' T G A G T C A 3' (137). D N A 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. Fos family proteins contain activation domains both amino-terminal and carboxy-terminal of 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 (138). 22 Although all possible combinations of Fos-Jun dimers wil l bind the consensus AP-1 target element, functional assays have revealed some difference in the ability of different dimer combinations to mediate transcriptional activation. AP-1 complexes also participate at multiple regulatory sites such as NF-AT and Oct-1 in a cooperative manner (139). IL-2 promoter also contains binding site for transcription factor N F - K B , although mutations in the N F - K B site are generally less deleterious than mutations at other sites of the promoter (140). The major nuclear factor binding to the IL -2 N F - K B site is probably the p50-p65 heterodimer (141). After T cell activation, the phosphorylation of p50-p60 heterodimer as well as the degradation of I K B inhibitor unmask the nuclear localization signal, and leads to the translocation of the heterodimer into the nucleus. At later times after activation, c-Rel may be a significant component for the complete binding to the N F - K B site. The levels of total and nuclear c-Rel in T cells increase considerably following stimulation. C-Rel may be more likely to maintain rather than to repress the late transcription of IL-2 mRNA (142). The p50 homodimer may also serve an important regulatory function, especially in untransformed T cells, by repressing IL-2 transcription (143). The IL-2 transcriptional apparatus integrates multiple signals to determine expression of the IL-2 gene by using many transcription factors that are activated or inhibited by different signaling pathways. Blocking the activity of just one or two of these transcription factors is sufficient to completely inhibit IL-2 expression. The explanation 23 behind this is that none of these transcription factors will interact stable with its target site in the IL-2 enhancer unless all the factors are present (144). ii) Transcriptional control of IL-2 R a chain gene expression The IL-2R comprises three subunits (IL-2Ra, IL-2RP and IL-2Ry), which are encoded by different genes. Il-2Ry is constitutively expressed on various populations of haematopoietic cells, The expression of both IL-2Rot and IL-2RP is restricted to lymphocytes and monocytes:macrophages. Three classes of IL-2 receptor proteins exist: high-affinity receptors (K& ~10"11 M) are heterotrimers containing a, p and y subunits; intermediate-affmity IL-2Rs (Kd ~10"9 M) contain p and y subunits, and low-affmity subunits contain only the a chain. Although both intermediate-and high-affinity IL-2Rs are capable of transducing mitogenic signals in response to IL-2, the high-affinity receptor is probably required to bind the relatively low concentrations of IL-2 produced physiologically (145). T lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of one of the components of the high-affinity IL-2R, namely the IL-2R a gene (146). Although specific antigens will induce expression of both IL-2 and its high-affinity receptor, IL-2 itself can also induce transcription of the IL-2R a gene (147). The IL-2R a chain promoter contains multiple regulatory sequence elements that bind several transcription factors, some of which are similar to those which control IL-2 gene 24 transcription in response to antigens. IL-2R a transcription in response to antigens is, at least in part, controlled by two regulatory regions in its promoter, termed PRRI and PRRII (148). PRRI is an inducible proximal enhancer that contains DNA-binding sites for SRF and N F - K B . Thus, N F - K B proteins play an important regulatory role in the transcription of both IL-2 and IL-2R a genes in response to antigens. PRRII is a T-cell-specific enhancer element which binds the T-cell-specific ETS protein, ETS-related transcription factor (ELF) (149) and certain high mobility group (HMG) proteins. The interactions between the proteins which bind to PRRI and PRRII probably result in the formation of a complex that regulates the transcriptional activity of the IL-2R a promoter upon stimulation with antigens. Interestingly, neither of these elements regulate IL-2R a transcription induced by IL-2 (148). This is mediated through PRRIII (148), a complex response element that lies upstream of PRRI and PRRII. PRRIII is composed of D N A -binding sites for the IL-2-inducible STAT proteins STAT5A and STAT5B, for the lymphoid :myeloid specific Ets family protein, Elf-1, for HMG-I(Y) and a gamma-aminon- butyrate (GATA)-like protein. Binding of all these factors appears to be required for optimal transcription of the IL-2R a gene in response to IL-2. iii) IL-2 signaling cascades Activation of the heterodimeric IL-2R by high-affinity binding of IL-2 is thought to activate three major signal transduction pathways. These are: the activation of She and consequent activation of Ras (150); activation of PI3-K, which activates S6 kinase through Ras independent M A P kinase pathways and some P K C isoforms through C a 2 + 25 and D A G independent pathways (151), and the activation of a JAK:STAT pathway (152). Janus kinases (Jaks) are cytoplasmic protein tyrosine kinases required for cytokine signaling. They play a central role in IL-2 signaling. Activation of the IL-2R induces the binding of J A K 1 and 3 to the intracellular domains of IL-2R p and IL-2R y (151). Jak phosphorylates several tyrosines in the cytoplasmic portion of the IL-2R p chain. The phosphotyrosines form part of docking sites for cytoplasmic signal transducers such as PI3K and She, as well as for the family of transcription factors STATs (signal inducers and activators of transcription). The physiologically more important STAT proteins for IL-2 receptor are probably STAT5. The STAT5 proteins contain functional domains. The N-terminus is a protein - protein interaction domain. The STAT SH2 domain is found between AA570-670 and serves as the docking site for phosphorylated tyrosine. The STAT D N A binding domain is located between AA400 and 500. It binds to gene promoters contained sequence AGTTTCNNTTTCNC/T or sequence T T N N N N N A A (N stands for any nucleotide). The transactivation (TA) domain is located at the carboxy-terminus. Phosphorylation of a single serine in the T A domain can enhance the transcription activity. The C-terminus is also important for the dephosphorylation of STAT5 (153). The activation of IL-2 receptor associated J A K induces the phosphorylation, subsequent dimerization, nuclear translocation and transcriptional activation of STAT5 (151). 26 1.2.4 Activation of signaling pathways downstream of the P T K cascade Signaling pathways are formed by a series of cytoplasmic molecules passing the activation signals from surface receptors to the nucleus for gene transcription and new protein synthesis. They are the information highways inside T lymphocytes. Several major pathways and their interactions have formed a vast and complicated signaling network, including Ras pathways, M A P K cascades, phosphatidylinositol 3 kinase (PI-3 kinase), PLC-y l , PKC, and the N F - K B pathways. They control the functions of T lymphocytes through molecular interaction. A great amount of research has been focused on understanding the signaling network in detail. T lymphocyte activation can be reduced by modifying key molecular interaction using specific inhibitors. Some of these inhibitors such cyclosporine and FK506 have been successfully used in the clinic to prevent acute allograft rejection. New immunosuppressive reagents could be developed by identifying other key signaling molecules, investigating the effects of their specific inhibitors on T lymphocyte activation in vitro and on preventing allograft rejection in vivo. i) Phospholipase C (PLC) - C a 2 + - calmodulin pathways One of the major events after T cell receptor cross-linking is tyrosine phosphorylation of the PLC-y (48). This occurs directly through ZAP-70 (154) or indirectly with the help of adapter protein L A T and SLP-76 (48, 75, 155). The Tec family kinases Itk and Rlk are 27 required for maximal PLC-y phosphorylation (156, 157). Phospholipase C (PLC) enzymes cleave the plasma membrane lipid phosphatidylinositol 4, 5-biphosphate [PtdIns(4,5)P2] into two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) (158). The Ptdlns (4,5)P2-derived second messengers play an essential role in TCR signaling. These include the entry of resting T cells into the cell cycle, induction of immediate early genes such as c-myc, c-fos, and egr-1 in T cells, and induction of IL-2, its high affinity receptor and interferon y gene transcription in T cells (159). Inositol 1,4,5-trisphosphate (InsP3) increases cytosolic free C a 2 + concentration by stimulating the release of Ca2 + from intracellular storage vesicles that possess an InsP3-regulated C a 2 + channel (755). This leads to the activation of C a 2 + / calmodulin-dependent protein kinases as well as the C a 2 + / calmodulin-dependent serine/threonine phosphatase calcineurin (phosphatase 2B). Calcineurin binds and dephosphorylates the regulatory domain of transcription factor NF-AT and moves it from the cytoplasm into the nucleus. Dephosphorylation also increases the binding ability of NF-AT to D N A and other transcription factors such as AP-1, N F - K B and Oct-1, thus facilitates the transcriptional activation of the IL-2 gene and other genes associated with T cell activation (160). Inositol 1,4,5-trisphosphate is also rapidly converted into a large number of other inositol phosphate species by the action of kinases and phosphatases. It is not known if these other inositol phosphate isomers also have signaling functions. 28 It has been described that CD28 regulates the tyrosine phosphorylation of PLC-y, Ptdlns hydrolysis and the cytosolic free calcium concentration (106). However, the relative ability of CD28 to elicit these responses appears to depend on the type of T cell model used. Some groups have reported that CD28 cross -linking with mAb is required for eliciting PLC activity in purified resting T cells and activated T cells (121). It is possible that the proximal pro line-rich region and /or the Tyr 1 8 8 and Tyr 1 9 1 residues in the cytoplasmic domain of CD28 molecule are involved in regulating the coupling of CD28 to C a 2 + signaling. However, the function of B7.1 and B7.2 in reducing C a 2 + signaling is very controversial. ii) Protein kinase C (PKC) pathways Protein kinase C (PKC) comprises a large family of serine/threonine kinases that have been important for several signaling pathways. At least 12 members have been identified to date. A l l P K C isoenzymes exist as a single polypeptide with two domains - an N -terminal regulatory region (20 - 40 kDa) and a C-terminal catalytic domain (~ 45 kDa). These two domains are linked by a flexible hinge region that may be proteolysed when the enzyme becomes membrane-bound. The catalytic domain contains binding sites for the nucleotide phosphate donor, ATP, and the protein phosphate acceptor. The regulatory domain contains binding sites for the various activators of the different P K C isoenzymes. PKCs have been classified into three families according to their cofactor requirements. The conventional PKCs (cPKCs) PKC-a , -01, -£2, -y, are activated by Ca 2 + , diacylglycerol (DAG) and other Ptdlns (4,5)P2-derived second messenger from 29 activation of PLC. These subspecies feature four sequence-conserved regions (C1-C4) and five variable regions (V1-V5). Novel PKCs (nPKCs) PKC-8, -s, -n, and -9, are distinguishable from the cPKCs in that they lack a functional C2 domain which allows them to function in a Ca2+-independent manner. nPKCs are optimally activated by combinations of lipids. Phosphatidylserine (PS) can activate nPKC in the presence of DAG/phorbol ester, and free fatty acids (FFA) can synergistically activate certain subspecies of nPKC (144). Atypical P K C (aPKC) P K C C, i , X, and p can be partially activated by PS, but DAG/phorbol ester and C a 2 + have no effect. These subspecies typically feature only one cysteine-rich repeat in C l region and do not exhibit phorbol ester binding ability. The tissue distribution of several P K C isoforms has been studied. Some subspecies of PKCs, including a, p i , P2, 8, and are expressed ubiquitously. PKCy is found only in central nervous system, PKCn is strongly expressed in skin and lung, and PKC9 is highly expressed in lymphocytes, thymus and skeletal muscle. P K C s is highly expressed in brain, and detectable in various other tissues. Following ribosomal synthesis, phosphorylation of P K C is required to prime the enzyme for activation. By binding to the second messengers such as C a 2 + or D A G , P K C isoenzymes are activated and their affinity to cell membrane has been greatly increased. The activation is usually accompanied by translocation of these enzymes from the cytosol to the membrane fraction of cells (144). 30 P K C plays a key role in T cell activation. There were observations that phorbol esters were able to induce sustained translocation of P K C in T cells and correlated with induction of IL-2 and IL-2 receptor expression (161). Anti-TCR/CD3 antibodies could also induce redistribution of different P K C isoenzymes in T cells (162-164). Studies have shown that many P K C isoenzymes could activate M A P kinase and stress - activated protein kinase pathways. Some isoenzymes also regulated the activity of transcription factors NF-AT and N F - K B (165, 166). The role of P K C activation in CD28 signaling is also unclear, since CD28 activation of Jurkat cells is not associated with P K C translocation, although levels of diacylglycerol are elevated following CD28 ligation by mAbs (167). Moreover, P K C inhibitors have been reported to either inhibit (167) or have no effect (168) on CD28-dependent IL-2 production. i i i) p21ras and M A P K pathways Ras plays a critical role in cytokine gene expression, particularly activation of the IL-2 promoter and T-cell proliferation (169). TCR ligation leads to a rapid accumulation of the active GTP-bound form of p21ras in the vicinity of the T-cell membrane (170, 171). The Ras guanine nucleotide binding cycle is controlled by the counter regulatory effects of guanine nucleotide exchange factors (GEFs) and GTPase - activating proteins (GAPs). GEF promote the activation of Ras by promoting the release of GDP, allowing GTP to bind in its place (172). GTPase - activating proteins stimulate the intrinsic GTPase 31 activity of Ras, thereby resulting in GTP hydrolysis and inactivation of Ras (172). Although there are several possible avenues for Ras activation in T cells, a well-characterized pathway is the involvement of the adapter protein growth factor receptor-bound protein (Grb2) and Son of Sevenless (Sos), a GEF (173). In this assembly the Grb2-SH2 domain is recruited to a consensus phosphotyrosine motif on L A T , whereas Grb2-SH3 domains bind to proline-rich regions in Sos (174). It has been suggested that Ras activation by phorbol esters involved P K C through the Ras guanyl releasing protein (RasGRP). RasGRP belongs to the CDC25 class of guanyl nucleotide exchange factors. One interesting feature of RasGRP is the presence of a C-terminal C l domain, which has high homology to the P K C C l domain and binds to diacylglycerol (DAG) and phorbol esters (175). It has been shown in B cells that the inhibition of P K C activity attenuated Ras activation and this attenuation correlated with an inhibition of RasGRP3 phosphorylation. RasGRP3 was phosphorylated in vitro by PKC-theta and PKC-beta2 and a dominant-activated mutant of PKC-theta phosphorylated RasGRP3 and enhanced Ras-ERK signaling (176). Norment et al also reported that RasGRPI was required for the efficient production of both CD4 and CD8 positive thymocytes under TCR stimulation (177). Once activated, Ras couples to multiple effector pathways, including activation of the mitogen-activated protein kinases (MAPK), also called E R K cascade (775, 179). There are over 50 diverse extracellular stimuli that have been shown to elicit activation of E R K cascade including engagement of antigen receptors, cytokine receptors and G protein coupled receptors. At lease four members have been identified in the M A P kinase 32 family, the best studied of which are p42 m a p k (ERK2) and p44 m a p k (ERK1). Phosphorylation on threonine and tyrosine residues is required for maximal activation of ERK1 and ERK2. Both of these modification are carried out by a dual-specificity (serine/threonine/tyrosine) kinase called M A P kinase kinase or M E K (for M A P or E R K kinase) (180). There are multiple pathways lead to the activation of M E K such as P K C phosphorylates and activates M E K kinase. p21 ras interacts directly with the serine-threonine kinase M A P 3 K (Raf-1), which is activated in a complex fashion at the surface membrane (179). Other kinases which have been reported to directly phosphorylate and activate Mek are Mos, Mekk and an insulin-activated 56-kDa M A P kinase kinase kinase (181). ERKs play an essential role in the expression of transcription factor AP-1, c-Fos, as well as c-myc (181). c-Fos is involved in transcriptional regulation of AP-1 response elements in the IL-2 promoter (139). This pathway is necessary, yet by itself not sufficient for induction of NF-AT:AP-1 transcription factor activity in T cells. Another parallel pathway implicated in NF-AT:AP-1 activation involves ras activation of the ras-related GTPase Rac-1. One potential pathway through which Rac-1 might increase transcription of Fos and Jun family members which form AP-1 is through its ability to regulate a kinase cascade phosphorylating Elk-1. Elk-1 is one of a family of proteins that can form a ternary complex with the transcriptional activator serum response factor (SRF). And Elk-1:SRF complexes are necessary and sufficient mediators of c-fos serum response element induction. 33 The CD28 p Y M N M site binds to the GRB-2-SOS complex via an SH2 domain (182). Anti-CD28 mAbs can activate the p21 ras pathway via the induction of RAS- GTP complexes and via the phosphorylation of Vav and the Grb-2/Sos-associated protein and activate ERK2 (755) In addition to the E R K cascade, the p38 M A P K and JNK cascades play a role in TCR signaling (757, 184, 185). In contrast to abundant E R K expression, the JNK1 and JNK2 isoforms are present in low quantities in primary T cells and require prior TCR engagement for their expression (186). Subsequent activation of JNK and p38 M A P K is CD28 dependent. JNK is required for activation of the IL-2 promoter, particularly the CD28 response element (CD28RE) (184, 185). In murine studies it has been shown that the major target of the JNK2 isoform is the U N - y gene and that knockout of JNK2 impairs TH1 development (187). In contrast, the major role of JNK1 is interference in TH2 development, as exemplified by increased IL-4 and IL-5 production in JNK1 knockout mice (755). This effect may be explained by the ability of JNK1 to enhance the nuclear export of NF-ATc, which is required for the activation of the IL-4 promoter (755). Similarly, the p38 M A P K cascade regulates IFN- y gene expression in TH1 cells but apparently does not affect TH2 cytokines (757). Resent research also indicated the role of p38 M A P K cascade in the induction of apoptosis in various T cells models (757, 759), though it might be controversial in other cell types. 34 iv) P I 3 - kinase ( P I 3 K ) pathways In addition to PLC-yl activation, PTKs affect PI turnover through the involvement of PI3-kinase (190). Three PI3K classes have been defined on the basis of their primary structure, regulation and in vitro lipid substrate specificity. The forms biochemically linked to lymphocyte activation are the class 1 PI3Ks. It comprises of a catalytic subunit with a molecular weight of 110 kDa (pi 10) and an adaptor regulatory subunit. There are three adapter subunits, p85a, p85p and p85y. The key to PI3K regulation is recruitment of the p i 10 subunit to the plasma membrane (191) where its phospholipid substrates are located. This is mediated via the SH2 domain of the adapter subunit p85. Activation of PI3-kinase in T lymphocytes was shown to occur following TCR engagement (190), costimulation by CD28 as well as mitogenic signals generated by cytokines such as IL-2 (190, 192). In TCR engagement, p85 subunit of PI3-kinase links with L A T , Src or other TCR -interacting adapters (193, 194). The p85 subunit has two SH2 domains which are able to bind to a core phosphotyrosine residue and adjacent residues within the p Y M N M motif of CD28. In this way, CD28 anchors the enzyme to the inner face of the plasma membrane, where it can act on target substrates (105, 195, 196). The p i 10 subunit is able to phosphorylate the D-3 position of the inositol ring, thereby converting PI, PI-4-P and PI-4,5-P2 to PI-3-P, PI-3,4-P2 and PI-3,4,5-P3, respectively (197). The PI-3-P binds to intracellular proteins such as Fablp, Y O T B , Vaclp and 35 early endosome antigen 1 (EEA1) which contain cysteine-rich, zinc-finger-like motif F Y V E and facilitates intracellular vesicular trafficking (198). PI-3,4-P2 and PI-3,4,5-P3 interact with the pleckstrin homology (PH) domains of protein tyrosine kinase families such as Tec, Btk as well as Vav and PLCy (199). PI3-kinase also exhibits serine kinase activity. A key serine/threonine kinase that mediates PI3K action is the 67 kDa, ubiquitously expressed phosphoinositide-dependent protein kinase 1 (PDK1). PI-3 kinase is also responsible for the recruitment and activation of serine- threonine kinases such as protein kinase B (PKB or Akt) through PDK1 (200). P K B - appears to lie upstream of another kinase called FRAP (FKBP12-rapamycin-associated protein). One of the down stream target of FRAP is serine/threonine S6 kinase which phosphorylates the 31-kDa S6 protein in the 40 S ribosomal subunit. S6 protein has been linked with protein synthesis and cell survival. v) N F - K B activation N F - K B is a transcription factor that plays an important role in IL-2 and IL-2 R a chain gene transcription. When activated, N F - K B binds specific D N A sequences in the promoter region upstream of IL-2 and IL-2 R a chain and activates transcription of these genes. N F - K B operates as a heterodimeric complex composed of members of the Rel/NF-K B family of polypeptides, including N F - K B 1 (p50/pl05), N F - K B 2 (p52/pl00), c-Rel, RelA/p50 and RelB/p65 (201). The primary mode of N F - K B activation involves modification of a pre-existing complex 36 rather than de novo synthesis. The modification process depends on a multisubunit 700 to 900-kd signaling complex called I K B kinases ( I K K ) (202, 203). I K K contains catalytic subunits of IKKCC, I K K P and a noncatalytic I K K subunit, IicKy (202-204). The activated I K K complex initiates the phosphorylation of the inhibitory proteins IKBCC and I K B P . I K B then becomes ubiquitinated and degraded by the proteasome. This leads to the release of N F - K B transcription factors, which are sequestered in the cytosol by IKBCC and I K B P . These factors enter the nucleus and initiate transcriptional activation of genes involved in cellular proliferation and survival (202-204). The signal which initiates N F - K B activation following antigen-specific T-cell activation is probably provided by the costimulatory molecule CD28 (205). The mechanism by which CD28 ligation induces N F - K B activation is not well understood but probably involves the activation of one or more tyrosine-specific protein kinases and the subsequent activation of phospholipase A2 and D5-lipoxygenase (206). The products of the action of these enzymes generate reactive oxygen species (ROS) which activate N F -K B , and in primary T lymphocytes, ligation of CD28 results in the rapid formation of ROS. The signals provided by CD28, including ROS, induce a rapid degradation of I K B molecules (207). The precise mechanism for this has not been elucidated but appears to involve a signal (ROS?)- induced site-specific phosphorylation by an as yet incompletely characterized serine/threonine protein kinase termed CFIUK (204), which appears to target I K B for covalent addition of multiple copies of the ubiquitin polypeptide. 37 One of the key gene target is the CD28RE in the IL-2 promoter (208) This is a combinatorial response element that requires c-Rel, as well as AP-1, transcription factors for full activity (208, 209). These costimulatory requirements are in excellent agreement with the key role of CD28 in IL-2 production in unprimed T cells. Another important target for the CD28 - N F - K B pathway is the Bcl-xL promoter, which expresses an N F - K B response element 860 bp upstream of the start site (128). Increased Bcl-xL expression plays a critical role in cellular survival during CD28 costimulation (119). The exact mechanism initiating the N F - K B cascade by TCR ligation is still unknown. It is dependent on the involvement of Vav and assembly of the cytoskeleton. PKC9 contributes to the activation of the I K K complex through its ability to phosphorylate hcKp (210-212). Interestingly, pharmacological interference in PKC0 activity abrogates N F - K B activation and IL-2 production (210). Based on inhibition by cyclosporine A and on transfection studies, the C a 2 + dependent phosphatase calcineurin is involved in the activation of N F - K B in T cells. However, the target of calcineurin is unknown (213). vi) Stat activation Stat (Signal Transducer and Activator of Transcription) proteins are important mediators of signaling by cytokine receptors (214). Recently, it has suggested that Stat proteins play a role in signaling through the TCR (215, 216). A transient phosphorylation of Stat5 by the TCR^ chain was observed after TCR crosslinking, as was phosphorylation of TCR<^ chain bound Stat5 by Lck (215). Furthermore, antigen receptor ligation induces 38 delayed but sustained phosphorylation of Statl on Ser727, which is dependent on phosphotidylinositol-3-kinase mediated signals (216). Although the ultimate function of the Statl and Stat5 involvement in T-cell activation remains unclear, it raises the possibility of cross-talk between antigen receptors and signaling components of cytokine receptors. vii) The sphingomyelinase /ceramide pathway Ceramide is generated intracellularly either by TCR-induced synthesis or by acid sphingomyelinase (aSmase)-mediated breakdown of sphingomyelin induced by CD28 or Fas triggering (217). It serves as a second messenger, and may activate downstream targets such as PKC<^ (218, 219), serine/threonine-specific protein kinases (220) and phosphatases (221). Ceramide is interconnected with T-cell biology at multiple levels. High levels of ceramide result in caspase-dependent TCR down-regulation. Prolonged treatment of T cells with exogenous ceramide or sphingomyelinase inhibits T-cell proliferation, Whereas low levels of ceramide result in TCR up-regulation (217). Ceramide can be generated as a result of Fas activation (222, 223), followed by inducing activation of a stress activated protein kinase (SAPK). Both Fas and S A P K play a key role in the control of apoptosis in lymphocytes. There are evidences that several 39 proteases may be involved in this process. CED-3 protein, a precursor of caspases, shows homology to the interleukin-ip-converting enzyme (ICE, also known as caspase 1) family, all of which are cysteine proteases (224). Activating Fas by binding to its ligand such as FasL leads to the recruitment of an adapter molecule, Fas-associated death domain protein (FADD), through the interface of complimentary regions on Fas and F A D D known as death domains (DDs) (225). The other end of F A D D contains a death effector domain (DED) that interacts with the DEDs on procaspase 8, drawing it into this complex which is known as the death-inducing signaling complex (DISC) (226). FasL-induced signal results in the activation of large amounts of caspase 8 by the DISC (227). Caspase 8 is then capable of activating downstream effector caspases such as caspases 3 and 7, leading to apoptosis (228). Recent studies suggest that caspases regulate the release of ceramide, which has been shown to play a positive role in apoptosis (229). Short-term and transient ceramide release by CD28-mediated aSmase stimulation induces T-cell proliferation (230). It has been shown that anti-CD28 mAbs induce the activation of acidic sphingomyelinase (231, 232). Moreover, this pathway seems to be required for costimulation since the cell permeable ceramide analogue C-6-cerainide mimicked the CD28 signal by inducing T cell proliferation and IL-2 gene transcription (232). The ability of CD28 to potentially activate both the PI3 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 (233), which lack any known PI3-kinase consensus binding motif. Thus, there may be synergy between the CD28- activated PBkinase and ceramide -mediated signaling pathways in protecting the cell from apoptosis and promoting cell survival and or IL-2 production. 40 Figure 1.2.1: T C e l l Act ivat ion Signaling Networks Signal 1 Signal 2 Signal 3 41 1.3 Blocking T lymphocyte activation Understanding of the key elements of TCR signaling pathways has led to the development of novel immunomodulators and promise to deliver new forms of immunotherapy for allergic diseases, autoimmunity, malignancy, and transplant rejection. These agents can be classified into different groups based on their targeting the TCR/CD3 complex, costimulatory receptors, or individual signaling components. It is important to consider that not all signaling components contribute equally to TCR signaling and that a considerable degree of redundancy exists in these pathways. The relative contribution of individual TCR pathways to global signal transduction has been addressed by calculating the amount of energy that is consumed by each pathway during mitogenic stimulation (234). With concanavalin A as the stimulus, it was calculated that 84% of the energy was spent on the PTK/PLC-yl /PKC pathway (234) This could be further subdivided into energy expenditures of 30% and 54%, respectively, toward the PLC-yl [Ca2 +]i/calcineurin and P K C pathways. Activation of mitogen-activated protein (MAP) kinase cascades was included in the PKC pathway and required 40% of the total energy expenditure. The remaining 16% of the energy was required for processes that could not be measured by the energetic approach, including assembly of macromolecular complexes (234). 42 1.3.1 Blocking T cell activation using cell surface antibodies Polyclonal and monoclonal antibodies have been developed against key signalling molecules on lymphocyte surface. Antithymocyte globulin is a purified polyclonal immunoglobulin prepared from hyperimmune serum of horse, rabbit, sheep, or goat after immunization with human thymic lymphocytes. Intravenous administration results in binding to the surface of circulating T lymphocytes, resulting in lymphopenia and profound suppression of cellular immune responses. Major toxicity includes serum sickness and arthralgia. It is used for the prevention and treatment of acute rejection of solid organ renal and cardiac transplants (235). Muromonab-CD3 (OKT3) is a monoclonal antibody against CD3 molecules. Because of the unique association between T cell receptor and CD3, OKT3 blocks TCR signaling, inhibits both the generation and function of cytotoxic T cells (236). OKT3 is approved for the treatment of acute allograft rejection in renal transplant. Most patients experience acute clinical syndrome (cytokine release syndrome) associated with the initial dose of OKT3. Manifestations range from a flu-like illness to capillary leak, hypotension, and multiorgan failure (237). CTLA-4Ig is an experimental monoclonal antibody, which shows promising results in preventing allograft rejection in animals. It blocks CD28 engagement by CD80/CD86 and induces anergy and long term tolerance by interfering in delivery of signal (238-240). Anti-CD4 monoclonal antibody (Keliximab) is designed to bind with CD4 molecule on 43 T lymphocytes and causes T cell depletion. It has been used to prevent graft-versus-host disease during human bone marrow transplantation (240). Basiuxiab and Daclizumab are monoclonal antibodies targeting the high-affinity interleukin-2 receptor that is expressed on activated T cells. They bind specifically to the a chain of the interleukin-2 receptor, thereby inhibiting interleukin-2 signalling. Both are now used clinically and have reduced the rate of acute rejection (241). 1.3.2 Src kinase inhibitor Brockdorff et al reported that pyrimidine derivatives PP1 and PP2 reduced IL-2 synthesis and T cell proliferation by inhibiting Src kinase activity (242). Given the key role of Lck in TCR activation, it is possible that agents that target Src PTKs might have been used as immunosuppressants in vivo. 1.3.3 Ca2+/calcineurin pathway inhibitors - Cyclosporine and FK506 The Ca2+/calcineurin inhibitors are the most widely used immunosuppressive agents in the transplantation clinic. The two most important members of this family are cyclosporine (CsA) and tacrolimus (FK506). Both inhibit T cell activation initiated by specific antigen. The initial targets of cyclosporine and tacrolimus are a group of intracellular proteins known as immunophilins. Immunophilins have the characters of strong binding affinity for natural product immunosuppressants and the ability to 44 catalyze the cis-trans isomerisation of cis-proline residues in proteins and peptides (PPIase activity). Cyclosporine targeted immunophilins are called cyclophilins. There are three isoforms of cyclophilins - cyclophilin A16 (CyPA), cyclophilin B17 (CyPB) and cyclophilin C18 (CyPC). Tacrolimus targeted immunophilins are called F K binding proteins (FKBP). There are many isoforms of F K B P such as FKBP12, 14 and 15 etc (243). After forming the complex with immunophilins, cyclosporine and tacrolimus interact with calcineurin, a calcium, calmodulin-dependent phosphatase. Calcineurin contains two subunits - calcineurin A and B. Calcineurin A has a catalytic domain and an auto inhibitory domain. When binding with protein calmodulin, together with the binding of C a 2 + to the calcineurin B subunit, the auto inhibitory domain is displaced and calcineurin A is activated (244). Cyclosporine and tacrolimus block the activation process of calcineurin A and therefore inhibit the dephosphorylation of the transcription factor NF-AT and prevent its migration from the cytosol into the nucleus. Thus they block the transcription of multiple genes, including the genes coding for the key cytokines such as the T-cell growth factor interleukin 2 (IL-2) (245). 1.3.4 Protein kinase C (PKC) inhibitors There are three areas on the P K C molecule which are obvious targets for rational drug design - the regulatory domain, the protein substrate binding region and the ATP-binding domain. The regulatory domain of PKC would appear to offer the best target for the design of a selective inhibitor of PKC since this region is unique to certain P K C isoenzymes and is not present in other serine:threonine kinases. However, greatest 45 progress has been made in the ATP binding site to design selective P K C inhibitors. Three nonselective ATP-competitive compounds have been used in isolated enzyme and cellular studies as inhibitors of PKC. They are the indolocarbazoles staurosporine, K252a, and the isoquinoline sulphonamide H7. Staurosporine and K252a are indeed potent P K C inhibitors and inhibit the enzyme in cellular systems, but these compounds are far from specific for PKC. Staurosporine has been shown to act as a broad inhibitor of serine:threonine kinases and to block both receptor and Src-family tyrosine-specific protein kinase activity (246). K252a exhibits a similar profile and has also shown to have some selectivity for the Ca.calmodulin-dependent phosphorylate kinase (247). H7 is a broad serine:threonine kinase inhibitor and is only weakly active against P K C in vitro (248). The newly designed bis-indolylmaleimides family such as Ro 31-8425 and Ro 32-0432 are more selective for P K C than for other serine: threonine kinases and are used widely as potent P K C inhibitors. Blocking P K C pathways had many influences on T cell activation. For instance, Ro 31-8425 and Ro 32-0432 inhibit the mixed lymphocyte reaction and T-cell proliferation in response to specific antigen in T cell clones (246). Another P K C inhibitor, the indolocarbazole CGP 41251, inhibited antigen-induced proliferation of human peripheral blood lymphocytes and murine Thl and Th2 clones equally well (249). There is also evidence that these PKC inhibitors block T-cell-mediated responses in vivo. Orally taken of Ro32-0432 could reduced T cell mediated inflammatory responses caused by injection of phorbol aster or adjuvant (Mycobacterium tuberculosis in liquid 46 paraffin) in mouse models (246). In a murine graft-vs-host model (250), both cyclosporine and Ro32-0432 significantly inhibited host immune response measured by the weight reduction of host lymph nodes. Other studies also demonstrated that Ro 32-0432 was able to prevent the activation of T lymphocytes in experimental allergic encephalomyelitis (EAE) (N. Lad et al., unpublished observations). It has also been reported that a nPKC inhibitor Rottlerin interferes with NF- K B activation and IL-2 production (210). More research needs to be done on its function to T cell activation. 1.3.5 MAP kinase cascade inhibitors Activation of mitogen-activated protein (MAP) kinase cascades required 40% of the total energy expenditure on T lymphocyte activation. E R K kinase (MEK), JNK and p38 are the three key elements which have been targeted by their specific inhibitors to block T cell activation. Some of these inhibitors have been tested as experimental drugs for immunosuppression. DeSilve and Jones reported that blocking M E K with its specific inhibitor U0126 could significantly reduced the proliferation and IL-2 production of mouse CD4 + T cell lines stimulated by APC or Con A. However, the expression of IL-2 receptor a chain and IL-2 induced cell proliferation were not affected. Blocking M E K did not induce cell anergy nor did it affect the induction of cell anergy by TCR crosslinking (251). Another M E K inhibitor PD98059 has been reported to block the production of other cytokines such as IL-3, 4, 5, 10, GM-CSF and INF- y (252). Jun N -terminal kinase (JNK) is a stress-activated protein kinase that can be induced by 47 inflammatory cytokines, bacterial endotoxin, osmotic shock, U V radiation, and hypoxia. SP600125 is a reversible ATP competitive inhibitor with >20-fold selectivity against a range of kinases and enzymes tested. Blocking Jun kinase (JNK) activity using inhibitor SP600125 also reduce IL-2 production through diminishing the activity of the transcription factor AP-1 and through influencing the N F - K B cascade at the level of I K B kinase p (127). Bennett and Sasaki showed that in peripheral human T cells, SP600125 dose -dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-y, TNF-a, and prevented the activation and differentiation of primary human CD4 + cell cultures. In animal studies, SP600125 blocked bacterial lipopolysaccharide induced expression of TNF-a and inhibited anti-CD3-induced apoptosis of CD4 + CD8 + thymocytes (253). P38 M A P K is another kinase activated during T cell activation. In the mouse primary T cell model, p38 M A P K was activated strongly and synergistically by either CD3/CD28 coligation or P M A / C a 2 + ionophore stimulation and correlated closely with T cell proliferation (254). However, there were controversial results of blocking p38MAPK on cytokine production. Zhang et al reported that mouse primary T cell proliferation and production of IL-2, IL-4, and IFN-y as well as the nuclear expression of the c-Jun and ATF-2 proteins were each blocked by the p38 M A P K inhibitor SB203580 (254). Using human peripheral CD4 + T cells, Koprak et al found that SB203580 had little effect on IL-2 production but strongly reduced the production and mRNA expression of IL-4, 5, 10, 13 and TNF-a (255). An orally active p38 M A P kinase inhibitor, RWJ 67657, has been used successfully to suppress endotoxin induced clinical effects (eg, fever) and cytokine (TNF-a, IL-8, and IL-6) production in human volunteers (256) Moreover, p38 M A P kinase inhibitors exert anti inflammatory effects in the 48 murine lung (257). E R K and J N K inhibitors interfere in I L - 2 production and might be useful to limit clonal expansion of activated T cells in vivo. 1.3.6 Blocking N F - K B signaling cascades Glucocorticoids (eg, prednisone) have an effect on N F - K B signaling cascades and are widely used as anti inflammatory and immunosuppressive agents. Although the major anti-inflammatory and immunosuppressive actions of glucocorticoids involve their ability to complex to steroid receptors that inhibit proinflammatory genes, occupied steroid receptors also interfere directly in the function of N F - K B and AP-1 transcription factors (258, 259). In addition, corticosteroids induce the expression of IKBOC, which prevents the intranuclear translocation of the N F - K B transcription factor p65 (260). This leads to decreased activation of NF-KB-dependent genes. Similarly, the nonsteroidal anti-inflammatory drugs aspirin and sodium salicylate are agents that exert inhibitory effects on the N F - K B pathway, although this is not the major mechanism of action of these drugs. More specifically, these drugs interfere in the phosphorylation of IKBCC through inhibition of the upstream kinase I K B kinase p (261). The use of specific drug inhibitors to interfere in the N F - K B cascades is still experimental and has not been used for immunomodulation in vivo, it has been demonstrated that cyclopentenone prostaglandins (eg, PGA and 15- deoxyprostaglandin J2) act as potent inhibitors of I K B kinase P and these lipid inhibitors, as well as sodium salicylate, can be 49 used to induce apoptosis in antigen-responsive T cells (128). Apoptosis is likely the result of interference in Bcl-xL expression, which is dependent on the N F - K B pathway (128). IL-10 inhibits the transcription of IL-2 as well as other cytokine genes associated with Thl cell responses. It was shown that IL-10 inhibited the induction of N F - K B by LPS in human P B M C (262). 1.3.7 Blocking IL-2 induced T ceil activation IL-2 is one of the key cytokines in T cell activation. After binding to its high affinity cell surface receptor, it evokes another series of interaction among cytosolic proteins and leads to cell cycle progression from the GI phase to S phase, cell proliferation and clonal expansion. Sirolimus (rapamycin), like CsA and tacrolimus, is a prodrug that complexes with an immunophilin to exert its immunosuppressive effects (263). Although sirolimus binds to FKBP-12, this drug-immunophilin complex interacts with the target of rapamycin (TOR) and forms a FKBP: R A P A : mTOR complex (264). TOR is a kinase-like protein that regulates enzymatic processes involved in Gl-to-S phase cell-cycle transition. The complex inhibits the mitogen-induced activation of p70 S6 kinase and its nuclear isoform p85 S6 kinase in activated cells. Therefore the major effect of sirolimus is to interfere in IL-2-and IL-4—induced T- and B-cell proliferation, rather than directly targeting TCR activation events (264). This agent is used as a maintenance immunosuppressant or as adjuvant therapy to calcineurin inhibitors. Because sirolimus has a relatively low index 50 of toxicity, its combination with CsA lowers the immunosuppressive dose of the latter drug, and this leads to a decrease in CsA toxicity (263). To this point, all of the kinases examined to block T lymphocyte activation are ubiquitously expressed in human tissues. For example, serine/threonine phosphatase calcineurin is expressed in neurons, cardiac and skeletal muscles, as well as in lymphoid tissue. Therefore, inhibiting calcineurin activity by cyclosporine and FK506 induces toxicities affecting renal, neural and hepatic tissues and causes sevious side effects in posttransplant patients. Future study needs to be focused on the molecules that are more selectively expressed in lymphoid tissue such as Jak3 and ZAP-70. 1.3.8 Induction of tolerance Tolerance is an actively maintained state of nonresponsiveness to a specific antigen in a human or an animal exposed to that antigen (265). It can be achieved in nature as central tolerance and peripheral tolerance. Central tolerance is the clonal deletion of activating lymphocytes to a specific antigen in the central lymphoid organs such as thymus and bone marrow. It is hard to achieve central tolerance in solid organ transplantation. Many efforts have been focused on achieving peripheral tolerance in transplantation. Peripheral tolerance is the nonresponsiveness of the mature lymphocytes in the peripheral lymphoid tissues to a certain antigen. The in vitro definition of peripheral tolerance, for which the term "anergy" is used, is defined as the inability of individual T cells or T-cell clones to produce IL-2 and proliferate on restimulation with an appropriate antigen and an APC 51 (266). Anergy can be induced in naive helper T cells during antigen occupancy in the absence of CD28 costimulation and is preventable by means of TCR/CD28 costimulation. This dual signaling requirement to prevent anergy is the cornerstone of the two-signal hypothesis of T-cell activation. The two signal concept is also applicable to in vivo immune modulation, as demonstrated by the fact that blockade of CD28 costimulation by a competitive ligand, CTLA-4Ig, can induce a tolerant state to bone marrow and solid organs (267). It is important to point out, however, that the correlation of in vitro anergy to in vivo immune tolerance is not clear as yet. Given the clinical importance of tolerance to the treatment of transplant rejection, autoimmunity, and allergy, there has been an intense focus on the biochemical events leading to or maintaining the anergic state. Although a lot remains to be learned about changes in TCR signaling in tolerant T cells, a number of studies show that anergic T cells exhibit poor phosphorylation of the TCR- i\ and TCR- s chains and are incapable of activating lymphocyte-specific protein tyrosine kinase, ZAP-70, Ras and JNK (5, 268, 269). TCR also fails to activate AP-1 and NF-AT binding sites in the IL-2 promoter. However, anergic T cells maintain their capacity to induce PLC-yl phosphorylation and [Ca2+]j flux, along with elevated inositol trisphosphate (IP3) levels and the phosphorylation and activation of CREB. These suggest that other kinases might be involved in C R E B / C R E M regulators in anergic T cells (270) It is interesting that the phenotypically distinct CD4 + /CD25 + T-cell subset, which is responsible for the maintenance of in vivo immune tolerance, exhibits constitutive 52 CTLA-4 expression (277). Cross-linking of CTLA-4 induces secretion of TGF-b\ which provides a possible explanation for the immunoregulatory capabilities of this cellular subset (272). Much remains to be learned, however, about the role of CTLA-4 and the mechanism of action of the CD4 + /CD25 + T-cell subset. 53 1.4 Hypothesis and objective T lymphocyte activation by donor antigen plays a key role in transplantation rejection. Signal 1 occurs through donor antigen binding with T cell receptor (TCR). Signal 2 occurs through costimulatory molecules such as CD28, and this can modulate TCR signals and augment the expression of essential proteins such as IL-2 and the IL-2 receptor a chain (CD25). Multiple signaling pathways transduce the message from the TCR and the CD28 molecules to initiate the gene transcription of IL-2 and IL-2 receptor a chain. The most important pathways known to date are the C a 2 + - calcineurin, protein kinase C (PKC), mitogen activated protein (MAP) kinase and the phosphatidyl inositol 3 kinase (PI3-kinase) pathways. Each of these contains several key kinases that can be activated in sequence. Together, these pathways form a complicated signaling network regulating T lymphocyte activation. Blocking recipient T lymphocyte activation can prevent graft rejection and increase graft survival and patient survival. This can be achieved by blocking one of the signaling pathways using specific kinase inhibitors. This theory has been proved by the successful clinical use of the specific calcineurin inhibitors cyclosporine and tacrolimus. The importance of other signaling pathways to T lymphocyte activation is now being investigated. Several studies have shown suppression of T lymphocyte proliferation by 54 the specific kinase inhibitors to the PKC, M A P kinase and PI3-kinase pathways. But most of these studies used murine or human T cell clones, in which the signaling pathways downstream of the TCR and CD28 molecules may have been altered and may not reflect the natural process of T lymphocyte activation. Some of these studies have also reported the inhibition of cytokine production, but few have demonstrated the influence on cytokine receptor expression and none of them have shown the inhibitor's effect on cytokine and cytokine receptor gene transcription at transcription factor level. We hypothesize in this study that: 1. Blocking M E K / E R K and p38 M A P kinase in the M A P kinase pathway or blocking the P K C pathway and PI3-kinase pathways by specific kinase inhibitors will inhibit human primary T cell activation. 2. This inhibition will be evident at the level of IL-2 production and IL-2 receptor a chain (CD25) expression and achieved by reducing the binding activity of the key transcription factors to their gene promoter region. 3. Combination of inhibitor of the CD28 and TCR pathways will increase the suppression of T cell proliferation. Novel immunosuppressant could be developed from these or related inhibitors tp reduce transplant rejection. The objective of this study was to block the P K C pathway, M A P kinase pathway and PI3-kinase pathway to T lymphocyte activation in primary human CD4 + T cells. First, we established a cell model in vitro to mimic the T lymphocyte activation process in graft rejection. This was achieved by stimulation of isolated human peripheral CD4 + T 55 lymphocytes through the T cell receptor and the costimulatory molecule CD28. The time courses of T lymphocyte proliferation, IL-2 secretion and IL-2 receptor a chain expression were established in this model. Specific inhibitors to P K C , PI3-kinase and to the key molecules in M A P kinase pathways - M E K and p38 M A P kinase, were used to check the effects of blocking these pathways on T lymphocyte proliferation, IL-2 secretion and expression of IL-2 receptor a chain. The specific inhibitors used in the investigation were P K C inhibitor Bisindolylmaleimide I, M E K inhibitor U0126, p38 M A P kinase inhibitor SB203580 and PI3-kinase inhibitor LY294002. Time effects and dose effects of these inhibitors were also checked. Furthermore, the specific regulatory changes at the NF-AT and AP-1 binding sites of the IL-2 promoter region, as well as the NF- K B and STAT5 binding sites of the IL-2 receptor a chain promoter region, were analyzed. Finally, combination effects of these inhibitors on T lymphocyte proliferation were examined. The ultimate goal was to examine new therapeutic targets within T lymphocyte signaling pathways and provide information for designing new immunosuppressive medications in the future. 56 CHAPTER TWO: MATERIALS AND METHODS 2.1 Solutions Cell culture medium RPMI 1640 medium (Irvine Scientific, CA) 10% FCS(lug/ml) 3% glutamine 3%> penicillin/streptomycin -Stored at 4°C Propidium Iodide (PI), Stock Solution Propidium Iodide 25mg ( sigma) PBS 50ml Dissolved and stored at 4°C Propidium Iodide (PD, Working Solution (1:10 dilution) PI stocked Solution 1ml 2% FCS-PBS-0.1 %NaN 3 9ml 57 7-Aminoactinomvcin D 20ug 2ml - Dissolved and stored at 4°C 7-AAD PBS Immunophenotyping Fixation Buffer, Stocked Solution 16% paraformaldehyde 100ml - Adjusted pH to 7.4-7.6 .- Aliquoted at 10ml quantities Stored at room temperature in the dark Immunophenotyping Fixation Buffer, Working Solution 16% paraformaldehyde 10ml PBS 150ml Stored at room temperature in the dark 58 Washing Solution 1 % heat inactivated FCS 1 Oml 0.1% sodium azide 1 Oml PBS 980ml Adjusted pH to 7.4-7.6 Thymidine ( 3rHl TdR\ Working Solution (1:50 dilution) thymidine 1ml sterile PBS 9ml - The bottle was labeled with radioactive tape and a label indicating 3[H] TdR concentration and date of preparation - Stored at 4°C 1.5% Agarose Gel agarose 1.2g 0.5X TBE 80ml - Boiled, stirring constantly Cooled to 50°C and 2.0ml ethidium bromide added 59 - Poured into a casting form, slot former ( 1mm ) inserted and cooled for 30 minutes - Poured in 0.5 X TBE to cover gel by 2mm, samples applied and electrophoresed Ammonium Acetate, Saturated - Added dH 2 0 to 500ml Stored at room temperature - Saturated solution - additional dH 2 0 added with a dropper until it went into solution without heating 10% Ammonium Persulfate 19.2 M N H 4 acetate* 740.0g ammonium persulfate lOOmg dH 2 0 1.0ml Made fresh weekly - Stored at 4°C 1% Ethidium Bromide ethidium bromide lO.Omg - d H 2 0 added to 1.0ml 60 - Stored at 4°C 0 . 1 M H C L Concentrated H C L 8.3ml - 800 ml dH 2 0 added, mixed and adjusted to 1.0L with dH 2 0 5X Loading Buffer bromophenol blue 50mM tris base 50nM Na 2 EDTA SDS sucrose - Tris and EDTA dissolved in 50ml dH20 - pH adjusted to 7.6 SDS dissolved, Bromophenol Blue, sucrose in Tris-EDTA Final volume adjusted to 100ml with d H 2 0 Stored at room temperature 0.10g 0.60g 1.68g 0.50g 40.0g I P X Loading Buffer 0.25% bromophenol blue 0.25g 25.0% ficoll type 400 25.0g 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 - Mixed until dissolved (sped up by mixing at 50°C for one hour with 40ml 10N NaOH added) - pH adjusted to 8.0 with NaOH dH 2 0 added to adjust for total volume of 1.0L and autoclaved or filter sterilized Aliquoted and stored frozen in 40ml quantities Na 2 EDTA 186.1g dH 2 0 800.0ml 62 O. lMNaOH NaOH 4.0g 1.0 d H 2 0 added and dissolved Stored at room temperature Nuclear Lysis Buffer lO.OmM Tris base 0.61g 400.0mMNacl 11.7g 2.0mM Na 2 EDTA 0.37g or 2.0ml of 0.5M Na2 EDTA stock - dH 2 0 added to 500ml - Stored at 4°C Protease K Stock Buffer 2.0mM Na2 EDTA 2.0ml ( 0.5M stock) 1.0% SDS (10% SDS stock) 50.0ml d H 2 0 448.0ml 63 Stored at 4°C or stored frozen in 50.0ml aliquots RBC Lysis Buffer 0 .144MNH 4 CL 15.2g 0.001M NaHCOs 0.17g - d H 2 0 added to 2.0L - Stored" at 4°C 10% SDS Stock SDS 50.0/100.0g - dH 2 0 added to 500ml/L 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 500ml of stock anhydrous citric acid ( 0.1M ) 64 Adjusted pH to 4.35 Aliquoted 11ml per vial and stored at -20°C I X TE 4.7mM Tris H C L 0.37g l.OmM Na 2 EDTA 1.0ml (0 .5M stock) - dH 2 0 added to 400ml - pH adjusted to 80 with 1 ON NaOH - dH 2 0 added to 500ml Autoclaved or filter sterilized Stored at room temperature 5 X T B E Tris base 54.0/108.0g Boric acid 27.0/54.0g Stock Na 2 EDTA (0.5M) 20.0/40.0ml - dH 2 0 added to 1.0/2.0L - pH adjusted to 8.3 - Stored at 4°C IPX TBE (Gel Buffer) 0.89M Tris base 215.6g 0.89 Boric acid llO.Og 0.02 Na 2 EDTA 80.0 ( from 0.5M stock) - d H 2 0 added to 2.0L - Stored at 4°C l M T r i s Tris base 60.9g - d H 2 0 added to 500ml - pH adjusted to 7.0 2.1.1 Oligonucleotides and E M S A Biotinylated dsDNA oligonucleotides were obtained from University Core D N A Services, University of Calgary, Canada. The following transcription factors of IL-2 and IL-2 R a chain derived oligonucleotides were used: 66 AP-1: 5' - T C G A G A A A T T C C A G A G A G T C A T C A G A A G A - 3' NFAT: 5' - T C G A A A G A G G A A A A T T T G T T T C A T A C A G A A G G -3' N F K B : 5 ' - A G T T G A G G G G A C T T T C C C A G G C - 3 ' STAT5: 5 ' - A G A T T T C T A G G A A T T C A A T C C - 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 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, Canada) Anti-CD28 anti-CD28 polyclonal antibody (Serotec, Kidlington, U K ) Anti-CD25 FITC conjugated anti-CD25 antibody (Becton Dickinson, California US) Anti-CD4 FITC conjugated anti-CD4 antibody (Becton Dickinson, California US) Control IgGi FITC conjugated mouse IgGi (Becton Dickinson, California) 2.1.3 Inhibitors (calBiochem, USA) LY294002 A cell-permeable, potent and specific 67 phosphatidylinositol 3 kinase (PI 3 kinase) inhibitor that acts on the ATP - binding site of the enzyme. U0126 A potent and specific inhibitor of M E K 1 and M E K 2 . SB203580 A highly specific, cell-permeable inhibitor of p38 kinase. Bisindolylmaleimide I A cell-permeable protein kinase inhibitor that is structurally similar to staurosporine, but is highly selective for the inhibition of protein kinase C. Cyclosporine A highly specific, cell-permeable inhibitor of calcineurin. A l l the inhibitors were dissolved in DMSO, aliquoted and stored in -20°C. Working solutions were made by diluting the stored solution with DMSO to the designed concentrations. 68 2.2 Methods 2.2.1 Cell culture A l l the procedures were performed in a sterile environment under a culture hood. i) Blood collection Used bags of fresh peripheral blood from healthy donors with E D T A anticoagulation were obtained from the Canadian 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) Peripheral blood mononuclear cells (PBMC) separation Whole blood was diluted with 1XPBS (1:1 volume dilution). A 50ml tube was filled with 15 ml of Ficoll-Hypaque and 30ml of diluted blood was carefully added to form a clear layer on top of Ficoll. The tube was then centrifuged at 1500 rpm for 30 min. The mononuclear cells layer (the top white layer) was carefully removed to another 50ml tube. The cells were washed by topping the tube with I X PBS, mixed and centrifuged at 1500 rpm for 10 min and decanted. After 3 washes, P B M C were resuspended in 1XPBS at concentration of 20X10 6/ml. 69 iii) C D 4 + T lymphocytes purification CD4 + T lymphocytes were purified from P B M C by negative selection using Lympho-Kwik (LK-50™, OneLambda). It contains complement, a density gradient and anti-RBC, anti-granulocyte, anti-monocyte, anti-B lymphocyte and anti-suppressor T lymphocyte monoclonal antibodies. P B M C were incubated in a 37°C water bath for 30 min. The tube was shaken every 10 min during the incubation to give a good interaction. After the incubation, 0.2 ml of 1XPBS was layered over the mixture and centrifuged for 2 min at 3500rpm. The interface and the supernatant were removed and the pellet was resuspended in 1XPBS. Cells were washed three times in PBS (centrifuged at 1500rpm for 5 min) and resuspended in cell culture media. The efficacy of purification was estimated by FACScan using FITC conjugated anti-CD4 monoclonal antibody and the purity was > 95%. iv) Cell activation and proliferation The method of activating human CD4 + T lymphocyte used in this study was mainly followed the procedure published by Baroga and ward (273, 274). However, cell concentration as well as the concentration of the stimulating antibodies was adjusted to maximize the effects of T cell activation and proliferation. In short, 96 well plates (Nunc) were coated with OKT3 (1 ug/ml) and anti-CD28 antibody (1 pg/ml) (Serotec) for 24 hours at 4°C. To activate the cells, antibody coated plates were washed 3 times with PBS. 200 pL of CD4 + T cells in RPMI-media (10% of FCS, 3% of glutamine and 3% of 70 penicillin/streptomycin) were added in each well at a concentration of 1 X 10° cells/ml. Cells were cultured in an incubator at 37°C with 5% CO2 and collected at different time periods. For measurement of proliferation, cells were pulsed with luCi/well [3H] thymidine (DuPont, Delaware) for 24 hrs. Cell proliferation was expressed as the mean cpm of quadruplicate wells. 2.2.2 Blockade of signaling pathways Specific kinase inhibitors were added at different concentration into the cell cultures at the starting point or 24 hrs later. Cell culture with or without inhibitors served as positive or negative controls. Cells were put into an incubator at 37°C with 5% C 0 2 and collected at different time periods. The kinase inhibitors were M E K 1 , 2 inhibitor U0126 (calBiochem), P38 kinase inhibitor SB203580 (calBiochem), protein kinase C (PKC) inhibitor Bisindolylmaleimide I (calBiochem), phosphatidylinositol 3- kinase (PI-3 kinase) inhibitor LY294002 (calBiochem) and Cyclosporine (V.G.H.) . 2.2.3 Detecting IL-2 receptor a chain (CD25) expression using flow cytometer IL-2 receptor a chain (CD25) surface expression was detected by FACScan (Coulter, U.S.A) using FITC conjugated anti-CD25 monoclonal antibody (Becton-Dickinson) according to the supplier's protocol. 71 i) Cell surface CD25 molecule labeling Cell cultures were removed from the CO2 incubator and cells were washed once with washing solution and once with 2% FCS - TCI99, then centrifuged at 1500 rpm for 10 min. Cells were resuspended in 2% FCS - TCI99. The cell concentration was adjusted to lX10 6 /ml. The cell suspension was then added into two polypropylene tubes with 0.2ml for each tube. lOul of FITC conjugated anti-CD25 antibody was added to one tube and the tube was marked as "sample"; lOul of FITC conjugated mouse IgGi was added to the second tube and the tube was marked as "control". Tubes were then vortexed thoroughly and were incubated at 4°C for 30 min. Cells were washed twice with washing solution and were centrifuged at 1500 rpm for 10 min. Cells were then resuspended in 200ul of washing solution and were analyzed with the flow cytometer within 24 hours. ii) Flow cytometer analysis Optical alignment of the flow cytometer was checked using polystyrene fluorospheres (Coulter PN6603488). Fluorescence standardization was checked using the fluorescent dye (Coulter, PN6604146). Instrument linearity and fluorescent intensity were standardized using Immuno - brite standard kit (Coulter, PN6603473). Voltages of the test panels were adjusted according to the voltage of standard brite. A l l the above steps, as well as the starting and the shutting down procedures were followed according to the Immunology Laboratory Fluorescent Standard Manual (VHHSC) and performed on a daily basis. 72 After standardization of the machine, all the tubes were vortexed and analyzed in order. As a pair, the control tube was analyzed first and the sample tube was analyzed second. After 100ml of cell suspension were sucked into the flow cytometer, cells staining with fluorescent dye showed on the screen as a green color. The cell groups were moved into FS -SS panel using a touch pad. The selected cell groups were surrounded with a circle and the dead cells at the lower left corner were eliminated. The percentage of positive cells and their mean florescent channel shift of the selected cell groups were counted by drawing a line crossing the mean fluorescent peak on the fluorescent density count -LFL1 panel. After finishing the analysis, remaining cells were washed off and the results were printed out automatically. 2.2 .4 Cell apoptosis analysis 1 X 106/ml of cells suspended in 200 uL PBS were stained with 1 uL of 7-AAD (lOug/ml, Sigma) at 4°C for 20 min. The control group was set up by adding the same amount of cells into the second tube without adding 7-AAD. The fluorescent signal was checked by FACScan at emission wavelength 655 using the same procedure as for the CD25 analysis. 73 2 . 2 . 5 I L - 2 E L I S A Sandwich ELISAs were used to detect IL-2 in 0.02ml supernatants removed from the cultures. 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 ug/ml in binding buffer (0.1M Na2HP04, pH 9.0) overnight at 4°C. Plates were then washed at least 4 times with PBS/Tween (0.5 mlTween-20 to 1 L PBS) and blocked non-specifically by adding 200ul of 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 (0.5ml Tween-20 and 100 ml Fetal Calf Serum in 900 ml PBS) and lOOul 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. lOOul/well of biotinylated anti-cytokine detection antibody (Pharmingen, San Diego, CA) was added at a concentration of 0.25-2ug/ml in blocking buffer/Tween. Plates were incubated at room temperature for 1 hr and washed at least 6 times in PBS/Tween. Avidin-Peroxidase (Vector, Burlingame, CA) was them diluted 1:2000 and added at lOOul/well. Plates were incubated at room temperature for 30 min and washed at least 8 times with PBS/Tween. Substrate buffer (0.1M 2,2'-Axino-bis in anhydrous citric acid, pH 4.35) was thawed within 20 min of use, mixed with lOul of 30% H2O2 per 11 ml of substrate buffer and immediately dispensed onto the plates at 100 ul/well. The color reaction was stopped by adding 50ul of 1% SDS solution. Plates were then read at 405nm. IL-2 production was expressed at the mean of triplicate wells. 74 2.2.6 Preparation of nuclear extracts Cultured cells were collected, washed in ice-cold PBS and lysed in cytosolic buffer (lO.OmM Tris base, 400.0 mm Nacl, 2.0mM Na 2 EDTA in 500ml of dH 2 0) containing 10 m M HEPES buffer (pH 7,9), 40 m M K C l , 3 m M M g C l 2 , 1 m M DTT, 5% glycerol, 0.2% Nonidet P-40, 1 pg/ml aprotinin, 1 pg/ml leupeptin, and 1 m M PMSF for 5 to 10 min on ice. Nuclei were removed by centrifugation at 14,000 rpm for 30s in an Eppendorf microcentrifuge. The nuclei were then washed once with ice-cold cytosolic buffer, pelleted and lysed in hypertonic buffer containing 20 m M HEPES (pH 7,9), 420 m M K C l , 1.5 m M M g C l 2 , 0.2 m M EDTA, 0.5 m M DTT, 25% glycerol, 1 pg/ml aprotinin, 1 pg/ml leupeptin, and 0.5 m M PMSF for 30 min on ice, and 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). 2.2.7 Oligonucleotides and E M S A Biotinylated dsDNA oligonucleotides were obtained from University Core D N A Services, University of Calgary, Canada. The transcription factor AP-1, NF-AT, N F - K B and STAT5 derived oligonuleotides were used. The sequences were described in page 67-68. 75 i) Co-incubation of oligonucleotides with DNA binding proteins Non-biotinylated homologous counterparts were obtained for each oligonucleotide. Both oligonucleotides were annealed by mixing them at a ratio of 1:1, heating them to 70°C and letting them cool down to room temperature slowly. To perform E M S A , 4 pg of nuclear extracts (4 X 106 cell equivalents) were incubated at room temperature for 15 min with 1 pg biotinylated oligonucleotide in the presence of 1 pg poly (dl/dC) and 5 pi 4-fold binding buffer (20 mM HEPES, pH 7.8, 0.1 m M EDTA, 1 m M DTT, 100 p M 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. 20ul of the binding mix was mixed with 5 ul of 5x loading buffer and applied to the gel. Binding mixtures were then separated on an agarose gel by the electrophoresis at 100V in 0.5 TBE buffer. D N A content was checked by illuminating the gel with U V light. Polaroid pictures of the D N A bands that had been made visible by the fluorescent dye ethidium bromide were taken. iii) Blotting After blotting on a Tropilon-Plus™ Nylon Membrane (Tropix, M A ) , bands were detected using a non-radioactive chemiluminescence method (Southern-Light™, Tropix, M A ) according to the protocol provided by the company. (Walker, R.G. et al. 1993; Weston, 76 S.A. et al. 1995). In short, the blots were washed 2x in a blocking buffer (0.2% I-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 room temperature prior to subsequent washing in blocking buffer, wash buffer (0.5% SDS in PBS), and assay buffer (provided in Southern-Light kit). After placing in Saran wrap, the blots were incubated in CDP-Star for 5 min before being exposed to standard x-ray film. 2.2.8 Quantitative Analysis of Dose-Effect Relationships - The Median Effect Equation The median effect equation was used to check the combination effects of inhibitors on T cell proliferation. The Median effect equation is a single and generalized method for analyzing dose -effect relationships in enzymatic, cellular and whole animal systems. It is based on the median effect principle of the mass action law and examines the problem of quantitating the effects of multiple inhibitors by providing definitions of summation of their effects. The median effect equation can be used without knowledge of conventional kinetic constants (i.e. Km, Vmax or Ki) and irrespective of the mechanism of inhibition (i.e. competitive, noncompetitive or uncompetitive). Furthermore, it is valid for multisubstrate reactions irrespective of mechanism. The equation states that: f a / f u =(D x /D 5 0 ) m ( l ) 77 where D is the dose, fa and fu are the fractions of the system inhibited and uninhibited respectively by the dose D x . D 5 0 is the dose required to produce the median effect of inhibition, m is a Hill-type coefficient signifying the sigmoidicity of the dose-effect curve, i.e., m=l for hyperbolic systems. m*l for sigmoidal system. Since by definition, fa + fu =1, fu = l-f a. The alternative form of median effect equation is D x = D 5 0 [fa / ( l - f a ) ] I / m The median effect equation may be linearized by taking the logarithms of both sides and forms the median effect plot, i.e. Log [fa/(l-fa)] = m Log (D x) - m Log (D 5 0) To analyze the combination effects of two inhibitors, a "combination index" (CI) needs to be calculated. For mutually exclusive inhibitors, CI = (D)i / (D x)i + (D) 2 / (D x ) 2 For mutually nonexclusive inhibitors, CI = (D)i / (D x)i + (D) 2 / (D x ) 2 + (D)i(D) 2 / (DX),(DX) 2 (D)i and (D) 2 are the calculated doses of inhibitor 1 and 2 needed to reach certain fraction of inhibition together. (D x)i and (D x ) 2 are the calculated doses of inhibitor 1 and 2 needed to reach the same amount of fraction of inhibition alone. When CI< 1 or Log(CI) < 0, synergism is indicated. This means that the combined effect of the two inhibitors is greater than the calculated additive effect. When CI = 1 or Log(CI) = 0, summation is indicated. This means that the combined 78 effect of the two inhibitors is the same of the calculated additive effect. When CI > 1 or Log(CI) > 0, antagonism is indicated. This means that the combined effect of the two inhibitors is smaller than the calculated additive effect. Requirements for analyzing multiple drug effects are essential for quantitating synergism, summation and antagonism of multiple drugs. They are: 1. A quantitative definition of summation is required. 2. Dose-effect relationships for drug 1, drug 2 and their mixture are required. 3. Measurements made with single doses of drug 1, drug 2 and their mixture can never alone determine synergism since the sigmoidicity of dose-effect curves and the exclusivity of drug effects cannot be determined from such measurements. 4. The dose - effect relationships should follow the basic mass-action principle relatively well (e.g. median-effect plots with correlation coefficients for the regression lines greater than 0.9). 5. Determination of the sigmoidicity of dose-effect curves and the exclusivity of effects of multiple drugs are necessary. The slope of the median-effect plot gives a quantitative estimation of sigmoidicity. When m=l, the dose-effect curve is hyperbolic; when m * l , the dose-effect curve is sigmoid, and the greater the m value, the greater its sigmoidicity; m<l is a relatively rare case which in allosteric systems indicates negative cooperatively of drug binding at the receptor sites. When the dose - effect relationships of inhibitor land 2 and their mixture are all parallel in the median effect plot, the effects of inhibitor 1 and 2 are mutually exclusive. If the 79 plot of inhibitor 1 and 2 are parallel but the plot of their mixture is concave upward with a tendency to intersect the plot of the more potent of the two drugs, their effects are mutually nonexclusive. If the plots for inhibitor 1 and 2 and their mixture are not parallel to each other, exclusivity of effects cannot be established. Alternatively, exclusivity of effects may not be ascertained because of a limited number of data points or limited dose range. In these cases, the data may be analyzed for the 'combination index" on the basis of both mutually exclusive and mutually nonexclusive assumptions. So, to determine the combination effects of the inhibitors in our study at any inhibition level, the procedure involves three steps: 1) . Construct the median effect plot using experimental data and linear regression coefficients. This will determine the m and D 5 0 values for inhibitor 1, inhibitor 2 and their combination. 2) . For a given degree of inhibition, calculate the corresponding doses of (D x ) i , (D x)2 and (D x)i,2 by using the alternative form of median effect equation D x = D50 [fa / ( l - f a ) ] ! / m . 3). Calculate the combination index (CI) using CI equations, where (D x ) i , and (D x)2 are from step 2). For the combination effect of inhibitor 1 and 2, the ratio is: inhibitor 1 / inhibitor 2 - P / Q. Thus (D)i = (D x ) , ; 2 x P/ (P +Q) and (D) 2 = (D x ) 1 > 2 x Q/ (P +Q). CI values that are smaller, equal to, or greater than 1, represent synergism, summation and antagonism, respectively. 80 2.9 Statistical Analysis Data were graphically displayed and statistically analyzed (paired T test and linear regression) using the Microsoft Excel Data analysis program. 81 C H A P T E R T H R E E : R E S U L T S 3.1 Building an in vitro human peripheral CD4 T lymphocyte activation model To mimic the actual in vivo human CD4 + T lymphocytes activation process, we stimulated lx l0 6 /m l of purified human peripheral CD4 + T lymphocytes with plate bonded monoclonal anti-CD3 antibody (OKT3) and anti-CD28 antibody, each at a concentration of lug/ml. The stimulation process was mainly followed the procedure published by Baroga and ward (273, 274). However, the cell concentration as well as the concentration of the stimulating antibodies was adjusted to maximize the effects of T cell activation in terms of CD25 expression. Figure 3.1. a, b and c show the kinetics of cell proliferation (a), extracellular IL-2 secretion (b) and cell surface IL-2 R a chain (CD25) expression (c) of this model within a 3-day culturing period. As measured by Thymidine ( [H] TdR) incorporation in figure 3.1.a, stimulation with OKT3 and anti-CD28 antibodies induced a continuous cell proliferation through the 3-day culture period. This process accelerated after day 1. At day 3, 3[H] TdR incorporation reached around 15,000 rpm, which is about 15 times more than that measured at dayl. Figure 3.1.b shows the level of extracellular secretion of IL-2 measured by ELISA. 82 Figure 3.1 .b shows the level of extracellular secretion of IL-2 measured by ELISA. Resting CD4 + T lymphocytes did not secrete extracellular IL-2. After activation, IL-2 secretion increased rapidly and reached the maximum level at day 1 with a concentration of more than 400 pg/ml, then gradually decreased. At day 3, the extracellular IL-2 declined to around 20-30 pg/ml, about 20 times lower than the maximum level. Figure 3.1.c demonstrated the kinetics of IL-2 R a chain (CD25) expression examined by flow cytometry. Resting CD4 + T Lymphocytes had a low level of CD25 expression with positive cells reflecting less than 10% of the cell population. The epitope density of the CD25 molecule - represented here as the mean log fluorescent channel shift (mlfc) - was around 5. After cell activation, CD25 expression increased and reached its peak level around day 3 of cell culture. At this time, 50-60% of the cells was CD25 positive and their epitope density (mlfc) was around 20. 83 • • Fill I S T - - » - - N o S T 20000 Day 0 Day 1 Day 2 Day3 C ii 1 tu rin g Tim e + 3 Figure 3.1.a: CD4 T lymphocyte proliferation measured by [H] TdR incorporation. lX10 6 /ml of C D 4 + T lymphocyte stimulated with (Full ST) or without (NO ST) OKT3 and anti- CD28 antibodies, each at lug/ml in RPMI - media and incubated at 37°C with 5% CO2. Cell proliferation were checked with 3[H]TdR incorporation at day 3 of cell culture. • • F u l l S T - • • • - N o S T 500 C u l t u r i n g T i m Figure 3.1.b: Kinetics of extracellular secretion of IL-2 measured by ELISA. CD4 T lymphocytes did not secrete extracellular IL-2 at resting stages. After activation, IL-2 secretion rapidly increased. And reached the maximum level at day 1 of cell culture then gradually decreased. 84 -» C D 2 5 % - - E - - mlfc 0 -| , , , \- 0 Day 0 Day 1 Day 2 Day 3 C u l t u r i n g T i m e Figure 3.1.c: Kinetics of IL-2 R a chain (CD25) expression examined by flow cytometry. Both the percentage of CD25 expression and the epitope density of CD25 molecule -represented here as the mean log fluorescent channel (mlfc) increased through out the 3 days of cell culture. 85 3.2 Inhibitor's toxicity to in vitro CD4 T lymphocytes culture Cell toxicity examined by apoptosis study and the result is shown on Figure 3.2. After 3 days of culturing CD4 + T lymphocytes with any of the four inhibitors at the maximum concentration used in all experiments, 2.5-7.5% of the whole cell population showed evidence of apoptosis compared to 8.5% of cell apoptosis in the culture without any inhibitors. It thus appeared that none of the inhibitors were toxic to CD4 + T lymphocyte culture. 1 2 3 4 5 6 1: No stimulation 2: O K T 3 + ant i -CD28 (ST) 3: S T + U 0 1 2 6 (50uM) 4: S T + S B 2 0 3 5 8 0 (50uM) 5: S T + Bis indolylmale imide I (2uM) 6: S T + L Y 2 9 4 0 0 2 (50uM) Figure 3.2: Cell apoptosis study. 1 X 106/ml of cells suspended in 200 pL I X PBS were stained with 1 pL of 7-AAD (lOug/ml, Sigma) at 4°C for 20 min. The control group was set up by adding the same amount of cells into the second tube without adding 7-AAD. The fluorescent signal was checked by FACScan at emission wavelength 655 using the same procedure as for the CD25 analysis. A l l inhibitors were not toxic to CD4 + T lymphocyte cell culture at tested concentration. 86 3.3 Effects of blocking signaling pathways under T C R and/or C D 2 8 complex to T lymphocyte activation at levels of T lymphocyte proliferation, I L - 2 secretion and I L -2 receptor a chain ( C D 2 5 ) expression To test the effects of blocking signaling pathways on T lymphocyte activation, we chose three pathways activated predominantly by the TCR complex and blocked them with specific inhibitors. They are 1. M A P kinase pathway blocked at the level of M E K level with specific inhibitor U0126 (lOuM); 2. p38 M A P kinase pathway blocked at the level of p38 M A P kinase using specific inhibitor SB203580 (50uM); 3. P K C pathway blocked at P K C level using specific inhibitor Bisindolylmaleimide I (2uM). To examine the effect of signaling pathways triggered by the costimulatory molecule CD28, we chose the PI3 kinase pathway and blocked it with the specific PI3 kinase inhibitor LY294002 (50uM). The concentrations of the inhibitors used here were closed to the IC50 in suppression CD25 expression tested in a one-day cell culture (Figure3.3.4). The concentration of cyclosporine was used at lug/ml as reported else where (275). Figure 3.3.1 shows that U0126, SB203580, Bisindolylmaleimide I and LY294002 significantly reduced CD4 + T lymphocytes proliferation throughout the cell culture period (P<0.0T). SB203580, Bisindolylmaleimide I and LY294002 almost completely eliminated any cell proliferation. The suppressive effect of U0126 was weaker than the other three inhibitors. However, it still reduced [H] TdR incorporation to around 4000 cpm at day 3, a 70% reduction in cell proliferation. Cyclosporine (CsA), a major IL-2 cascade inhibitor 87 was used here as a comparison. It produced around 60% reduction in cell proliferation at day 3, which was significantly higher than U0126 (P>0.05) and was the weakest of all inhibitors. Figure 3.3.2 shows that all inhibitors significantly reduced the secretion of extracellular IL-2 throughout the period of cell culture. (P<0.01). P<0.01 18000 -> 16000 NO ST Full ST L Y UO SB Bil CsA NO ST=No Stimulation Full ST=OKY3+ anti-CD28 LY=Full ST+LY294002(50uM) UO=Full ST+U0126(10uM) SB=Full ST+SB203580(50uM) BiHFul l ST + Bisindolylmaleimide I(2uM) CsA=Full ST+cyclosporine(lug/ml) Figure 3.3.1: Effects of the inhibitors on CD4 + T lymphocytes proliferation examined by 3[H] TdR incorporation. A l l inhibitors significantly reduced CD4 + T cell proliferation throughout 3 days of cell culture (P<0.01). Cyclosporine had the weakest inhibitory effect among all inhibitors. 88 P<0.01 NO ST Full ST UO SB Bil L Y CsA NO ST=No Stimulation Full ST=OKY3+ anti-CD28 LY=Full ST+LY294002(50uM) UO=Full ST+U0126(10uM) SB=Full ST+SB203580(50uM) Bil=Full ST + Bisindolylmaleimide I(2uM) CsA=Full ST+cyclosporine(lug/ml) Figure 3.3.2: Effect of inhibitors on extracellular IL-2 secretion. A l l inhibitors significantly reduced the secretion of extracellular IL-2 throughout the period of cell culture. (PO.01). 89 The effects of the inhibitors on CD25 expression are shown in figure 3.3.3. CD25 expression (CD25%, Figure 3.3.3.a) and the mean log fluorescent channel shift of CD25 expression (mlfc, Figure 3.3.3.b) were checked. The mlfc represents the density of CD25 molecules on the surface of CD4 + T lymphocytes. SB203580, Bilsidilymilomide I and LY294002 significantly reduced the percentage of CD25 expression from the beginning of cell activation. At day 1, the percentage of positive CD25 cells (CD25%) dropped to less than 10% for all three inhibitors. This was an approximately 70% reduction compared to the full stimulation (P<0.01). The inhibition continued to day 3 of cell activation. The effect of U0126 on CD25 expression was weaker. At day 1 of the cell activation, the percentage of positive CD25 cells dropped to around 20%, which is around 35% of reduction compared to full stimulation (P=0.07). At day 3, the effect of U0126 picked up and very significantly reduced the percentage of positive CD25 cells (PO.01). Cyclosporine also significantly reduced CD25% expression at day 3 (P=0.041). Compared to all four other inhibitors, the inhibitory effect of cyclosporine was the weakest and the difference was significant at day 3 of cell activation (P=0.047). U0126, SB203580, Bilsidilymilomide I and LY294002 very significantly reduced mlfc of CD25 expression from day 1 (PO.01). The mlfc dropped to under 5 for all four inhibitors, which was more than a 50% reduction compared to the full stimulation. The inhibition effects lasted to day 3 of cell activation, which mlfc remained under 5 for all 90 four inhibitors. It was a more than75% reduction compared to the full stimulation. Cyclosporine also had a very significant reduction on mlfc at day 3 (P<0.01). Comparing to the other four inhibitors, cyclosporine had the weakest reduction on mlfc from the day 1 of cell activation. The difference among them was significant. (PO.01) 2.P=0.07 NO ST Full ST UO S B Bil LY C s A NO ST=No Stimulation Full ST=OKY3+ anti-CD28 LY=Full ST+LY294002(50uM) UO=Full ST+U0126(10uM) SB=Full ST+SB203580(50uM) Bil=Full ST + Bisindolylmaleimide I(2uM) CsA=Full ST+cyclosporine(lug/ml) Figure 3.3.3.a: A l l inhibitors suppressed CD25 expression (CD25%) in activated CD4 + T lymphocytes. SB203580, Bilsidilymilomide I and LY294002 very markedly reduced the expression of CD25. The inhibition started from day 1 (l.P<0.01) and lasted to day 3 of 91 cell activation. For inhibitor U0126, inhibition was not significant at day 1 (2.P=0.07) but gradually increased and became significant at day 3 of cell activation (3.PO.01). Cyclosporine also significantly reduced CD25 expression at day 3 (4.P=0.041). Compared to all four other inhibitors, the effect of cyclosporine was the weakest (5.P=0.047). 1.PO.01 NO ST Full ST UO L Y CsA NO ST=No Stimulation Full ST=OKY3+ anti-CD28 LY=Full ST+LY294002(50uM) UO=Full ST+U0126(10uM) SB=Full ST+SB203580(50uM) Bil=Full ST + Bisindolylmaleimide I(2uM) CsA=Full ST+cyclosporine(lug/ml) Figure 3.3.3.b: A l l inhibitors suppressed CD25 expression (mlfc) in activated CD4^ T lymphocytes. U0126, SB203580, Bilsidilymilomide I and LY294002 very markedly reduced mlfc of CD25 expression from day 1 (1. PO.01) and last to day 3 of cell activation. Cyclosporine also significant reduced mlfc at day 3 of cell culture (2. P<0.01). Comparing to the other four inhibitors, cyclosporine had the weakest reduction on mlfc from the day 1 of cell activation (3. P<0.01). 92 Figure 3.3.4 showed that the suppression of CD25 expression occurred in a dose dependent response. Ix 106/ml of CD4 + T lymphocytes was stimulated with OKT3 and anti-CD28 antibody at lmg/ml for 24 hours with inhibitor at different dosages. For the MEK1/2 inhibitor U0126 (upper, left), less than O.luM had no significant effect on the percentage and mlfc CD25 expression. The effect of U0126 became more prominent with increasing doses. The IC50 for suppression CD25% expression was 5uM - lOuM. The IC50 for suppression of the mlfc CD25 expression was l u M - lOuM. For the P38 M A P kinase inhibitor SB203580 (lower, left), the effect was very prominent on both the percentage and the mlfc CD25 when the dosage of the inhibitor was higher than O.luM. The IC50 for suppression of the percentage CD25 expression was lOuM - lOOuM. The IC50 for suppression of the mlfc CD25 expression was l u M - lOuM. The protein kinase C inhibitor Bisindolylmaleimide I (upper, right) sharply reduced both the percentage and the mlfc of CD25 expression even at a concentration below O.luM. The IC50 for suppression of the percentage CD25 expression was l u M - 2uM. The IC50 for suppression of the mlfc CD25 expression was O.luM - l u M . The PI3 kinase inhibitor LY294002 suppressed both the percentage and the mlfc of CD25 expression starting at O.luM. The IC50 for suppression of the percentage CD25 expression was lOuM - lOOuM. The IC50 for suppression of the mlfc CD25 expression was around 0. l u M . The effect of all four inhibitors decreased in a time dependent manner (Figure 3.3.5). When adding inhibitors one day after T cell activation, the suppression of both the percentage and mlfc CD25 expression diminished greatly. This suggested that suppression by these inhibitors was more active on the early phase of CD25 expression. 93 -+-CD25% mlfc +OuM +0.1uM +luM +10uM +100uM Dosages of U0126 Added into Cell Culture +OuM +0.1uM +luM +2uM +10uM Dosages of BIOSINDOLYLMALEIMIDEI Added into Cell Culture 60 50 40 S30 p ° 2 0 10 0 14 12 10 8 6 4 2 0 +0uM +0.1uM +luM +10uM +100uM Dosages of SB203580 Added into Cell Culture 60 50 40 :20 V > — - — — * \ i i i i 14 12 T6 1 4 Full ST O.luM luM lOuM lOOuM NO ST Dosages of LY294002 Added into Cell Culture Figure 3.3.4: The suppression of inhibitors on CD25 expression showed a dose dependent response. 94 Figure 3.3.5: Suppressive effect of all four inhibitors was time dependent. When inhibitors were added one day after T lymphocytes activation, suppression of both the percentage and the mlfc of CD25 expression diminished greatly. It suggested that the action of these inhibitors was principally on the early phase of CD25 expression. 95 3.4 The suppression of CD25 expression is partially independent of the reduction of extracellular IL-2 Previous research provided evidence that IL-2 itself has a positive feedback on its receptor a chain (CD25) expression [1, 2]. In our T lymphocyte activation model, all five inhibitors substantially reduced IL-2 secretion. To separate the influence of the reduction of IL-2 from direct suppression of CD25 expression, we added lOOU/ml per day of extracellular human recombinant IL-2 into the cell culture. The concentration of extracellular IL-2 added into the cell culture was adjusted to the concentration of observed during in vitro cell activation. We checked the rescue effect of extracellular IL-2 using the percentage and the mlfc of CD25 expression separately. Figure 3.4.1 showed that extracellular IL-2 could only partially attenuate the suppression of CD25% expression by SB203580, U0126, Bilsidilymilomide I, LY294002 and cyclosporine (p=n.s). But this effect of CD25% expression was limited. When compared with the positive control, the suppression of CD25% expression by all inhibitors despite adding IL-2 was still marked. With extracellular IL-2, SB203580, Bilsidilymilomide I and LY294002 (p<0.01) as well as U0126 and cyclosporine (p<0.05) still significantly reduced CD25% expression. Extracellular IL-2 had a more potent rescue effect on the mlfc of CD25 expression (Figure3.4.2). Extracellular IL-2 significantly attenuated the suppression of mlfc CD25 96 expression by U0126, SB203580, Bisindolylmaleimide I and LY294002 (p<0.05) and partially attenuate the suppression by cyclosporine (p^n.s). Nonetheless, IL-2 did not eliminate the inhibition of mlfc CD25 expression caused by Bisindolylmaleimide I and LY294002. Their inhibitory effects were still significantly (p<0.05) comparing to the positive control with extracellular IL-2. On the other hand, extracellular EL-2 could convert most of the suppression by U0126, SB203580 and cyclosporine since none of these inhibitors could still significantly suppress the mlfc of CD25 expression after adding extracellular IL-2 (U0126 p=0.067, SB203580 p=0.058 and cyclosporine p=0.199). These results demonstrated that extracellular EL-2 could attenuate, but not totally eliminate, the suppression on CD25 expression by the inhibitors employed, indicating that they exerted a direct suppressive effect on CD25 expression. 97 U O S B Bi l LY C s A I Figure3.4.1: Adding lOOU/ml/day of extracellular IL-2 didn't significantly reduce the suppressive effects of all inhibitors on the percentage of CD25 expression (p=n.s). 98 * P<0.05 P=n.s 90 80 70 o <*- 60 E c o 50 c o 40 Inhil 30 4-o 20 10 0 u o T . • Day 3 HDay 3 + IL-2 SB Bil LY CsA Figure3.4.2: Effects of extracellular IL-2 on CD25 expression (mlfc). Adding lOOU/ml/day of extracellular IL-2 for 3 days significantly reduced the suppressive on mlfc of CD25 expression due to U0126, SB203580, Bisindolylmaleimide I and LY294002 (*=P<0.05), but did not significantly reduce the suppression made by cyclosporine (p=n.s) to the mlfc of CD25 expression. 99 3.5. Role of transcription factors N F - A T and A P - 1 in IL-2 gene expression and transcription factors N F - K B and S T A T 5 in IL-2 R a chain ( C D 2 5 ) gene expression After checking the influence of inhibitors on T lymphocyte proliferation, cytokine secretion (IL-2) and cytokine receptor expression (CD25), we examined the effects of blocking each signaling pathway at the transcription factor level. Transcription factors NF-AT, AP-1, N F - K B and STAT5 are among the most important D N A binding proteins regulating T lymphocyte activation. NF-AT and AP-1 are important for regulating IL-2 gene expression and N F - K B and STAT5 are important for regulating IL-2 receptor a chain (CD25) gene expression. Transcription factor nuclear binding activity represents the degree of transcription factor activation in T lymphocyte activation. To check the change of transcription factor nuclear binding activity, CD4 + T lymphocytes were stimulated with OKT3 + anti-CD28 antibody. Specific protein kinase inhibitors were added at the beginning of cell culture. T lymphocytes stimulated with or without antibodies were used as positive and negative controls. Nuclear extracts were prepared and binding reactions performed using a transcription factor specific biotinylated oligonucleotide probe. The kinetics of NF-AT and AP-1 nuclear binding activity during three days of T lymphocyte activation were displayed in Figure 3.5.1. The maximum binding activity of NF-AT occurred at day 1 of cell activation and decreased with cell culture time. AT day 100 3, N F - A T nuclear binding activity almost totally diminished. The nuclear binding activity of transcription factor AP-1 reached a maximum at day 2 and was sustained up to day 3 of cell culture. The kinetics of nuclear binding activity for transcription factors N F - K B and STAT5 are shown in figure 3.5.2. N F - K B had a low level of nuclear binding activity in resting CD4 + T lymphocytes. After T cell activation, it increased and reached the maximum level at 48 hrs and this was sustained to 72 hrs of the cell culture. Transcription factor STAT5 did not demonstrate its nuclear binding activity until around 24 hrs after T lymphocyte activation. At 24 hrs, it started to increase and reached the maximum level at 48 hrs and maintained at the same level at 72 hrs of cell culture. The kinetics of N F - A T nuclear binding activity correspond with the kinetics of EL-2 secretion throughout the first 3 days of T lymphocyte activation. However, the kinetics of AP-1 nuclear binding activity seems to have a reverse trend compared to the kinetics IL-2 secretion. This indicates that N F - A T may play a more important role than AP-1 in EL-2 gene transcription. AP-1 may help to maintain the low level of IL-2 gene transcription after the initial stage of T cell activation. The kinetics of both N F - K B and STAT5 nuclear binding activity correspond with the kinetics of CD25 expression during 3 days of T lymphocyte activation, and both of these transcription factors play important roles in transcription of the CD25 gene. However, since STAT5 did not show nuclear binding activity until day 1 of cell culture, it may not play an important role in CD25 expression 101 during the early phase of T cell activation. Time Oday l d a y 2day 3day < NF-AT Oday l d a y 2day 3day AP-1 Figure 3 . 5 . 1 : Kinetics of NF-AT and AP-1 binding activity. This figure represents one out of three independent experiments. Nuclear extracts were incubated with NF-AT and AP-1 specific biotinylated oligonucleotides respectively. The maximum binding for NF-A T occurred at day 1 of cell activation and decreased with culturing time. The binding activity of transcription factor AP-1 increased to a maximum level at day 2 of cell activation, was sustained until day 3 of cell activation. Other experiments that checked AP-1 binding activity showed clear AP-1 D N A binding on day one of cell culture with the same trend through the three-day culture period. 102 Time Ohr 12hr 24hr 36hr 48hr 60hr 72hr N F - K - B Ohr 12hr 24hr 36hr 48hr 60hr 72hr ^ STAT5 Figure 3.5.2: Kinetics of N F - K B and STAT5 binding activity. CD4 T -lymphocytes were stimulated with OKT3 + anti-CD28 antibody for Oh, 12hs, 24hs, 36hs, 28hs, 60hs and 72hs. This figure represents one out of three independent experiments. Resting CD4 + T -lymphocytes had low N F - K B binding activity. The binding strength increased along with cell activation, reached the maximum at 48 hours, and sustained until 72 hours of cell culture. There was no STAT5 binding activity for resting CD4 + T -lymphocytes. The binding strength started to pick up at 24 hours, reached the maximum at 48 hours, and sustained until 72 hours of cell culture. 1 0 3 3.6. Blocking either TCR or CD28 signaling pathways dramatically reduced NF-AT and AP-1 nuclear binding activity CD4 + T lymphocytes were stimulated with OKT3 and anti-CD28 antibody with or without the addition of specific protein kinase inhibitors at the beginning of cell culture. Figure 3.6.1 (column A and B) shows that blocking the TCR signaling pathways using the M E K inhibitor U0126, P38 M A P kinase inhibitor SB203580 and P K C inhibitor Bisindolylmaleimide I markedly reduced NF-AT nuclear binding activity throughout the 3 days of T cell activation. Figure 3.6.2 demonstrated that blocking the CD28 signaling pathway by inhibiting PI3 kinase activity using the inhibitor LY294002 dramatically reduced NF-AT nuclear binding activity in 3 days of T cell activation (Lane 1). The result in Lane 2 also showed the reduction in NF-AT nuclear binding activity by cyclosporine throughout 3 days of T cell activation. Figure 3.6.3 demonstrated the effects of blocking TCR.signaling pathways on AP-1 nuclear binding activity. M E K inhibitor U0126 (Lane 1), P38 M A P kinase inhibitor SB203580 (Lane 2) markedly reduced AP-1 binding activity. In Lane 3, though AP-1 binding activity did not show a increase in the first two days of cell activation in this set of experiments, it clearly showed that in day 3, P K C inhibitor Bisindolylmaleimide I markedly reduced AP-1 binding activity. Figure 3.6.4. Lane 1 showed that blocking CD28 signaling pathway using specific PI3 kinase inhibitor LY294002 markedly reduced AP-1 nuclear binding activity during 3 days of T cell activation. Meanwhile, results 104 showed in Lane 2 demonstrated that though AP-1 binding activity did not increase in the first two days of cell activation in this set of experiments, cyclosporine markedly reduced AP-1 nuclear binding activity at day 3 of cell activation. These results indicated that all of the inhibitors examined had a profound inhibitory effect in IL-2 gene transcription during T cell activation by markedly reducing the nuclear binding activity of transcription factor NF-AT and AP-1. Column A: Column B: OKT3 + anti-CD28 OKT3 + anti-CD28 + Inhibitor Time Time lday 2day 3day lday 2day 3day NF-AT NF-AT NF-AT 1: + U 0 1 2 6 2: + SB203580 3: +Bisindolylmaleimide I Figure 3.6.1: Effects of blocking signaling pathways downstream of TCR molecule on NF-AT binding activity. CD4 + T -lymphocytes were stimulated with OKT3 + anti-105 CD28 antibody with or without adding the specific protein kinase inhibitor at the beginning of cell culture (column B and A). M E K inhibitor U0126, P38 M A P kinase inhibitor SB203580 and P K C inhibitor Bisindolylmaleilide I markedly reduced NF-AT binding activity through out 3 days of cell activation. Column A: Column B: OKT3 + anti-CD28 OKT3 + anti-CD28 + Inhibitor Time Time lday 2day 3day lday 2day 3day Lane L + LY294002 NF-AT Lane 2: + Cyclosporine Figure 3.6.2: Effects of blocking PI3 kinase signaling pathways downstream of CD28 molecule on NF-AT binding activity (Lane 1). LY294002 markedly reduced NF-AT binding activity throughout 3 days of cell activation. Line 2 shows that cyclosporine also reduced NF-AT binding activity from the beginning of cell activation. 106 Column A: Column B: OKT3 + anti-CD28 OKT3 + anti-CD28 + Inhibitor Time Time Oday lday 2day 3day lday 2day 3day AP-1 Lane 1: + U0126 AP-1 Lane 2: + SB203580 AP-1 Lane 3: +Bisindolylmaleimide I Figure 3 . 6 . 3 : Effects of blocking signaling pathways downstream of TCR molecule on AP-1 binding activity. M E K inhibitor U0126 (Lane 1), P38 M A P kinase inhibitor SB203580 (Lane 2) markedly reduced AP-1 binding activity throughout 3 days of cell activation. In Lane 3, though AP-1 binding activity did not show a clear increase in the first two days of cell activation in this set of experiments, it clearly showed that in day 3, PKC inhibitor Bisindolylmaleimide I markedly reduced AP-1 binding activity. 107 Column A: Column B: O K T 3 + anti-CD28 O K T 3 + anti-CD28 + Inhibitor Figure 3.6.4: Effects of blocking PI3 kinase signaling pathways downstream of the CD28 molecule on AP-1 binding activity (Lane 1). LY294002 markedly reduced AP-1 binding activity throughout 3 days of cell activation. In Lane 2, though AP-1 binding activity did not show a clear increase in the first two days of cell activation in this set of experiments, it clearly showed that in day 3, cyclosporine markedly reduced AP-1 binding activity. 108 3.7. The effects of blocking T C R or CD28 signaling pathways of the nuclear binding activity transcription factor N F - K B and STAT5 Figure (3.7.1-3) demonstrates the effects of blocking signaling pathways on IL-2 receptor a chain (CD25) gene expression at the transcription factor level. As results shown in Figure 3.7.1, none of the M E K inhibitor U0126 (Lane 2), P38 M A P kinase inhibitor SB203580 (Lane 3) nor P K C inhibitor Bisindolylmaleilide I (Lane 4) had any markedly effects on N F - K B nuclear binding activity throughout 3 days of T cell activation. Adding 100 U/ml of extracellular IL-2 into the cell culture for 3 days with any of the above inhibitors had no influence on N F - K B nuclear binding activity. However, blocking PI3 kinase signaling pathways downstream of the CD28 molecule profoundly reduced the N F - K B binding activity. Figure 3.7.2 showed that the PI3 kinase inhibitor LY294002 dramatically reduced N F - K B nuclear binding activity throughout 3 days of T cell activation. Adding 100 U/ml of extracellular IL-2 into the cell culture for 3 days partially increased the strength of N F - K B nuclear binding activity. This indicated that LY294002 had a direct suppression effect on the nuclear binding activity of transcription factor N F - K B . Figure 3.7.3 showed that STAT5 nuclear binding activity was highly reduced by U0126 and SB203580 throughout 3 days of T cell activation (Lane 2 and 3). The reduction could only be partially overcome by adding lOOU/ml of extracellular IL-2 for three days. These 109 results indicated that U0126 and SB203580 had a direct suppressive effect on STAT5 nuclear binding activity. The P K C inhibitor Bisindolylmaleimide I (Lane 4) cause no important reduction of STAT5 nuclear binding activity at day 3 of cell activation and the reduction could be fully overcome by extracellular IL-2. This indicated that Bisindolylmaleilide I had no direct influence on transcription factor STAT5 binding activity. Blocking PI3 kinase signaling pathways downstream of CD28 molecule reduced STAT5 nuclear binding activity. Figure 3.7.4 demonstrated that PI3 kinase inhibitor LY294002 markedly reduced N F - K B binding activity throughout 3 days of T cell activation. Adding 100 U/ml of extracellular IL-2 into the cell culture for 3 days had a partial effect in increasing STAT5 nuclear binding activity (Lane 2), indicating that LY294002 had a direct suppressive effect on the binding activity of transcription factor STAT5. Figure 3.7.5 showed that cyclosporine did not markedly reduce the nuclear binding activity of N F - K B , but reduced the binding activity of STAT5 through 3 days of T cell activation. However, the reduction on STAT5 binding activity could be fully overcome by adding extrcellular IL-2 for 3 days (Lane c). This indicated that cyclosporine had no profound direct influence on transcription factor N F - K B and STAT5 binding activity. 110 3day + T ime l d a y 2day 3day IL-2 Figure 3.7.1: Effects of blocking signaling pathways downstream of TCR molecule on N F - K B binding activity. CD4 + T -lymphocytes were stimulated with OKT3 + anti-CD28 antibody with (Line 2,3,4) or without (Lanel) adding the specific protein kinase inhibitor at the beginning of cell culture. One out of three independent experiments is shown here. None of M E K inhibitor U0126 (Lane 2), P38 M A P kinase inhibitor SB203580 (Lane 3) or P K C inhibitor Bisindolylmaleilide I (Lane 4) substantially reduced N F - K B binding activity throughout 3 days of cell activation. Adding 100 U/ml/day of extracellular IL-2 into the cell culture for 3 days with the inhibitor had no influence on N F - K B binding activity. I l l 3day + T ime Oday l d a y 2day 3day I L - 2 Figure 3.7.2: Effects of blocking PI3 kinase signaling pathways downstream of CD28 molecule on N F - K B binding activity. CD4 + T -lymphocytes were stimulated with OKT3 + anti-CD28 antibody with (Lane 2) or without (Lanel) adding the specific PI3 kinase inhibitor at the beginning of cell culture. PI3 kinase inhibitor LY294002 markedly reduced N F - K B binding activity throughout 3 days of cell activation. Adding 100 U/ml/day of extracellular IL-2 into the cell culture for 3 days partially increased the strength of N F - K B binding. This indicated that LY294002 had both direct and indirect suppression effect on the binding activity of transcription factor N F - K B . 112 3day + T i m e l d a y 2day 3day I L - 2 STAT5 Lane 1: 0KT3 + anti-CD28 STAT5 — • STAT5 — • STAT5 — • Lane 4: +Bisindolylmaleimide I Lane 2: + U0126 Lane3: + SB203580 Figure 3.7.3: Effects of blocking signaling pathways downstream of TCR molecule on STAT5 binding activity. Lane 2 and Lane 3 show that STAT5 binding activity was highly reduced by U0126 and SB203580 throughout the 3 days of cell activation. The reduction could only be partially overcome by adding lOOU/ml/day of extracellular IL-2 for 3 days. The PKC inhibitor Bisindolylmaleimide I (Lane 4) slightly reduced STAT5 binding activity at day 3 of cell activation and the reduction could be fully overcome by extracellular IL-2. This indicated that Bisindolylmaleilide I had no direct influence on the binding activity of transcription factor STAT5. 113 3day + T ime l d a y 2day 3day I L - 2 STAT5 Lane 1: 0KT3 + anti-CD28 STAT5 Lane 2: + LY294002 Figure 3.7.4: Effects of blocking PI3 kinase signaling pathways downstream of CD28 molecule on STAT5 binding activity. PI3 kinase inhibitor LY294002 markedly reduced STAT5 binding activity throughout 3 days of cell activation. Adding extracellular IL-2 into the cell culture for 3 days partially increased the strength of STAT5 binding activity. This indicated that LY294002 had a direct suppressive effect on the binding activity of transcription factor STAT5. 114 OKT3 + anti-CD28 OKT3 + anti-CD28 +CsA Time Oh 12h 24h 36h 48h 60h 72h 12h 24h 36h 48h 60h 72h Figure 3.7.5: Effects of cyclosporine on kinetics of transcription factor N F - K B and STAT5 binding activity. Cyclosporine had no influence on N F - K B binding activity throughout 3 days of cell activation and only slightly reduced the binding activity on STAT5 at day 3. The reduction on STAT5 could be fully overcome by adding extrcellular IL-2 for 3 days (Lane c). This indicated that cyclosporine had no directly influence on transcription factor STAT5. 115 In summary (Figure 3.7.6): Blocking the M E K pathway, P38 M A P kinase pathway and PKC pathway with their specific inhibitors followed by activation via the TCR/CD3 complex markedly reduced the nuclear binding activity of transcription factors NF-AT and AP-1 - the key players for IL-2 gene transcription. For IL-2 receptor a chain gene expression, blockade of M E K and P38 M A P kinase pathways reduced the nuclear binding activity of transcription factor STAT5 but had no influence on transcription factor N F - K B nuclear binding activity. Extracellular IL-2 could only partially overcome these inhibitory effects. However, blocking PKC pathway had no influence on either the STAT5 or N F -K B binding activity. Blocking the PI3 kinase pathway with inhibitor LY294002, followed by CD28 activation reduced the nuclear binding activity of all transcription factors examined, showing the importance PI3-kinase pathway in both IL-2 and IL-2 receptor a chain gene expression. Extracellular IL-2 only partially attenuated the inhibitory effects. These results also indicated that transcription factor N F - K B was activated mainly through PI3 kinase pathways in response to CD28 activation. i c R L -2 g e n e e x p r e s s i o n I L - 2 R g e n e e x p r e s s i o n 116 3.8 The quantitative analysis of dose-effect relationships between inhibitors using median effect equation To check the effects of simultaneously blocking TCR and CD28 pathways signaling pathways on T cell activation, we combined the specific CD28 pathway inhibitor LY294002 with each of the TCR pathway inhibitors examined in this study at a 1:1 molar ratio. They were U0126, SB203580, Bisindolylmaleimide I and cyclosporine. The inhibitory effect was checked on in vitro cell proliferation represented by 3[H] TdR incorporation. The dose - effect relationships were analyzed using the median effect equation: fa / fu=(Dx /D 5 0 ) m The median effect equation was then linearized by taking the logarithms of both sides and forms the median effect plot: Log [fa/(l-fa)] = m Log (D x) - m Log (D 5 0) m was a Hill-type coefficient signifying the signoidicity of the dose-effect curve. D50 was the dose required producing the median inhibition effect. Table 1 -4 were data of individual and combination effects of inhibitors examined on T cell proliferation measured by 3[H] TdR. Figure 3,8.1-4.a were the median effect plots generated from the above data. Using linear regression, we generated trend lines and 117 calculated median effect equations for the inhibitory effect of each of the inhibitor as well as their combination effects. The median effect equation for LY294002 is: yO.1128x+0.1341, the Hill-type coefficient is m=0.1128, the dose required producing the median inhibition effect is D5o=0.06474uM, the regression coefficient is r = 0.9874. The median effect equation for U0126 is: y=0.5131x-0.0885. m=0.5131, D50=1.487582uM, r=0.7584. For the combination effect of LY294002 and U0126, the median effect equation is: y=0.6384x+0.4328. m=0.6384, D5o=0.357001uM, r=0.9284. The median effect equation for Bisindolylmaleimide I is: y=0.1803x+0.6597. m-0.1803, D5o=0.000219uM, r=0.9705. For the combination effects of LY294002 and Bisindolylmaleimide I, the median effect equation is: y=0.6865x+1.0571. m=0.6865, D5o=0.02885 l u M , r=l. The median effect equation for cyclosporine is: y=0.2553x+01.118. m=0.2553, D50=4.1767E-5 uM, r=0.999. For the combination effects of LY294002 and cyclosporine, the median effect equation is: y=0.5672x+1.1874. m=0.5672, D50=0.008064 uM, r=0.999. The median effect equation for SB203580 is: y=0.7746x-0.3379. m=0.7746, D5 0=2.7304uM, r=0.9705. The median effect equation for LY294002 in the study with SB203580 is: y=0.2465x+0.313. m=0.2465, D5 0=0.05373uM, r = 0.8352. For the combination of LY294002 and SB203580, the median effect equation is: y=0.7746x-0.3379. m=0.7746, D5o=2.7304uM, r=0.9705. The slope of the median-effect plot (m) gives a quantitative estimation of sigmoidicity. When m=l, the dose-effect curve is hyperbolic; when m ^ l , the dose-effect curve is sigmoid, and the greater the m value, the greater its sigmoidicity; m<l indicated negative 118 cooperatively of inhibitor binding with kinase. Since all the slopes of the median effect plots (m) in our studies were smaller than 1, this indicated that when dose of inhibitors increased, the proportion of inhibitors binding of these inhibitors to their kinase decreased We also noticed that the regression coefficient (r) for studies of U0126 alone and LY294002 alone were 0.7584 and 0.8352 respectively, which were less than 0.9. The median-effect analysis requires that the dose - effect relationships follow the basic mass-action principle relatively well. This means that the correlation coefficients of median-effect plots for the regression lines should be greater than 0.9. In our study, the regression coefficient could be increased by adding more experimental data plotted for the linear regression. To analyze the combination effects of two inhibitors, a "combination index" (CI) was calculated. For mutually exclusive inhibitors, CI = (D)i / (D x)i + (D)2 / (Dx)2 For mutually nonexclusive inhibitors, CI = (D)i / (D x)i + (D) 2 / (Dx)2 + (D)i(D) 2 / (Dx)l(Dx)2 When CI < 1 or Log(CI) < 0, synergism was indicated. This means that the combined effect of the two inhibitors was larger than the sum of their individual inhibitory effects. When CI = 1 or Log(CI) = 0, summation was indicated. This means that the combined effect of using the two inhibitors was the same as the sum of their individual inhibitory effects. 119 When CI > 1 or Log(CI) > 0, antagonism was indicated. This means that the combined effect of the two inhibitors was smaller than the sum of their individual inhibitory effects. To determine the exclusivity of effects of inhibitors, median effect plots were drawn. When the dose - effect relationships of inhibitor land 2 and their mixture are all parallel in the median effect plot, the effects of inhibitor 1 and 2 are mutually exclusive. If the plot of inhibitor 1 and 2 are parallel but the plot of their mixture is concave upward with a tendency to intersect the plot of the more potent of the two drugs, their effects are mutually nonexclusive. If the plots for inhibitor 1 and 2 and their mixture are not parallel to each other, exclusivity of effects cannot be established. Alternatively, exclusivity of effects may not be ascertained because of a limited number of data points or limited dose range. In these cases, the data may be analyzed for the "combination index" on the basis of both mutually exclusive and mutually nonexclusive assumptions. In our studies, since Figure 3.8.1-4.a showed that the equations for inhibitor alone and their mixture were not parallel to each other, exclusivity of effects cannot be established. The causes were limited number of data points or limited dose range. In these cases, these data were analyzed for the 'combination index" on the basis of both mutually exclusive and mutually nonexclusive assumptions. Figure 3.8.1-4.b showed the calculated Log (CI) by assuming the inhibitors were mutually nonexclusive. These figures showed that Log (CI) stayed high above 0 within the lowest 120 range of total inhibition. For example, at 10% inhibition of cell proliferation, the LY294002 -U0126 combination had a Log (CI) of around 8, the LY294002 - SB203580 combination had a Log (CI) of around 3, the LY294002 - Bisindolylmaleimide I combination had a Log (CI) of around 12 and the LY294002 - cyclosporine combination had a Log (CI) of around 10. The higher the Log (CI) indicated the more antagonistic the two inhibitors. With increasing inhibition, all of the Log (CI) started to decrease, indicating that the two inhibitors were less antagonistic at higher combined doses. At certain percentages of total inhibition, all of the Log (CI) in our studies dropped below 0 and continuously dipped into the negative zone. It indicated that after a certain degree of inhibition, the two inhibitors exhibited increasing synergy. The total percentage of inhibition where Log (CI) began to drop below 0 as shown in figure 3.8.1-4 b were 50-60% for the LY294002 - U0126 combination, 60-70% for the LY294002 - SB203580 combination, 70-80% for the LY294002 - Bisindolylmaleimide I combination and around 90% for the LY294002 - cyclosporine combination. When we assumed that the inhibitors were mutually exclusive, the results of calculated Log (CI) had the same trend as of the results showed here (results and figures were not showed). 121 Table. 3.8.1: U0126 and LY294002 combination effects on CD4 + T lymphocyte proliferation 3[hj]TcR lixorporation Dose(iM) UO10JV1 5 1 0.5 0.1 0 LY OuM 1094 2120 5051 5010 4058 6545 LY0.1 2AT7 3490 3204 LY0.5 1609 1362 2906 2858 LY1 1288 1513 1991 2798 Figure 3.8.1.a Figure 3.8.1.b ro ro U) o Median Effect Plots LY : UO =1 : 1 Log Dose The Combination Index (CJ) LY:UO=1:1 Calculated Data FfealData o to o Antagonist NTT"1 r " 0 10 20 30 40 50 60 yif> 80- 90 1C» — * Synergistic % Inhibition Figure 3.8.1: Combined effects of LY294002 and U0126 on CD4 + T lymphocyte proliferation. Figure a was the median effect plots of the data presented on Table 3.8.1. The median effect equation was analyzed using linear regression. For LY294002, y=0.1128x+0.1341. m=0.1128, D5o=0.06474uM, the regression coefficient of r = 0.9874. For U0126, y=0.5131x-0.0885. m=0.5131, D50=1.487582uM, r=0.7584. For LY294002 and U0126 in combination (molar ratio 1:1), y=0.6384x+0.4328. m=0.6384, 122 D5o=0.357001uM, r=0.9284. Figure b was the computer generated graphical presentation of the Log (CI) with respect to fraction of inhibition. With increasing the fraction of inhibition, Log (CI) had a tendency to decline and dropped below 0 between 50-60% of inhibition. This indicated that at the lower range of inhibition, LY294002 and U0126 were antagonist to each other. Their antagonism gradually decreased with the increasing level of inhibition. Above 50-60% inhibition, these two inhibitors started to become synergistic. Table. 3.8.2: SB203580 and LY294002 combination effects on CD4 + T lymphocyte proliferation 3[hJ]TcR IrrxfLTrsticn DteefUVj LYIOlM 5 1 0.5 0.1 0 SBOiM 2366 4680 4531 4131 7311 14322 0.1 7580 13204 0.5 6723 10731 1 1574 2902 6738 11374 5 2788 1275 953 4435 10 598 1661 4G48 Figure 3.8.2.a Figure 3.8.2.b M ed ian Ef fect P lo ts LY : SB = 1:1 j/^*-^ LY 5 1 ^ rt A 6-L o g D o s e T h e C o m b i n a t i o n Index (Cl) - Calculated Data Real Data 3.5 3 2.5 2 at 1.5 O o 1 0.5 -1 -1.5 X Antagonistic A 0.5 fi in ?n 3Q 4n sn B Q u a an m n L 10 11 TO 0 ' Synergistic °/oZnhibition 123 Figure 3.8.2: Combined effects of LY294002 and SB203580 on CD4 + T lymphocyte proliferation. Figure a was the median effect plots of the data presented on Table 3.8.2. The median effect equation was analyzed using linear regression. For LY294002, y=0.2465x+0.313. m=0.2465, D5o=0.05373uM, the regression coefficient of r = 0.8352. For SB203580, y=0.7746x-0.3379. m-0.7746, D5 0=2.7304uM, r=0.9705. For LY294002 and SB203580 in combination (molar ratio 1:1), y=0.7319x+0.3274. m=0.7319, D5o=0.357001uM, r=0.9606. Figure b showed that with increasing the fraction of inhibition, CI had a trend to declination and dropped below 0 between 60-70% of inhibition. This indicated that at the lower range of inhibition, LY294002 and SB203580 were antagonist to each other. Their antagonism gradually decreased with the increasing level of inhibition. Above 60-70%) of inhibition, these two inhibitors started to become synergistic. 124 Table. 3.8.3: Bisindolylmaleimide I and LY294002 combination effects on CD4 + T lymphocyte proliferation 3[H]]TdR Incorporation Dose (uM) LYOuM LY0.1 LY0.5 LY 1 Bil 0 uM 6545 3204 2858 2798 Bil 0.1 1608 1368 1329 Bil 0.5 1364 819 530 373 Bil 1 1136 396 337 Figure 3.8.3.a Figure 3.8.3.b at o 0 Median Effect Plots LY : Bil = 1 :1 Log Dose Bil 14 12 10 8 § 6 e> o 4 _i 2 0 -2 -4 The Combination Index (Cl) Calculated Data Real Data \ Antagonistic ~ \ 6 10 20 30 40 50 60 70 ^ 9 0 100 % Inhibition Synergistic Figure 3.8.3: Combined effects of LY294002 and Bisindolylmaleimide I on CD4 + T lymphocyte proliferation. Figure a was the median effect plots of the data presented on Table 3.8.3. The median effect equation was analyzed using linear regression. For LY294002, y=0.1128x+0.1341. m=0.1128, D5o=0.06474uM, the regression coefficient of were antagonist to each other. Their antagonism gradually decreased with the increasing 125 level of inhibition. Above 60-70% of inhibition, these two inhibitors started to become synergistic. Table. 3 .8.4: Cyclosporine and LY294002 combination effects on CD4 + T lymphocyte proliferation 3[H]TdR Incorporation Dose (uM) LYOuM LY0.1 LY0.5 LY 1 CsA 0 uM 6545 3204 2858 2798 CsA 0.05 898 1278 692 CsA 0.1 759 917 471 CsA 0.5 700 601 391 CsA1 384 221 279 Figure 3.8.4.a Median Effect Plots o L Y : C s A = 1 :1 CsA Log Dose Figure 3.8.4.b The Combination Index (Cl) CaaiatedCaa • RslQia 12 10 8 § 6 O O 4 2 0 -2 V Arfcgxistic 0 10 2 0 3 0 4 0 5 0 6 0 7 0 ao 90 1^00 110 %lnhibrtiori Synergstic 126 Figure 3.8.4: Combined effects of LY294002 and cyclosporine on CD4 T lymphocyte proliferation. Figure a was the median effect plots of the data presented on Table 3.8.3. The median effect equation was analyzed using linear regression. For LY294002, y=0.1128x+0.1341. m=0.1128, D5o=0.06474 uM, the regression coefficient of r = 0.9874. For cyclosporine, y=0.2553x+01.118. m=0.2553, D50=4.1767E-5 uM, r=0.999. For LY294002 and cyclosporine in combination (molar ratio 1:1), y=0.5672x+1.1874. m=0.5672, D5o=0.008064 uM, r=0.999. Figure b shows that with increasing the fraction of inhibition, CI had a trend to declination and dropped below 0 around 90% of inhibition. This indicates that LY294002 and cyclosporine were antagonist to each other. Only when their combination effect reached to 90% of inhibition, these two inhibitors started to become as synergistic. 127 CHAPTER FOUR: DISCUSSION Understanding the process of T cell activation has led to the development of novel immunomodulators for transplant rejection. Several essential signaling pathways in T cell activation have been identified in recent years, including the C a 2 + - calcineurin pathway, P K C pathway, M A P K pathway and PI3-kinase pathway. They are regulated by activation signals through the TCR and /or the costimulatory molecule CD28. Calcineurin inhibitors Cyclosporine and FK506 reduce T cell activation by blocking C a 2 + - calcineurin pathway and have been used successfully as clinical immunosuppressants in transplant rejection. However, Cyclosporine and FK506 are associated with side effects, including infections, malignancies, nephrotoxicity and metabolic disorders (276). The long-term morbidity and mortality of these agents still remain a substantial problem, and only 50% of cadaver renal allograft surviving the first year are still functioning after 10 to 13 years (277). The principle objective of the current study was to examine the effects of blocking the P K C pathway, the M E K / E R K pathway, the p38 M A P kinase pathway and the PI3-kinase pathway in T cell activation using specific kinase inhibitors. Detailed reports of their effects on T cell proliferation, cytokine production and cytokine receptor expression, as well as on modulating transcription factor activation, together with the combination effects of these inhibitors on T cell proliferation, have been documented. These results may provide useful information for their future development as potential immunosuppressants. 128 As a first step, an in vitro T cell activation model was established. This was achieved by isolating human peripheral CD4 + T lymphocyte and stimulating them through TCR/CD3 complex with the costimulatory B7/CD28 signal. Most of the existing in vitro T cell activation models used mouse or human T cell clones as the primary system and stimulated them with mitogen such as PHA, Con A or P M A . There are at least two disadvantages of these models. Firstly, signaling pathways in T cell clones could have been modified, and therefore may not reflect the natural signal process of T cell activation. Secondly, although mitogens can evoke progressive T cell clonal expansion, the signaling networks they activated are not clearly defined. Meanwhile, signaling pathways in T cell activation could also be bypassed in mitogen stimulation as suggested in P M A activated T cells (278). Stimulating human peripheral CD4 + T cells with monoclonal antibodies against TCR/CD3 complex and costimulatory molecule CD28 is based "the two signal model" and mimics the in vivo T cell activation process during rejection. A body of literature has reported the use of cytokine secretion and cell surface molecule expression as indicators of T cell activation. These include the cytokines IL-2, IL-4, IL-5, IL-6, IL-12, IFN-y, TNF-a, and TNF-P and cell surface molecule CD25, CD69 and CD40L. Since IL-2 secretion and IL-2 receptor a chain (CD25) expression have been described as being prominent and consistent in T cell activation, these two criteria, as well as T cell proliferation measured by [HJTdR incorporation, were used to demonstrate the activation status of T cells in our model. Furthermore, besides the percentage of CD25 expression, we included a second criterion measured as the mean 129 log fluorescent channel of CD25 (mlfc). This represents the average density of CD25 molecule on each CD25 positive cell. The combination of CD25% and mlfc could provide more information about T cell activation status. During the 3-day T cell activation process, T cell proliferation and CD25 expression were increased and their kinetics corresponded to each other, as confirmed by other T cell activation models (279-281). However, the kinetics of the extracellular IL-2 secretion differed markedly from those of T cell proliferation and CD25 expression, as has been observed in APCs activated B6 spleen cells (281). These results showed that CD25 expression was an important marker throughout the T cell activation process while IL-2 secretion represented only the early phase of T cell activation. The relationship of IL-2 secretion to T cell proliferation and CD25 expression underlined the paradoxical function of IL-2 and its relationship with its high affinity receptor. IL-2 is a growth factor for T cell activation, but prolonged exposure of the T cells to high-dose IL-2 leads to activation-induced cell death (AICD) and down-regulation of the T cell population (282). IL-2 conveys its signal through its high affinity receptor, of which a chain (CD25) is the key component. There is evidence to suggest that CD25 is critical for the sensitivity of IL-2 induced T cell proliferation and AICD (283). Our results indicated that to facilitate T cell activation and proliferation, IL-2 needed to provide a steady but not an exclusive signal through its high affinity receptor. This could be done by regulating the time and/or the magnitude of IL-2 and CD25 production. As described by Krauss S et al (234), the proportion of energy spent on signaling 130 pathways in T cell activation differs. In a T cell activation model stimulated with mitogen ConA, the PTK/PLC-y/PKC pathway downstream of the TCR signal consumed about 84% of the energy employed. This could be further divided into 30% on Ca calcineurin pathway and 54% on the P K C pathway including the M A P kinase cascade. This suggested that blocking different signaling pathways could induce substantially different scales of inhibition to T cell activation. Meanwhile, a CD28 signal is required for IL-2 secretion and full activation of T cells (106). The activation signal provided by CD28 is Cyclosporine resistant (121). It is therefore interesting to investigate the effects of inhibitors to the CD28 signaling pathways as well as their combination with inhibitors to the TCR signaling pathways in T cell activation and transplant rejection. While CD28 couples to a plethora of signaling molecules including PTKs such as Lck, Fyn and itk, PLC, sphingomyelinase, P21 r a sand PI3-kinase (106, 190), PI3-kinase has been suggested to play a pivotal role in CD28-mediated T cell activation and IL-2 secretion (190). Thus, an inhibitor specific for PI3-kinase was included in our investigation. In light of these considerations, we selected the P K C inhibitor Bisindolylmaleimide I, the M E K inhibitor U0126, the p38 M A P kinase inhibitor SB203580 and the PI3 kinase inhibitor LY294002 as the agents to block T cell activation. We have shown that the effects of blocking the M E K / E R K pathway, the p38 M A P kinase pathway, the P K C pathway and the PI3-kinase pathway have translated into decreased proliferation of T cells, and that proliferation was blocked at the level of IL-2 and IL-2 receptor a chain (CD25) production. Our results are in agreement with the data shown in T cells or T cell clones using ERK, P K C and PI3 -kinase inhibitors in terms of IL-2 131 production and T cell proliferation (246, 251, 284, 285). Though the p38 M A P kinase inhibitor has been reported not to influence T cell proliferation and IL-2 production in an IL-2 dependent T cell clone (251), our results have been confirmed elsewhere using U0126 in normal T cells (274). The inhibition of p38 M A P kinase dependent production was further demonstrated in a study showing that pretreatment of cells with SB203580 suppressed the transcriptional activation of the IL-2 promoter (285). Significant reduction of CD25 expression by all inhibitors in our study demonstrated the importance of E R K pathway, p38 M A P kinase pathway, P K C pathway and PI3-kinase pathway in the expression of CD25. Few studies directly, illustrated the signal transduction pathways linked to CD25 expression upstream of its gene transcription. One study confirmed our results, showing reduced IL-2 receptor expression using P K C inhibitor H-7 in PHA stimulated T cell activation (286). IL-2 can induce CD25 expression through activating the transcription factor STAT5 (148). Our results in Figure 3.4.1-2 demonstrated that the reduction of CD25 expression was not entirely due to the reducing of IL-2 in the cell culture. Of the two CD25 expression measures checked in this study, the reduction in CD25 positive cells was largely independent of IL-2, indicating that proportion of cells expressing CD25 molecule was largely dependent on the activating signals downstream of TCR/CD28. Yet the suppression of mlfc CD25 was more IL-2 dependent, indicating that the concentration of the CD25 molecule on CD25 positive T cells was dependent on signals from both TCR/CD28 and from the IL-2 cascade. Results in Figure 3.1.c showed that though these inhibitors reduced T cell activation 132 and proliferation, none of them is inherently toxic to activated T cells through either cell apoptosis or cell necrosis (checked by cell viability, data not shown). In fact, the PT3-kinase pathway and the M E K / E R K pathway are essential for cell survival in tumor cell lines stimulated with growth factors, cytokines or cultured with cytotoxic agents. One of the immediate downstream signaling events of PI3-kinase is the activation of protein kinase B (PKB), the human homolog of the transforming v-Akt, which is essential for cell survival (287, 288). Intriguingly, one substrate for PKB/Akt is the Bcl-2 family member Bad; phosphorylation of Bad prevents its pro-apoptotic ability. The same site in Bad also can be phosphorylated by p90 r s k, a downstream effector of the E R K 1/2 pathway (289). It has been reported that the PI3-kinase inhibitor LY294002 totally suppressed IGF-1-mediated protection from Fas killing in activated tumor T cell line (290), and another specific M E K inhibitor PD98059 dramatically enhanced the rate of apoptosis induced by vinblastine (291). The role of the P K C pathway in cell survival and apoptosis is not very clear. It might support cell survival through the E R K pathway (165). One study also demonstrated that the cytotoxic effect of a P K C inhibitor (PKC412) is mediated via downregulating PI3K/Akt pathway (292). However, our results have shown that blocking P K C , M E K and PI3-kinase pathways did not induce apoptosis, indicating that none of these three pathways is critical for survival of normal T cell after activation through the TCR/CD28 signals. Since all these pathways are activated in T cell proliferation, the loss of some survival signals by blocking one of the pathways could be compensated by the activation of other pathways. The role of P38 M A P kinase pathway in cell survival and apoptosis has been reported 133 to differ from the other three pathways. It seemed to play a pro-apoptotic role since many cytotoxic agents activated p38 M A P kinase (228). This was supported in our study by the anti-apoptotic function of the p38 M A P kinase inhibitor SB203580. Figure 3.3.4 showed that the reduction of CD25 expression by the inhibitors employed was dose dependent. The concentration that inhibits the activity of its targeted protein kinase is usually much lower than that required blocking T cell activation. Data provided by CalBiochem showed that U0126 inhibits MEK1 activity with IC 5 0=72nM, inhibits M E K 2 activity with IC 5 0=58nM; SB203580 inhibits p38 M A P kinase activity with IC 5 0=600nM; Bisindolylmaleimide I inhibits P K C subsets with IC5 0=8-200nM; LY294002 inhibits PI3-kinase activity with IC5o=1.4uM. However, when used to block T cell activation, these inhibitors must be present in cell culture for hours to days at much higher doses (25J, 274, 293). Used at these higher doses, inhibitors might lose their kinase specificity. One example of this is observed with SB203580. Though it does not significantly inhibit I N K or p42 M A P kinase activity even at dose of lOOuM (Data provided by CalBiochem), it starts to inhibit the activity of the p54 M A P kinase isoform c-Jun N-terminal kinase 2 at dose higher than l u M (294). Therefore, SB203580 used in our study might also block kinase activity other than p38 M A P kinase. However, U0126, LY294002 and Bisindolymaleimide I had not been reported to affect other kinase activity at the dose used in this study. Another interesting phenomenon shown in our study is the time dependence of the response. Adding of all four inhibitors 24 hours after stimulation reduced their 134 inhibitory effects on both the percentage and mlfc of CD25 expression. This suggested that all inhibitors blocked principally the early events in human primary T cell activation. A similar results have been demonstrated in a study where addition U0126 24 hours after T cell activation diminished its inhibition of T cell proliferation and prevented PBLs from entering into the S phase of the cell cycle (251). Since IL-2 is mainly secreted in the first 24 hours of T cell activation, addition of these inhibitors 24 hours after stimulation may not prevent the activation of IL-2 signaling cascade. By binding to its high affinity receptor, IL-2 can activate the PI3-kinase pathway (295, 296), M A P kinase pathway (297) and some protein kinase C isoforms via calcium-and DAG-independent pathways (298). Yet our study showed that inhibitors to these pathways did not significantly reduce T cell activation. This indicated that these pathways might not be exclusive for the IL-2 cascade in normal T cell activation. Other signaling pathways such as the JAK-STAT pathway might play a more essential role in IL-2 induced T cell activation (152). The results of our study also showed that inhibition of EL-2 production and IL-2 receptor a chain (CD25) expression by blockade of the E R K pathway, p38 M A P kinase pathway, P K C pathway and PI3-kinase pathway occurred at the gene transcription level. It has been suggested that N F - A T and AP-1 are two of the important transcription factors for IL-2 gene transcription (126) and N F - K B and STAT5 are two of the important transcription factors for CD25 gene transcription (148, 149). Our study showed that the trend of N F - A T binding activity paralleled the kinetics of IL-2 secretion. In fact N F -135 AT has also been reported to bind to the CD25 promoter region (299). The reverse trend of NF-AT to CD25 expression indicated that NF-AT induced predominantly the early stage of CD25 gene transcription. AP-1 binding activity in this study was in parallel with the kinetics of T cell proliferation but did not mimic the kinetics of IL-2 production. This reflected that AP-1 was not only important for IL-2 transcription but might also regulate the expression of other proteins in T cell activation as suggested by others (300). The trend of N F - K B binding activity corresponded with the kinetics of CD25 expression. Yet, STAT5 binding activity did not increase until 24 hours after stimulation, which lagged behind the CD25 expression. This suggested that although N F - K B was important for the whole process of CD25 expression, STAT5 played its role in the late stages of CD25 expression. In fact, STAT5 is mainly activated by IL-2 signaling through J A K kinase (152) after IL-2 secretion in the late stages of T cell activation. Our study showed that blocking M E K , p38 M A P kinase, P K C and PI3-kinase inhibited NF-AT and AP-1 binding activity. Signaling pathways that lead to NF-AT activation have been extensively investigated in T cells. TCR activation results in calcium mobilization from internal stores by opening IP3R channels. A later, long-lasting calcium influx from external stores leads to a sustained activation of calcineurin (CN), dephosphorylates NF-AT and promotes NF-AT nuclear translocation (132). P K C could regulate NF-AT activity through its calcineurin dependent pathway (301). In fact, one 136 study has shown that Bisindolylmaleimide I inhibited the reaccumulation of NF-ATp in the cytoplasm and its re-phosphorylation after ionomycin removal (301). This confirmed our finding of blocking NF-AT activation by using Bisindolylmaleimide I. The direct role of PI3-kinase in regulating NF-AT activity is not clear in the T cells. Research has shown that downregulating activity of the protein kinase, GSK-3, correlated with increasing NF-AT activity in T cells (302). However, the mechanism of GSK-3 regulation in T cells remains unclear. The current paradigm worked out in non-T cells (303) showed that phosphatidylinositol 3-kinases (PI3K) down regulated GSK-3 activity by activating P K B . Further investigation needs to be done to demonstrate the kinase linking between PI3-kinase and NF-AT activation in our T cell model. The functions of E R K and p38 pathways in regulating NF-AT activation are also not clearly defined. Genot et al reported that the expression of dominant negative M E K 1 , an upstream kinase of ERK-2, prevents NF-AT induction (178), whereas, Wu et al showed that inhibition of p38 M A P K led to selective inactivation of NF-AT in T cells (304). These studies supported our finding that M E K / E R K and p38 M A P kinase played a role in regulating NF-AT activation. Signaling pathways regulating AP-1 activity are relatively clearly defined in T cells. Studies have shown that activated E R K phosphorylated ELK-1 and led to the transcriptional activation of the cFos and cJun genes, which are involved in transcriptional regulation of AP-1 activation (139, 181). p38 M A P kinase also regulates 137 transcription factor cFos through ELK-1 (254). An isoform of P K C , PKCG was reported to activate I N K (301), which subsequently activated c-Jun, therefore influencing AP-1 activity (305). PI3-kinase may regulate AP-1 through activation of ERK, INK and p38 M A P kinase pathways (306). Thus, blocking ERK, P38 M A P kinase, P K C and PI3-kinase will reduce the activation of AP-1, as demonstrated in our study. The primary activation of N F - K B involves modification of a pre-existing complex rather than de novo synthesis dependent on a signaling complex called I K B kinases ( I K K ) (202, 203). The activation of I K K complex initiates the phosphorylation of the inhibitory proteins IxBa and I K B P , which leads to the release of N F - K B transcription factors. These factors enter the nucleus and initiate transcriptional activation of genes involved in cellular proliferation and survival (202-204). Our study showed that blocking PI3-kinase but not ERK, p38 M A P kinase or PKC, reduced N F - K B binding activity. This indicated that the regulation of N F - K B activity was through CD28 signaling rather than through TCR signaling in T cells. The role of CD28 signal on N F - K B activity was also reported elsewhere (205). The mechanism by which CD28 ligation induces N F - K B activation is not well understood. In primary T lymphocytes, ligation of CD28 may induce a rapid formation of the oxygen species (ROS) by activating phospholipase A2 and D5-lipoxygenase(20r5). ROS could then rapidly degrade I K B molecules (207) through an as yet incompletely characterized serine/threonine protein kinase termed CHUK. Whether the blocking of 138 N F - K B activation by PI3-kinase inhibitor in our study is through phospholipase A2 and D5-lipoxygenase still need more investigation. There are also reports showing the influence of P K C pathways in N F - K B activation. PKC9 contributes to the activation of the I K K complex through its ability to phosphorylate I K K P (210-212), and pharmacological interference in PKC0 activity abrogates N F - K B activation and IL-2 production (210). Based on inhibition by Cyclosporine A and on transfection studies, the C a 2 + dependent phosphatase calcineurin is involved in the activation of N F - K B in T cells. However, the target of calcineurin is unknown (213). Yet, using the specific P K C inhibitor Bisindolylmaleimide I in our study did not reduce N F - K B activation, which indicated that P K C pathway was not essential in regulating N F - K B in this primary human T cell model. Stat5 protein is mainly activated through cytokine receptors (214). However, our study suggested that it might also be regulated by TCR/CD28 signals through the M A P kinase cascade and PI3 -kinase pathway. Only a few studies investigated in this area. A transient phosphorylation of Stat5 was observed after TCR crosslinking, as was phosphorylation of the TCR£, chain bound Stat5 by Lck (215). Furthermore, antigen receptor ligation induced delayed but sustained phosphorylation of Statl on Ser727, which was dependant on phosphatidylinositol-3-kinase mediated signals (216). More studies need to be done to illustrate the pathways link to STAT protein activation under TCR/CD28 molecules. 139 Since blocking either TCR or CD28 signals reduces T cell activation, combination of inhibitors affecting these two pathways may exaggerate their inhibitory effects. CTLA-4 Ig blocks The CD28 signal and has been tested in combination with Cyclosporine in transplantation rejection (238). Our data showed that the combination of LY294002 with U0126 or SB203580 or Bisindolylmaleimide I increased the inhibition of T cell proliferation. This can be understood since CD28 signals enhance TCR signals in T cell activation and proliferation. Review of the raw data suggested a minor degree of antagonism at certain lower concentrations of both agents. When the median effect equation was used to examine the relationship of the two inhibitors, LY294002 appeared to operate as an antagonist to its paired partners at low level of T cell inhibition and became synergistic as inhibition increased. This discrepancy could be caused by the small number of replicates and the relatively large variations of the experimental data and correspondingly low r values in the study. The mathematical model described by the median effect equation may not totally reflect biological events and further studies are required to confirm or refute this interaction. In addition, since the median effect equation are derived from generalized mass action considerations, they do not define specific mechanisms. Therefore, the dose-relationships of LY294002 with TCR signal inhibitors reported here can only be considered as a reference for the dose to be used in future combination studies. They do not indicate any mechanism by which these inhibitors interact to influence T cell activation and proliferation. The inhibitory effects of Cyclosporine were the weakest among the inhibitors tested in our study in terms of T cell proliferation and CD25 expression. This indicated that in 140 our T cell activation system, a greater proportion of energy was spent in the M E K / E R K , p38 M A P kinase, P K C and PI3-kinase pathways than in the Ca2 +-calcineurin pathway. In fact, P K C and PI3-kinase can activate both calcineurin dependent and calcineurin independent pathways. It has also been reported that Cyclosporine blocks anergy induction in T cells (307) and has been shown to interfere with the deletion of thymocytes bearing self-reactive TCRs in vivo (308). However, inhibitors such as U0126 had no influence on anergy induction (251). Therefore, they may function as a more efficient immunosuppressant than Cyclosporine in transplant rejection. B lymphocytes also play an important role in transplant rejection. B cell activation requires antigen binding to the B cell receptor (BCR) and costimulatory signaling through binding of cytokines (such as IL-2) and cell surface molecules (such as CD40 ligand) from activated T helper cells. After activation, B lymphocytes proliferate and differentiate into antibody secreting plasma cells. Antibodies facilitate the destruction of the allograft by activating the complement cascade and by acting as tags that promote endocytosis, phagocytosis and /or killing by macrophages, neutrophils, and other cells (25). The signaling networks in B lymphocyte activation are complicated and involve many signaling pathways similar to those described in T lymphocyte activation such as C a 2 + - calcineurin, P K C , M E K - E R K , p38 Map kinase, JNK and PI3 kinase pathways. Many studies have examined the effect of existing immunosuppressants such as cyclosporine and FK506 on B lymphocyte antibody secretion in animal models and in transplant patients. However, none has indicated whether inhibition was direct acting 141 solely on the B lymphocyte or indirect reflecting the T lymphocyte suppression. Yamaoka and K i m et al described in one study that cyclosporine and FK506 directly reduced in vitro primary B cell proliferation at high concentration. Yet, there is no report on the direct effect of these immunosuppressants on B cell antibody secretion. In recent years, researchers have started to investigate the effects of blocking PKC, M E K - E R K , p38 Map kinase as well as PI3 kinase pathways on B lymphocyte activation using various kinase inhibitors. The results have shown a direct influence of these pathways on B cell activation on certain downstream molecules. For example, blocking PI3 kinase pathway using LY294002 inhibited the activation of cyclin D (309) and N F -K B (310) as well as inducing apoptosis (311) in primary B cell or B cell clones. However, few studies have shown a direct influence of blocking PI3 kinase or other signaling pathways on B cell proliferation and antibody secretion. 142 C H A P T E R F I V E : S U M M A R Y T cell activation requires signals from both TCR and CD28 molecules. Several essential signaling pathways need to be activated to transduce the activation messages from cell surface to the nucleus. Key among these are the C a 2 + - calcineurin pathway, P K C pathway, and M A P K pathway downstream from the TCR molecule and PI3-kinase pathway downstream from the CD28 molecule. T cell activation is the key immunological process in transplant rejection. Blocking recipient T cell activation can prevent graft rejection and increase graft survival and patient survival. This can be achieved by blocking one of the signaling pathways using specific kinase inhibitors, as has been proven by the successful clinical use of the specific calcineurin inhibitor cyclosporine and tacrolimus. This study was designed to examine the effects of blocking the P K C pathway, M E K / E R K pathway p38 M A P kinase pathway and PI3-kinase pathway using specific kinase inhibitor Bisindolylmaleimide I, U0126, SB203580 and LY294002, respectively, on T cell activation and proliferation. We first established a cell model in vitro to mimic the T lymphocyte activation process in graft rejection. This was achieved by stimulation of isolated human peripheral CD4 + T lymphocytes through the T cell receptor and the costimulatory molecule CD28. The data 143 reported here have confirmed that blocking PKC pathway, M E K / E R K pathway and p38 M A P kinase pathway and PI3- kinase pathway greatly reduced T cell proliferation. This inhibition was demonstrated at the level of IL-2 production and IL-2 receptor a chain (CD25) expression and exhibited a dose dependent and a time dependent response. The CD25 expression in our study was largely independent of IL-2. The PKC, M E K / E R K , p38 M A P kinase and PI3-kinase pathways were important to regulate CD25 expression after TCR/CD28 stimulation. These pathways were not essential to IL-2 induced CD25 expression. The suppression of IL-2 production and CD25 expression was evident at the transcription factor level. NF-AT and AP-1 are two of the important transcription factors for IL-2 gene transcription and N F - K B and STAT5 are two of the important transcription factors for CD25 gene transcription. Blocking all four pathways markedly reduced the binding activity of NF-AT and AP-1, yet blocking only the PI3-kinase pathway caused a dramatic reduction in N F - K B binding activity. STAT5 binding activity was partially reduced by blocking the M E K / E R K , p38 M A P kinase and PI3-kinase pathway. This information was also summarized in Figure 3.7.6. Combination of the CD28 pathway inhibitor LY294002 with one of the TCR pathway inhibitors Bisindolylmaleimide I, U0126 or SB203580 magnified the suppression of T cell proliferation. The dose relationship study showed that LY294002 worked as an antagonist to the TCR pathway inhibitor at the lower end of inhibition and became 144 more and more synergistic as T cell inhibition increased. Our study also showed that none of the inhibitors were toxic to activated human T cells, and all induced more profound suppression to T cell proliferation and CD25 expression than cyclosporine. These data indicated that the PKC, M E K / E R K , p38 M A P kinase and PI3-kinase pathways are important for T cell activation. 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