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The role of B7-H4 in islet transplantation Wang, Xiaojie 2011

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THE ROLE OF B7-H4 IN ISLET TRANSPLANTATION  by  XIAOJIE WANG M.Sc., The University of British Columbia, 2000  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2011  © Xiaojie Wang, 2011  ABSTRACT Allogeneic pancreatic islet transplantation has the potential to cure type 1 diabetes. One of the barriers to islet transplantation is the alloreactive T-cell response between donors and recipients. Co-stimulatory molecules, which play a major role in regulation of the immune response during graft rejection, may be used to inhibit allograft destruction and generate tolerance. B7-H4 is one such member in the co-stimulatory family to negatively regulate T-cell responses. However, its role in the transplantation field has not been investigated. The aim of this study is to determine the function of B7H4 in modulating an alloreactive immune response and its signal transduction pathway. The role of B7-H4 in alloimmunity was assessed using a fully major histocompatibility complex mismatched mouse islet transplantation model. Expression of B7-H4 by a recombinant adenovirus in donor allograft islets significantly improved their survival. Moreover, prolonged graft survival was observed in the absence of immunosuppression after secondary transplant, suggesting development of donor specific tolerance. B7-H4 controlled alloreactive immune response by limiting infiltrating cells and increasing Foxp3 expressing cells in the local graft. B7-H4 treatment significantly modulated naturally occurring and inducible regulatory T cells in the periphery and generated hyporesponsiveness to alloantigen. Study of the mechanism by which B7-H4 inhibits T cells revealed interference with activation of ERK, JNK, and AKT, but not through CD3/TCR proximal signal ZAP70 or LCK. This study advances our understanding of the role of the novel co-inhibitory molecule B7-H4 in alloimmune responses. Local expression of B7-H4 prolongs islet  ii  allograft survival and generates donor-specific tolerance in vivo, suggesting translational potential for use in reducing immune injury during islet transplantation.  iii  PREFACE List of publications: 1. Wang X, Hao J, Metzger DL, Mui A, Ao Z, Verchere CB, Chen L., Ou D., Warnock GL. B7-H4 induces donor specific tolerance in mouse islet allografts. Cell Transplantation in press. 2. Wang X., Ou D., Ao Z., C.B. Verchere, Meloche M., Johnson JD., Mui A., Warnock GL. Hope and challenges for islet transplantation in the treatment of type 1 diabetes. Handbook of type 1 diabetes mellitus: etiology, diagnosis, and treatment. Nova science publishers Inc. 2009 Chapter 15:403-418. 3. Wang X, Hao J, Metzger DL, Mui A, Ao Z, Verchere CB, et al. Local expression of B7-H4 by recombinant adenovirus transduction in mouse islets prolongs allograft survival. Transplantation 2009 Feb 27;87(4):482-490. 4. Warnock GL, Liao YH, Wang X, Ou D, Ao Z, Johnson JD, et al. An odyssey of islet transplantation for therapy of type 1 diabetes. World J.Surg. 2007 Aug;31(8):1569-1576. A version of Chapter 3 has been published (#3). I designed and wrote the first draft of the manuscript. The surgical work of islet transplantation and part of data analysis was performed by Dr. Jingqiang Hao. I conducted all the experiments and analyzed data. Drs. Metzger, Chen, Ou, Mui, and Warnock edited the manuscript. Chapter 4 was accepted by Cell Transplantation (#1). Dr. Hao performed islet transplantation and part of data analysis. I designed, conducted all the experiments, analyzed data, and wrote manuscripts. Drs. Metzger, Hao, Ao, Verchere, Chen, Ou, Mui, and Warnock participated in designing experiments, analyzing data, and editing manuscript. UBC animal care committee approved Animal Care Certificate (#A06-0008) on April 1st, 2006 to Dr. Garth Warnock. UBC biohazards committee approval Biohazard Approval Certificate (#H07-0037) on Jan 22nd, 2008 to Dr. Garth Warnock.  iv  TABLE OF CONTENTS ABSTRACT ............................................................................................................ii  PREFACE............................................................................................................... iv  TABLE OF CONTENTS........................................................................................ v  LIST OF TABLES ................................................................................................. ix  LIST OF FIGURES ................................................................................................ x  LIST OF ABBREVIATIONS ..............................................................................xii  ACKNOWLEDGEMENTS .................................................................................. xv  CHAPTER 1: BACKGROUND AND AIMS OF THE STUDY ......................... 1  1.1 TYPE 1 DIABETES.................................................................................................2 1.1.1 Pathogenesis of type 1 diabetes ........................................................................2 1.1.2 Clinical aspects of type 1 diabetes ....................................................................3 1.1.3 Therapies for type 1 diabetes ............................................................................3 1.2 ISLET TRANSPLANTATION...............................................................................4 1.2.1 History of islet transplantation..........................................................................5 1.2.2 Barriers in islet transplantation .........................................................................6 1.2.3 Limitations of current immunosuppressive drugs.............................................8 1.3 TRANSPLANTATION REJECTION ...................................................................9 1.3.1 Types of transplantation....................................................................................9 1.3.2 Types of rejection ...........................................................................................10 1.3.3 Mechanisms of graft rejection ........................................................................10 1.3.3.1 Role of the major histocompatibility complex (MHC)........................11 1.3.3.2 Role of T cells in transplant rejection ..................................................12 1.4 COSTIMULATION BLOCKADE IN ISLET TRANSPLANTATION ...........14 1.4.1 Rationale for using costimulatory molecules in islet transplantation .............14 1.4.2 B7 family ........................................................................................................15  v  1.4.3 CD28/CTLA-4:B7 pathway............................................................................18 1.4.3.1 CD28 and CTLA-4: positive and negative costimulators....................18 1.4.3.2 CTLA-4 in transplantation...................................................................18 1.4.3.3 Mechanisms of action for CTLA-4......................................................19 1.4.3.4 Treg and CTLA-4 ................................................................................20 1.4.4 PD-1:B7-H1 pathway......................................................................................21 1.4.5 ICOS:B7-H2 pathway.....................................................................................22 1.4.6 B7-H4 pathway ...............................................................................................23 1.4.6.1 Gene organization of B7-H4................................................................24 1.4.6.2 Expression pattern of B7-H4 ...............................................................25 1.4.6.3 B7-H4 orphan receptor ........................................................................26 1.4.6.4 Mechanisms of B7-H4 engagement.....................................................26 1.5 TOLERANCE STRATEGIES..............................................................................27 1.5.1 Central tolerance .............................................................................................27 1.5.2 Peripherial tolerance .......................................................................................28 1.5.3 Tregs and tolerance .........................................................................................28 1.6 TCR SIGNAL TRANSDUCTION .......................................................................30 1.7 THESIS OBJECTIVES.........................................................................................33  CHAPTER 2: METHODS AND MATERIALS  ...............................................35  2.1 ANIMALS...............................................................................................................36 2.2 ISLET ISOLATION ..............................................................................................36 2.3 PRIMARY AND SECONDARY ISLET TRANSPLANTATIONS ..................36 2.4 GENERATION OF RECOMBINANT B7-H4 (Ad-B7-H4) ..............................37 2.5 RNA ISOLATION AND REAL-TIME PCR ANALYSIS .................................37 2.6 WESTERN BLOT .................................................................................................38 2.7 GLUCOSE STIMULATION ASSAY ..................................................................40 2.8 INTRAPERITONEAL GLUCOSE TOLERANCE TEST (IPGTT) ................40 2.9 MIXED LYMPHOCYTE REACTION ASSAY (MLR) ....................................40 2.10 HISTOLOGICAL ANALYSIS...........................................................................41 2.11 FLOW CYTOMETRY........................................................................................42 2.12 T CELL STIMULATION ...................................................................................42 vi  2.13 GENERATION AND PURIFICATION OF B7-H4.Ig FUSION PROTEIN .43 2.14 [3H]-THYMIDINE INCORPORATION ASSAY .............................................43 2.15 CYTOKINE DETECTION.................................................................................44 2.16 STATISTICAL ANALYSIS ...............................................................................44  CHAPTER 3: THE ROLE OF B7­H4 IN ISLET ALLOGRAFT REJECTION45  3.1 BACKGROUND ....................................................................................................46 3.2 RESULTS ...............................................................................................................47 3.2.1 B7-H4 expression in Ad-B7-H4–transduced primary islets ...........................47 3.2.2 B7-H4 recombinant adenovirus transduction does not affect β cell function.51 3.2.3 Local B7-H4 expression in islets prolongs allograft survival.........................53 3.2.4 B7-H4 is expressed transiently in the islet grafts............................................55 3.2.5 Immune responses and β-cell function in the islet grafts................................57 3.2.6 Lymphocyte responses to alloantigen stimulation in B7-H4 recipients .........59 3.3 SUMMARY OF FINDINGS AND DISCUSSION ..............................................60  CHAPTER 4: THE ROLE OF B7­H4 IN GENERATING TOLERANCE .....62  4.1 BACKGROUND ....................................................................................................63 4.2 RESULTS ...............................................................................................................64 4.2.1 A regulatory phenotype develops in B7-H4 treated grafts and periphery ......64 4.2.2 MLR demonstrates attenuated proliferation in B7-H4 treated recipients.......66 4.2.3 The presence of CD4+CD25+ T Cells is required for hyporesponsiveness.....66 4.2.4 B7-H4 inhibits expression of pro-inflammatory cytokines in the grafts ........69 4.2.5 Second-set islet allografts survive after removal of long-term surviving Ad-B7-H4–transduced islet grafts ...........................................................................70 4.2.6 Inflammation, beta cell function, and immune infiltrates are distinct in the secondary transplantation recipients. ..............................................................72 4.3 SUMMARY OF FINDINGS AND DISCUSSION ..............................................76  CHAPTER 5: B7­H4 SIGNAL PATHWAY.....................................................81  5.1 BACKGROUND ....................................................................................................82  vii  5.2 RESULTS ...............................................................................................................84 5.2.1 B7-H4 engagement inhibits T Cell .................................................................84 5.2.2 B7-H4 engagement inhibits IL-2 production and CD25 expression .............90 5.2.3 B7-H4 engagement inhibits expression of CD69 ...........................................93 5.2.4 ERK activity is reduced upon B7-H4 engagement .........................................95 5.2.5 JNK activity is reduced upon B7-H4 engagement..........................................95 5.2.6 AKT activity is reduced upon B7-H4 engagement .......................................96 5.2.7 B7-H4 does not affect early parameters of TCR triggering............................99 5.3 SUMMARY OF FINDINGS AND DISCUSSION ............................................101  CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS ................... 104  REFERENCES ................................................................................................... 111   viii  LIST OF TABLES Table 1.1 Expression patterns, function, and receptors of B7 family ligands.................17 Table 2.1 PCR primer pairs.............................................................................................38  ix  LIST OF FIGURES Figure 1.1 Genomic organization of human and mouse B7-H4 genes ……....………..24 Figure 1.2 TCR-mediated signal transduction ……………………….……....………..32 Figure 3.1 B7-H4 expression in primary mouse islets transduced by Ad-B7H4………48 Figure 3.2 Expression of B7-H4 on isolated islets after recombinant Ad-B7-H4 transduction………...………...…………....…………....…………....…………....……..50 Figure 3.3 Influence of Ad-B7-H4 transduction on glucose-stimulated insulin secretion in islets…...………...…………....…………....…………....…………....……..51 Figure 3.4 Blood glucose of recipients transplanted with syngeneic donor islets which were treated with adenovirus………………………………………………………….....52 Figure 3.5 Prolongation of allograft survival in streptozotocin-induced diabetic recipient C57BL/6 mice transplanted with Ad-B7-H4–transduced Balb/c mouse islets...54 Figure 3.6 B7-H4 expression in the transplanted islet grafts transduced with Ad-B7H4………………………………………………………………………………………...56 Figure 3.7 Inflammatory infiltration and residual β cells in the islet grafts transplanted with Ad-LacZ– or Ad-B7-H4–transduced islets..………………………….58 Figure 3.8 Allogeneic mixed-lymphocyte reaction of transplanted mice with Ad-B7-H4–treated islets in comparison with that of non-transplanted wild type mice…59 Figure 4.1 High levels of Foxp3, Tregs, and IL-10 were found in the grafts and periphery……………………………………………. …………………………………..65 Figure 4.2 Mixed lymphocyte reaction for lymphocytes from long-term surviving recipients with Ad-B7-H4–transduced islets showed hyporesponsiveness…………...…68 Figure 4.3 Cytokine RNA expression in the grafts……...……………………………..69  x  Figure 4.4 B7-H4 induces unresponsiveness to second-set donor islet allografts, but not third party islets…………………………………………………….. ..…………71 Figure 4.5 Histology of rejected and surviving allografts after secondary transplant shows distinct patterns.………………………………………………………….……….74 Figure 4.6 FACS analysis of Foxp3+, Tregs, and Teffs in the spleens and renal lymph nodes in 2 failed and 3 surviving recipients after secondary transplantation……….…...75 Figure 5.1 B7-H4 inhibits T-cell proliferation…………………………………………86 Figure 5.2 Expression of B7-H4 receptor on activated T-cell subsets………...….........87 Figure 5.3 B7-H4 inhibits phosphorylation of AKT on different T cell subsets similarly……………………………………..…………………………………………...89 Figure 5.4 B7-H4 inhibits IL-2 and IL-2 receptor  chain (CD25) expression......……92 Figure 5.5 B7-H4 inhibits CD69 exprssion…………………………………....………94 Figure 5.6 B7-H4 suppresses activation of ERK, JNK, and AKT…………......………98 Figure 5.7 B7-H4 does not affect phosphorylation of LCK or ZAP70………….........100 Figure 6.1 The effects of B7-H4 on alloreactive responses in transplantation…..…...106 Figure 6.2 The role of B7-H4 in islet transplantation……………..………….............108  xi  LIST OF ABBREVIATIONS aa  amino acid  AP-1  activator protein-1  Ab  antibody  APC  antigen presenting cells  ATCC  American type culture collection  BB rat  bio-breeding rat  BTLA  B- and T-lymphocyte attenuator  BM  bone marrow  BrdU  5-bromo-2-deoxyuridine  BSA  bovine serum albumin  CD  clusters of differentiation  cDNA  complementary deoxyribonucleic acid  CFA  complete Freund’s adjuvant  CSA  cyclosporine A  CTL  cytotoxic T lymphocytes  DC  dendritic cells  DC-SIGN  dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin  DM  diabetes mellitus  EAE  experimental autoimmune encephalomyelitis  EC50  half maximal effective concentration  ELISpot  enzyme-linked immunosorbent assay  ERK  extracelluar signal-regulated kinase  FACS  fluorescence-activated cell sorter  FBS  fetal bovine serum  Foxp3  forkhead box protein 3  GAD  glutamic acid decarboxylase  Grb2  growth factor receptor-bound protein 2  HLA  human leukocyte antigen  HRP  horseradish peroxidase  xii  HVEM  herpes virus entry mediator  IA-2  protein tyrosine phosphatase-2  IDO  indoleamine 2,3-dioxygenase  IEQ  islet equivalents  IFN  interferon  Ig  immunoglobulin  IL  interleukin  ITAM  immunoreceptor tyrosine-based activation motif  ITIM  immuno tyrosine-based inhibitory motif  ICOS-L  inducible co-stimulator ligand  LAK  lymphokine-activated killers  LPS  lipopolysaccharide  MAPK  mitogen-activated protein kinase  MEK  mitogen-activated protein kinase kinase  mTOR  mammalian target of rapamycin  MLR  mixed lymphocyte reaction  MHC  major histocompatibility complex  H&E  haemotoxylin and eosin  NFAT  nuclear factor of activated T cells  NK  natural killer  NKG2D  NK cell group 2D  NKT  natural killer T  NOD  non-obsese diabetic  NS  natural suppressor  PBMC  peripheral blood mononuclear cells  PBS  phosphate-buffered saline  PCR  polymerase chain reaction  PE  phycoerythrin  PI3K  phophoinositide 3-kinase  PLC  phospholipase C  PMA  phorbol myristate acetate  xiii  PD-L1  programmed death-1 ligand  RAE-1  retinoic acid early inducible-1 gene  RAG  recombinantion-activating gene  RBC  red blood cells  RNA  ribonucleic acid  RORγt  Retinoic acid receptor-related orphan receptor γ  RT  room temperature  SDS  sodium dodecyl sulfate  SH2  src homology 2  Syk  spleen tyrosine kinase  T1D  type 1 diabetes  T2D  type 2 diabetes  TCID  tissue culture infective dose  TCR  T cell receptor  Th  T helper  TLR  toll-like receptor  TNF  tumor necrosis factor  Y  tyrosine  xiv  ACKNOWLEDGEMENTS This project was supported by Canadian Institutes of Health Research (CIHR), Michael Smith Foundation for Health Research, and Canadian Diabetes Association. The completion of this thesis was only possible with tremendous support of the Vancouver General Hospital Research Pavilion and Jack Bell Animal Facility at every level. To have Dr. Garth Warnock as my supervisor has been a constant source of pride and inspiration to excel at the bridge of laboratory research and clinical application. Along with that came the privilege of presenting my findings at local Immunity and Infectious Research Centre (IIRC), international scientific meetings, such as International Congress of Transplantation Society, American Diabetes Association Scientific Conference and Canadian Diabetes Association Scientific Conference and publications. To have Drs. Bruce Verchere, Janet Chantler, and Alice Mui as my committee members allows me to hypothesize new ideas and resolve problems. The professionalism and compassion of Dr. Alice Mui was greatly appreciated. I will never forget our meeting every Friday morning starting at eight. She provided delicate care for my detailed subjects, especially on B7-H4 signalling transduction pathway. I would also like to thank Dr. Megan Levings and her lab members for assistance on flow cytometry machine. I would like to thank Drs. Jingqiang Hao, Dawei Ou, Ziliang Ao, Longjun Dai, Zehua He, Theresa Liao, and Crystal Robertson who provided crucial surgical assistance, immunological assays, and day-to-day operations of the lab. Finally and perhaps most importantly, I acknowledge the constant support from my family and friends, particularly my father, who has infectious enthusiasm to meet life’s challenges. As a medical doctor, basic researcher, and medical journal editor, he guides me to this path with his passion. Unconditional supporters that I always appreciate include my mother and siblings.  xv  CHAPTER 1                              BACKGROUND AND AIMS OF THE STUDY   1  1.1 TYPE 1 DIABETES  Diabetes is one of the most common endocrine disorders involving the failed production or the failed use of insulin in the body. There are two main forms of diabetes, type 1 and type 2. Both types are characterized by hyperglycemia and progressive loss of β cells. In type 1 diabetes, the body fails to produce insulin as a result of an autoimmune assault against the β cells. It features absolute insulin deficiency. Type 2 diabetes refers to non-insulin dependent diabetes. It is associated with obesity and insulin resistance. Both genetic and environmental factors trigger the development of diabetes. It is estimated that almost 6% of the world population is affected by this disease. Diabetic patients will exceed 350 million by 2010 [1], 10% of whom will have type 1 diabetes. With the increased number of patients, the direct health care cost for this disease is tremendous. In Canada alone, it is expected to cost approximately $6 to 7 billion in 2011 and over $8 billion by 2016 [2]. This significant global financial and social burden drives both clinicians and scientists to explore improved therapies for type 1 diabetes.  1.1.1 Pathogenesis of type 1 diabetes  The pathogenesis of type 1 diabetes has been extensively studied using two animal models, the nonobese diabetic (NOD) mouse and the diabetes-prone BioBreeding (DP-BB) rat, which have facilitated our understanding in etiology of human type 1 diabetes. Several factors including β cell autoantigens, antigen presentation cells (APCs), and T cells contribute to the beta cell-specific autoimmune response [3–5]. β Cell autoantigens such as glutamic acid decarboxylase (GAD), insulin, and protein tyrosine phosphatase-2 (IA-2) are processed by APCs including macrophages, dendritic cells, and B cells in the local pancreatic islets and subsequently 2  stimulate autoreactive CD4+ T cells to proliferate in the peripheral lymphoid tissue. These activated CD4+ T cells secrete cytokines which can then induce β cell-specific cytotoxic CD8+ T cells. The activated T cells are recruited back to the islets and produce cytokines which further activate APCs and T cells, leading to the destruction of β cells.  1.1.2 Clinical aspects of type 1 diabetes  Complications of type 1 diabetes are severe. High blood glucose causes retinopathy, heart disease, neuropathy and nephropathy. These conditions lead to vision loss, myocardial infarction, stroke, gangrene of extremities, amputations, and dependence on renal dialysis. Current treatment options for type 1 diabetes include exogenous insulin. Intensive glycemic control is well established to reduce retinopathy by 34% to 76%, decrease microalbuminuria by 35%, and cut neuropathy by 65% [6]. However, the risk of hypoglycemia due to excess insulin is present and the intensive injections required impair quality of life. Best medical therapy combines the control of blood glucose using insulin with other treatments to control blood lipid levels, blood pressure and protect renal function. Nevertheless, progressive microvascular and other severe complications remain prevalent, and require a transplantation therapeutic option [6–9].  1.1.3 Therapies for type 1 diabetes  Apart from insulin injection, current therapies include restoration of β cell mass by pancreas/islet cell transplantation. Both whole organ pancreas and isolated islet transplantations offer better glycemic control compared with exogenous insulin injection and avoid life-threatening hypoglycemia.  3  Pancreas transplantation offers a source of physiologically regulated insulin secretion. Unfortunately, pancreas transplantation is a major surgical procedure with risks of pancreatitis due to the excessive release of harmful digestive enzymes from the exocrine tissue. Currently, whole pancreas transplant is primarily used for end-stage diabetic patients either through simultaneous pancreas-kidney or pancreas after kidney transplantation [10]. In contrast, pancreatic islet transplantation is much less invasive and relatively safe.  1.2 ISLET TRANSPLANTATION  Islet cell transplantation is a promising treatment option for type 1 diabetes. Isolated islets are gently infused into the liver via the portal vein with fluoroscopic cannulation. The procedure is completed under local anesthesia during the infusion and patients are observed overnight and discharged home with ambulatory clinic follow-up. The 82% rate of maintained insulin/cpeptide secretion after islet transplantation at one-year follow-up matches that of pancreas transplantation but patients are frequently still dependent upon reduced amounts of insulin injections to supplement the islet graft function [11]. The Edmonton protocol yielded 100% insulin-independence rates at median follow-up of seven months in seven patients and emphasized sufficient infused islet mass and glucocorticoidfree immunosuppressive regimens [12]. Under this protocol, an average of 11,000 islet equivalents (IE) per kilogram bodyweight was used. In order to achieve this number, at least two deceased-donor pancreata were needed. The immunosuppressive regimen included combinations of rapamycin, tacrolimus and daclizumab. Islet transplantation centers around the world conducted co-ordinated trials thereafter. Unfortunately, it has been observed that there is an inexorable decline of graft function, leaving merely 10% of patients with insulin independence after five years [11]. Despite this, persistent C-peptide secretion is observed in more than 80% 4  and patients suffer substantially reduced episodes of hypoglycemia. Islet transplantation is superior to intensive medical therapy in terms of improved HbA1c level and reduced development of retinopathy nephropathy after 3-years follow-up [13, 14]. Therefore, islet transplantation is well established to achieve improved metabolism and control of debilitating hypoglycemia for prolonged durations of time, and emerging data suggests a beneficial effect on some microvascular complications [15, 16].  1.2.1 History of islet transplantation  Investigation of the pancreas and its function was enlightened 140 years ago when Langerhans first described islets in 1869. In humans, these “Islets of Langerhans” constitute about 1-2% of the pancreas volume and receive at least 10% of the pancreatic arterial blood flow [17]. The different cell constituents have a variable distribution: 28-75% are β cells, 10-65% are α cells, and 1.2-22% are δ cells [18, 19]. The discovery that insulin originated from the pancreatic islets was a milestone for diabetes therapy, yielding the 1923 Nobel Prize for Canadian scientists Banting and McLeod, who shared their recognition with Best and Collip. Exogenous insulin achieved a legacy of remarkable preservation of life for patients with type 1 diabetes; however, it was not a cure. The notion of using islets to treat hyperglycemia was first published in 1894 by Dr. Watson-Williams who reported using isolated small fragments of sheep’s pancreas to treat a patient with diabetic ketoacidosis [20]. No purification of islets was made. The discovery of insulin attracted interest away from islet transplantation. Subsequent pioneering work done in rats proved that insulin-producing pancreatic beta cell mass could be restored by transplantation [21, 22]. Since then, the concept of islet transplantation as a treatment for diabetes has motivated great effort toward islet isolation and purification from the larger, more compact pancreas of 5  mammals and humans [23]. Islet transplantation outcomes were predicted by developing the concept of providing a critical mass of highly purified islets per unit body weight [23]. Islet mass was inversely correlated to the time to glycemic normalization [24].  1.2.2 Barriers in islet transplantation  Based upon the experience of the Edmonton protocol, it is widely accepted that sufficient numbers of islets and use of immunosuppressive regimens that do not damage islets are crucial determinants for success of islet transplantation. Despite this, the major challenges in islet transplantation include both a limited number of available donor islets and islet allograft rejection. The challenge of recurrent autoimmunity is an additional immunlogical issue in islet transplantation. The recipients with an autoimmune background of type 1 diabetes face challenges of both autoimmune and alloimmune mediated attack on donor islets. Transplanted islets, even between identical twins, are subject to autoimmune destruction. Recurrent autoimmunity is characterized by targeted loss of insulin producing β cells but not other types of cells such as α cells in the islets after transplantation. The molecular mechanisms involved in this process may include long-lived autoreactive memory T cells whose TCR is selected on one or more β cell restricted epitopes presented on APC class II MHC, or generation of β cell reactive thymocytes after transplantation. At the cellular level, it is still unclear how to distinguish the process of the autoreactivity against the transplanted islets from alloreactivity [25–27]. Recurrence of autoimmunity can be prevented by complete Freund's adjuvant (CFA) treatment in autoimmune diabetic NOD mice, but no comparable treatment exists for humans [28]. In order to expand the number of islets, alternative sources may be considered, such as xenografts and stem cells. Porcine islets are the preferred donor source in xenotransplantation for 6  several reasons. First, pig insulin is almost the same as human insulin, with only one amino acid variation. In fact, it has been used successfully in patients for many years. Secondly, porcine glucose homeostasis is comparable to human. In addition, pig islets can be isolated in a similar manner as human ones and yields are high. Moreover, the size of pig islets are appropriate for human. However, the spectre of transferring diseases from animals to humans and hyperacute rejection across species keeps xenotransplantation at the experimental stage [29]. Other alternatives have been addressed, such as producing humanized insulin from stable lines of transgenic tilapia, a teleost fish. These fish could be used as the alternative xenogeneic donors for clinical islet transplantation [30]. The other alternative source is to use a renewable supply of cells. Stem cells are clonogenic cells capable of differentiation into single and/or multiple lineages. Production of β cells through regeneration/neogenesis or transdifferentiation demonstrates the potential to genetically modify cells ex vivo for transplantation. However, ethical issues and inconsistent reproducible results obstruct stem cell research. To maximize usage of the current limited supply of islets, many attempts have been made to optimize islet isolation/preservation techniques, reduce apoptosis, limit pro-inflammatory cytokines during isolation and increase proliferation or neogenesis of β cells [15, 31]. Lifetime use of immunosuppressive drugs poses the serious challenge of unwanted side effects. Therefore, basic research to improve islet allograft survival with reduced immunosuppression may optimize achievement of the ultimate goal of a cure for type 1 diabetes.  7  1.2.3 Limitations of current immunosuppressive drugs  Despite initial success, most patients experience graft deterioration or loss after one year and only 10% maintain insulin independence after five years using current immunosuppressive regimens. Basiliximab which is commonly used to induce the immunosuppressive regimen, thereby minimizing glucocorticoids, is an anti-CD25 monoclonal antibody drug. The current immunosuppressive regimen for islet transplantation includes tacrolimus and either of sirolimus or mycophenolate mofetil. Tacrolimus, also named FK506, binds to the immunophilin FK506 binding protein-12 (FKBP-12). The complex of FK506-FKBP-12 inhibits T cell proliferation and IL-2 transcription [32]. Sirolimus, also known as rapamycin, binds to the same cytosolic protein as tacrolimus does but inhibits the mammalian target of rapamycin (mTOR) instead of calcineurin [33]. Mycophenolate mofetil (MMF) inhibits a key enzyme for purine synthesis that is used by proliferating B and T lymphocytes [34]. Generalized immunosuppression by these drugs increases risks of infection and of malignancy. The major side effects of these drugs also include kidney damage, hypertension, diarrhea, anemia, and leucopenia. Acute and chronic effects of FK506, Rapamycin, and MMF on β-cell function were evaluated by our group and many others. We found that chronic exposure to FK506 or MMF reduced glucose-stimulated insulin secretion in human islets [35]. FK506, but not MMF, impaired human islet graft function in diabetic NOD.SCID mice [35]. All three drugs increased apoptosis in islets, as evidenced by caspase-3 cleavage and activity [35]. Collectively, these results indicate that current immunosuppressive drugs induce cytotoxicity to islets and reduce β-cell function. The side effects of current drugs and their life-time usage after transplantation hamper application of islet transplantation. Reversing complications of diabetes while inducing a disorder of generalized immunosuppression is an unsatisfactory option. Novel therapeutic  8  immune modulation after islet transplantation is needed to control islet allograft loss with an acceptable level of toxicity or side effects.  1.3 TRANSPLANTATION REJECTION  Since few recipients remain insulin independent after 5 years, many explanations may account for allograft loss, including hypoxia, recurrence of β-cell autoimmunity and destruction by alloreactive T cells. It is estimated that majority of transplanted islets are lost during the first week after transplantation [36]. Rejection of donor grafts occurs as a result of humoral and cellmediated reaction to major histocompatibility complex (MHC), antigens in humans known as human leukocyte antigens (HLA).  1.3.1 Types of transplantation  Transplantation is the surgical procedure of transferring cells, tissues, or organs from one to another, for the purpose of replacing the recipient’s damaged or absent organ. Kidneys are the most commonly transplanted organs. The first successful identical twin transplant of a human kidney was performed by Joseph E. Murray in 1954 in Boston, followed by the first successful liver transplant by Dr. Thomas E. Starzl in 1967, the first heart transplantation by Christian Barnard in 1967, and the first successful bone marrow transplant by E. Donnall Thomas in 1968. Transplanted grafts can be divided into different types. Autografts refer to transplantation of one part of the body to another (eg, skin grafts). Isografts are grafts between identical individuals (eg, monozygotic twins). Allografts are grafts between members of the same species with different genetic signatures. This is the most common form of transplantation. Xenografts are transplanted tissues between different species, such as from pigs to humans. 9  1.3.2 Types of rejection  Hyperacute rejection is a humoral-mediated response in recipients with pre-existing antibodies to the donor (for example, ABO blood type antibodies). It occurs within minutes resulting in rapid thrombosis. Hyperacute rejection typically occurs in xenotransplanted organs. Acute rejection usually begins weeks to months after transplantation. It is caused by mismatched HLA, which are present on all cells of the body. A perfect match between the donor and the recipient is extremely rare since there are many different alleles of each HLA. The diagnosis of acute rejection is based on both clinical observation and laboratory testing. Three typical measurements are considered. First, lymphocytes infiltrate the transplanted tissue; these may be accompanied by other cell types including eosinophils, plasma cells and neutrophils. Secondly, characteristic structures are altered in the transplanted tissue; the degree of this injury will depend on the type of tissue being transplanted. Lastly, the blood vessels of the transplanted tissue are damaged [37]. Chronic rejection happens months after transplantation. It is associated with fibrosis of the internal blood vessels of the transplanted organ which results in loss of graft function. Both immune and non-immune factors contribute to chronic rejection.  1.3.3 Mechanisms of graft rejection  Rejection is a normal adaptive immune reaction to foreign antigens. The degree of mismatched alleles determines the level of rejection. It is mediated through both T cell mediated and humoral immune mechanisms. Transplant rejection can be reduced through the use of immunosuppressive drugs. 10  1.3.3.1 Role of the major histocompatibility complex (MHC)  Graft rejection occurs as a result of alloreactive T cell immune response. MHC molecules are expressed on the surface of APCs such as macrophages and dendritic cells. Their normal function is to bind foreign peptides and present them to TCR. This recognition step initiates Tcell–mediated immune responses. In other words, a host T cell response can only be initiated when the T cell recognizes a foreign antigen that is presented to its TCR by an MHC molecule. There are two different MHC antigens, the MHC class I and class II. In general, CD8+ T cells binds to antigenic peptides presented on MHC class I molecules, while CD4+ T cells respond to antigen fragments on MHC class II molecules [38]. Rejection of donor grafts is the result of humoral and cell-mediated reaction to major MHC antigens, known in humans as human leukocyte antigens (HLA), H2 in mice and RT1 in rats. MHC molecules play a primary role in graft rejection. Identical MHC alleles are unusual except in genetically identical twins because alleles of MHC are highly polymorphic. The more closely that MHC types are matched between donor and host, a greater degree of acceptance is observed [38, 39]. In addition to major MHC class I and II antigens, the immune system can also respond to minor histocompatibility antigens, such as the male Y antigen.  11  1.3.3.2 Role of T cells in transplant rejection  The recognition of alloantigen presented on MHC molecules by TCR is believed to occur via two pathways of antigen presentation [39]. In the direct pathway, recipient T cells recognize intact alloantigens on MHC molecules expressed on donor APCs. In the indirect pathway, T cells interact with processed donor alloantigens presented by recipient APCs. The direct and indirect pathways may be responsible for the acute and chronic rejection, respectively [39, 40]. T cells play a central role in transplantation rejection [40]. They can be either CD4+ or CD8+ subsets depending on which surface glycoprotein is expressed. CD4+ cells, also called helper T cells (Th), play a dominant role for initiating graft rejection. CD8+ T cells trigger graft rejection through direct cytotoxic lysis of donor tissues and can also damage the transplanted organ through secreted proinflammatory cytokines [41]. The function of CD4+ T cells includes the production of cytokines. Cytokines can act either on themselves (autocrine) or on others (paracrine). CD4+ Th cells can differentiate into one of several subtypes, including Th1, Th2, Th17, or Treg, which secrete different cytokines to facilitate a different type of immune response [41]. Differentiaton into Th1, Th2, Th17 or Treg cells is directed by interleukin-12 (IL-12), IL-4, transforming growth factor (TGF)-β plus IL-6 or TGF-β, respectively. The differentiated cells are characterized by expression of particular transcription factors, T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for Tregs and the retinoic acid receptor-related orphan receptor γ (RORγt) for Th17 cells. Once differentiated, each lineage secretes a specific cytokine profile, with interferon γ (IFN-γ) for Th1 cells and IL-4, IL-10 for Th2 cells. The subsets of T cells, Th1, Th2, Th17, and Tregs, cooperate and shape the outcome of transplantated organs and cells. The Th1 cytokine profile is previously thought to be associated 12  with allograft damage and rejection, while the Th2 profile favors the acquisition of protection and tolerance. The proinflammatory cytokines, such as IFN-γ, IL-2, and IL-6, are increased locally by production from infiltrating lymphocytes during acute graft rejection [42, 43]. However, this paradigm may not be sufficient to explain redundant effects of cytokine networks in vivo. The newly described T subset derived cytokine IL-17 has been recognized as an important cytokine in transplantation. Upregulated IL-17 mRNA and protein levels were observed in the allograft as early as day 2 post-transplant [44]. Similar results were observed in humans. IL-17 protein is elevated in human renal allografts during rejection but not in nonrejecting recipients. Moreover, IL-17 mRNA was only detectable in the urine of these patients but not in non-rejecting patients [44]. Accumulating data demonstrate that IL-17 is a proinflammatory cytokine and functions to augment inflammatory conditions in humans and mice. The other newly-characterized T cell subsets, Tregs, have contradictory functions. Unlike Th17, Treg plays an anti-inflammatory role and maintains tolerance to self-antigens. Tregs suppress proliferation and production of cytokines by T cells of CD4+ CD25– and CD8+ in response to polyclonal stimuli in an antigen-non-specific manner [45]. CD4+ CD25+ Tregs can be derived from the thymus (referred to as natually occuring, nTregs) or generated in the periphery (referred to as inducible Tregs, iTregs) [46, 47]. Tregs play an important role in preventing transplant rejection and generating tolerance [47]. Graft rejection is determined by the interaction between forces that maintain tolerance to the graft and those that promote rejection. CD4+ T cells can be programmed into Th1, Th2, Th17 or Tregs lineage. However, the subsets exhibit lineage plasticity and can be re-programmed upon antigen stimulation. For example, Th17 cells can express the Treg specific transcription factor Foxp3 whereas Tregs can be induced to produce IL-17 [48, 49]. Tregs can be reprogrammed to  13  Th2 if Foxp3 expression is suppressed [50]. T cell subsets retain a great degree of plasticity in order to accommodate different stimuli and maintain acquired functions.  1.4 COSTIMULATION BLOCKADE IN ISLET TRANSPLANTATION  T cells have been considered a main player in graft rejection. The requirement of two separate signals for full activation of T cells suggests a critical role of co-signalling pathways in determining the fate of transplantation. Costimulation blockade has been targeted to effectively control allograft rejection and to induce tolerance in animal models. The most exciting example is Belatacept ( a mutant form of CTLA-4) which is under phase III clinical trials in human renal transplantation.  1.4.1 Rationale for using costimulatory molecules in islet transplantation  Activation of T cells requires two independent signals. Signal 1 is the antigen-specific engagement of the TCR-peptide bound to an MHC molecule on the surface of APCs. Signal 2 is provided by co-stimulatory molecules. After receiving the first signal, the naïve T cells must obtain a second verification signal from costimulatory molecules to ensure response to a foreign antigen. Upon recognition of donor antigens by the recipients’ T-cells, an alloreactive response is initiated. In the presence of signal 2, alloreactive CD4+ T cells trigger alloantigen-specific clonal expansion, secrete cytokines such as interleukin 2 (IL-2), and differentiate into T helper 1 (Th1) and T helper 2 cells (Th2). Differentiation of CD4+ T cells into Th1 and Th2 lineages determines whether humoral or cell-mediated immunity will predominate after initial sensitization [51]. Transplanted organs can be destroyed in several ways. CD8+ T cells are up-regulated by primed CD4+ T cells and become armed cytotoxic CD8+ T cells. These cytotoxic effector CD8+ 14  T cells lyse the donor graft through targeted release of perforin and granzyme B. In addition, B cells are also involved in destruction of transplanted organs through opsonization and complement binding. This alloreactive response is a normal reaction to protect self from destruction by foreign pathogens and tissues [51, 52]. Based on our understanding of transplantation immunology, it is widely accepted that T cells play a fundamental role in transplantation [41]. Therefore, T cells are a logical target to harness for limitation graft rejection and to generate tolerance. The requirement of one cell to deliver both recognition signal (signal 1) and verification signal (signal 2) is crucial to control alloreactive immune responses. In fact, without signal 2, T cells will undergo anergy or apoptosis [53]. This observation suggests the attractive prospect that allografts could become tolerated if signal 2 is blocked. After transplantation, recipient T cells receive signal 1 through the complex of TCR-donor-derived peptides on MHC and signal 2 through “positive” co-stimulatory molecules such as CD28, leading to full activation in response to donor alloantigen stimulation [51, 54]. At the same time, expression of a negative co-signaling molecule CTLA-4 is induced by activated T cell crosslinking. CTLA-4 is a negative co-stimulatory signal which functions to terminate activated T cell response [55, 56]. Manipulation of signal 2 is crucial to terminate T cell response or to induce tolerance.  1.4.2 B7 family  Positive and negative co-stimulatory molecules belong to one of the two different superfamilies: the immunoglobulin (Ig) superfamily and the tumor necrosis factor (TNF) superfamily. The B7 molecules belong to the Ig superfamily and consists of structurally related ligands and receptors [57, 58]. Ligands have both IgV and IgC extracellular domains whereas receptors contain only a single IgV. IgV domain in both ligands and receptors is responsible for 15  binding of these two molecules. The fate of T-cell response is determined by B7-mediated modification response delivered by antigen-specific TCR on T cells. Expression levels or patterns for ligands or receptors are coordinated and result in up- or down-regulation of the immune response (Table 1.1) [57-59]. There are seven known members of the B7 family: B7.1 (CD80), B7.2 (CD86), B7-H1 [also known as programmed death-1 ligand (PD-L1)], B7-H2 [also known as inducible costimulator ligand (ICOS-L)], programmed death-2 ligand (PD-L2), B7-H3, and B7-H4. The B7 family has been widely studied and plays critical roles in transplantation immunology. The engagement of B7 and its receptors maintains T cell homeostasis by either activation or inhibition. The interaction of B7.1 and B7.2 with the CD28 receptor promotes T cell differentiation and clonal expansion. This proliferation is then attenuated by negative cosignaling molecule CTLA-4. Other B7 homology molecules show similar positive and negative co-stimulation paradigms but in a different fashion. Instead of the “on-off switch” mediated by the B7.1/B7.2:CTLA-4/CD28 pathway, B7 homology molecules may fine-tune immune responses [58, 59]. Abundant data shows that a combination of co-stimulation blockade with delivery of negative signals to T cells improves allograft survival [59]. An ultimate goal in solid organ transplantation is to induce antigen-specific tolerance to foreign histocompatibility antigens; if antigen-specific tolerance were achieved, a recipient’s immune system would not reject a graft, while responding to all other foreign antigens normally.  16  Table 1.1 Expression patterns, function, and receptors of B7 family ligands Ligand  Expression  B7.1 (CD80)  T, B, DC, monocytes (induced)  B7.2 (CD86)  Interaction  B, DC, monocytes (constitutive);  Receptor  Expression  CD28  T (constitutive)  CTLA-4  T (activated)  PD-1  T, B, monocytes (activated)  PD-1  T, B, monocytes (activated)  ICOS  T (activated)  Function  T (induced) B7-H1 (PDL1)  T, B, DC, monocytes (induced); *  B7-DC (PDL2)  B, DC, monocytes (induced); *  B7-H2 (ICOSL, B7RP-1, B7h, GL-50)  B7-H3  B, DC, monocytes (constitutive);  * T, DC, monocytes (induced); *  ?  T, B, DC, monocytes B7-H4 ? NK(induced); * * They are also expressed on non-lymphoid tissues. Co-stimulatory ( ) or co-inhibitory signal ( ) is shown in green or red, respectively.  17  .  1.4.3 CD28/CTLA-4:B7 pathway  The classic B7.1/B7.2:CTLA-4/CD28 pathway plays an important role in controlling transplantation rejection. The fact of the dual specificity of B7.1 and B7.2 for the activating receptor CD28 and the inhibitory receptor CTLA-4 makes for some controversial results and adds complexity to this pathway [60].  1.4.3.1 CD28 and CTLA-4: positive and negative costimulators  B7.1 and B7.2 each can engage two different receptors on T cells, the positive cosignaling molecule CD28 resulting in T-cell activation and the negative co-signaling receptor CTLA-4. CD28 is constitutively expressed on the surface of T cells. B7.1 is inducible and expresses later after activation. B7.2 is constitutively expressed at low levels and is rapidly upregulated. CD28 engagement delivers a positive signal by promoting T cell survival through enhanced expression of anti-apoptotic genes, such as Bcl-XL. It also augments transcription and stability of IL-2 mRNA [61, 62]. Upon T cell activation, CTLA-4 is expressed. The engagement of B7.1/B7.2 and CTLA-4 results in termination of activated T cell response. CTLA-4 inhibits T cell proliferation by arresting cell cycle progression and inhibition of IL-2 synthesis [63, 64].  1.4.3.2 CTLA-4 in transplantation  CTLA-4 is one of the most important co-inhibitory molecules. A profound negative regulatory role of CTLA-4 is well demonstrated in CTLA-4 deficient mice that develop severe lymphoproliferative disorders and die 3 to 4 weeks after birth [65]. The effects of CTLA-4 blockade on islet transplantation have been investigated in several studies. Local expression of 18  CTLA-4 by gene gun transfection prolonged mouse islet allograft survival from 13 days to 67 days [66]. Systemic administration of CTLA-4.Ig prolonged human islet xenograft survival [67]. The ability of CTLA-4 to inhibit T-cell proliferation is further enhanced in a mutant form of CTLA-4, called Belatacept (LEA29Y) which has greater binding but lower dissociating avidity to B7.1 and B7.2. Belatacept inhibited T cell proliferation in vitro 10-fold more strongly than wild-type CTLA-4 [68]. Belatacept provided better efficacy in preventing acute rejection compared with unmodified CTLA-4.Ig in nonhuman primate renal transplant studies [68]. It prolonged allograft function with reduced cytotoxicity in human renal transplant studies [69]. This line of investigation is currently at the phase III clinical trial stage.  1.4.3.3 Mechanism of action for CTLA-4  CTLA-4 can inhibit T cell proliferation through different mechanisms. One mechanism involves antagonism of B7-CD28-mediated positive co-signalling [56]. Both CTLA-4 and CD28 bind B7.1/B7.2 through a conserved MYPPPY (methionine-tyrosine-proline-proline-prolinetyrosine) recognition motif in their extracellular domain. Unlike CD28, CTLA-4 forms two different homodimer lattices with B7 which can only cooperate with a single B7 dimer at a time [70, 71]. The formation of this stable structure results in higher binding affinity between CTLA-4 and B7 which is 500 to 2500 times more avid than CD28. Therefore, the complex of CTLA-4/B7 is more stable than CD28/B7 [58, 72]. CTLA-4 retains its inhibitory properties in the absence of CD28 through involvement of other mechanisms. CTLA-4 could inhibit CD28-deficient T cell responses in vitro and in vivo [73, 74]. This CD28-independent inhibition can be explained by modulation of T cell motility [75]. Upon TCR ligation, CTLA-4 localizes to the immunological synapse [76]. This re-  19  localization and expression of CTLA-4 decrease T cell proliferation and cytokine production by reducing contact periods between T cells and APCs [75]. Another mechanism involves in delivery of negative intracellular signals. In addition to its passive inhibition of T cell activation, CTLA-4 can actively inhibit TCR-mediated signals. The localization of CTLA-4 to the immunological synapse re-programs the proteins involved in downstream signalling after TCR activation. The re-route of intracellular signal transduction pathway results in deactivation of the T cell response [77–80].  1.4.3.4 Treg and CTLA-4  A role for CTLA-4 in Treg function arises from constitutive expression of CTLA-4 on Treg populations [81]. CTLA-4 is not expressed in resting T cells but is induced upon T-cell activation [82]. CTLA-4 is exclusively expressed by CD4+CD25+ Tregs [81, 83] and is considered a marker for Tregs. Despite the signature expression of CTLA-4 on Treg, its precise role in Treg remains controversial. The controversy surrounding the function of CTLA-4 in Treg is due to the fact that CTLA-4 inhibits T cell proliferation largely through competing with CD28 which is essential for Treg survival and function [84]. Whether CTLA-4 expression is required for Treg generation or intrinsic regulatory capacity remains unresolved. Some studies report an essential role for CTLA-4 in Treg function but others suggest a dispensable role. For example, studies reveal that administration of anti-CTLA-4 monoclonal antibody results in a loss of Treg-mediated suppression both in vitro and in vivo [81, 83]. Also, polymorphisms in CTLA-4 have also been associated with autoimmune disorders in humans [85] and CTLA-4 up-regulation is also associated with enhanced Treg activity in inflammatory bowel disease [86]. However, CD4+Foxp3+ Treg can be generated in CTLA-4-deficient mice, and these Treg can suppress T 20  cell responses in vitro and in vivo [86]. CTLA-4 expression is not completely absent in Foxp3 knockout mice, suggesting that Foxp3 is not required for CTLA-4 expression. 1.4.4 PD-1:B7-H1 pathway  Program death-1 (PD-1) molecule was isolated through substract hybridization from a Tcell hydridoma undergoing programmed cell death [87]. PD-1 is expressed on activated T, B, and myeloid cells. PD-1 can engage two ligands: B7-H1 (PD-L1) and PD-L2. The complexity of PD-1:B7-H1/PD-L2 results in much conflicting data depending on the experimental conditions. PD-1 deficient mice develop multiple autoimmune diseases, suggesting a role in establishment and/or maintenance of peripheral tolerance [88]. The ligands of PD-1 (B7-H1, also called PD-L1 and PD-L2) are members of the B7 costimulatory family. Engagement of PD-1 by B7-H1 leads to the inhibition of T cell receptormediated lymphocyte proliferation and cytokine secretion [89]. B7-H1 is broadly expressed on resting B, T, myeloid, and dendritic cells and is upregulated on activation [90]. B7-H1 is also expressed by non-lymphoid tissues, suggesting B7-H1 may regulate self-reactive T and B cells in peripheral tissues and determine the extent of inflammatory responses in the target organs. PD-1 ligand 2 (PD-L2) was identified as PD-1 second immunoregulatory receptor [91]. Interaction between PD-1 and PD-L2 results in downregulating TCR-mediated proliferation and cytokine production. Furthermore, B7-H1 or PD-L2.Ig inhibited T cell proliferation and cytokine production stimulated by anti-CD3 mAb [89, 91]. This effect of negative regulation by B7-H1 is mediated by PD-1 since inhibition is abrogated in PD-1-/- T cells. B7-H1 transduces a bidirectional signal through interaction with either PD-1 or B7.1 [92]. Endogenous expression B7H1 in a tissue-specific transgenic mouse promotes organ-specific autoimmune diabetes and transplant rejection [93]. 21  PD-1 deficient mice develop multiple autoimmune diseases, suggesting a role in maintenance of peripheral tolerance. PD-1 deficient mice develop arthritis and lupus-like glomerulonephritis on the C57BL/6 background , whereas a dilated cardiomyopathy develops on BALB/c background [88, 94]. Neither B7-H1.Ig nor PD-L2.Ig alone prolonged cardiac allograft survival. However, B7-H1.Ig, but not PD-L2.Ig, plus Cyclosporine A (CSA) significantly enhanced allograft survival over that of CSA or PD-L1.Ig alone. B7-H1.Ig and anti-CD154 similarly synergized and induced long-term survival of islet allografts, whereas either alone failed to prolong islet allograft survival [95]. B7-H1.Ig also promoted long-term cardiac graft survival in CD28-/- recipients, suggesting a CD28 independent pathway controlled by B7-H1 [96]. B7-H1 is expressed on Tregs. Depletion of CD25+ T cells abrogated the effect of B7-H1 [97]. B7-H1 blockade inhibited Treg suppressive activities in vitro. Anti-B7-H1 abrogated the protective effects of anti-CD25 antibody administration to prevent lethal GVHD in mouse. AntiB7-H1 inhibited Treg-mediated alloimmune response in skin transplantation [98]. B7-H1-/- but not from PD-L2-/- donors displayed accelerated rejection [99]. 1.4.5 ICOS:B7-H2 pathway ICOS was first reported on activated human T cells [100]. ICOS protein is not expressed constitutively on naïve T cells, but is induced rapidly after TCR engagement [101-103], indicating that ICOS might provide a positive signal in T cell responses. Indeed, ICOS promotes T cell proliferation and cytokine production [100]. ICOS mRNA can be detected constitutively in lymphoid and non-lymphoid tissue [104-106]. ICOS expression is upregulated by both TCR and CD28 stimulation [104, 105]. However, ICOS expression is not solely dependent on CD28 because blockade of ICOS in CD28-/- mice further inhibits Th1/Th2 differentiation [106].  22  The ligand for ICOS, named B7-H2 (also known as ICOSL, B7Rh-1), is expressed on B cells and macrophages [99]. T cell activation and proliferation are defective in the absence of ICOS [107]. In addition, ICOS-/- T cells fail to produce interleukin-4 (IL-4) but remain fully competent to produce IFN-γ, suggesting a role in Th2 differentiation [107, 108]. ICOS-/- mice are deficient T-cell-dependent B cell responses, geminal centre formation, and immunoglobulin class switching [108]. B7-H2-/- and ICOS-/- mice have similar phenotypes [107-109]. The engagement of ICOS and B7-H2 modulates early but not late phases of IgG1 maturation and is essential for primary but not secondary helper T cell responses [109]. Anti-ICOS mAb or an ICOS.Ig prolong cardiac graft survival [110]. ICOS.Ig plus CTLA-4.Ig result in long-term survival of cardiac allografts and donor-specific tolerance in islet allografts [111, 112]. The complex function of ICOS: B7-H2 pathways is well demonstrated in early versus late treatment in a mouse cardiac transplantation model. ICOS blockade prolonged allograft survival but did more effectively in the late treatment group and it was associated with suppression of CD8+ T cells. ICOS blockade is still effective in regulating allograft rejection in the absence of CD28 costimulation, suggesting that it regulates independently from the B7:CD28 pathway [112]. ICOS-ICOSL pathway provides key positive second signals that promote T cell activation, differentiation, effector responses and T cell-dependent B cell responses. ICOS does not upregulate IL-2 production. Thus, ICOS stimulates T effector function but not T cell expansion [113]. 1.4.6 B7-H4 pathway In 2003, a new negative co-signalling molecule B7-H4 (B7x or B7S1) was reported by three independent groups [114-116]. B7-H4 has about 25% homology in the extracellular region with other B7-family members. B7-H4 inhibits T cell proliferation, cytokine production and 23  cytotoxicity. Administration of B7-H4.Ig impairs antigen-specific T cell responses in mice [114]. In concordance with this inhibitory effect, injection of monoclonal antibody that blocks endogenous B7-H4 expression promotes T cell responses and exacerbates experimental autoimmune encephalomyelitis (EAE) [114,115]. Collectively, these results suggest that B7-H4 is a novel negative regulator in the B7 family. 1.4.6.1 Gene organization of B7-H4  The genes for human and mouse B7-H4 are located on chromosome 1 and 3, respectively. Genomic DNA of B7-H4 consists of six exons and five introns, and mature proteins encoded by 849 bp region are spanned exon III, IV, and part of V. In both genes, exons I and II form a signal peptide, V encodes transmembrane and intracellular regions, and IgV-IgC domain is comprised of the extracellular region (Fig. 1.1) [114, 117].  Figure 1.1 Genomic organization of human and mouse B7-H4 genes Exons from human B7-H4 (A) and mouse B7-H4 (B) are shown. The coding region of B7-H4 ( ) and 5' ( ) and 3' ( ) UTR are represented. Numbers in the boxes represent the length of exons in base pairs, and numbers in kilobases mark the length of introns between exons. Adapted from [114].  24  1.4.6.2 Expression pattern of B7-H4  B7-H4, also known as B7x and B7S1, is a member of the B7 family of immune costimualtory proteins. Mature B7-H4 is a 50 kDa – 80 kDa glycosylated molecule with a 28 kDa protein core. The 230 amino acid (aa) extracellular region of B7-H4 contains one Ig V and one Ig C domain. Within the extracellular domain, mouse B7-H4 shares 90% and 99% aa sequence identity with human and rat B7-H4, respectively. It shares 21% - 29% aa sequence identity with B7.1, B7.2, B7-H1, B7-H2, B7-H3, and PD-L2 [114]. B7-H4 mRNA is ubiquitously expressed in both lymphoid and non-lymphoid tissues including placenta, kidney, liver, lung, ovary, testis, and spleen [114]. Protein expression appears to be more restricted. Although B7-H4 cell-surface protein was expressed in normal human epithelial cells of the female genital tract, kidney, and lung, B7-H4 protein was generally undetectable in other normal human tissues. B7-H4 expression is induced on mitogen- or LPS-activated B cells, T cells, dendritic cells, monocytes, and macrophages [114–117]. B7-H4 is highly expressed in the human cancer microenvironment [118–122]. For example, elevated levels of B7-H4 protein are expressed in human ovarian cancers and low levels of soluble B7-H4 protein were found in the sera. In addition to tumour cells and sera, tumour-infiltrating macrophages and endothelial cells of small blood vessels in the cancer microenvironment are also expressed B7-H4 [118–122].  25  1.4.6.3 B7-H4 orphan receptor The IgV domain of the B7 family is primarily involved in the binding with the counterreceptor. In B7.1 and B7.2, the V region contains a conserved A’GFCC’C face for CTLA-4/CD28 binding. B7-H4 is not predicted to be a ligand for CTLA-4/CD28 based on the fact that there is no such conserved binding region on B7-H4. In fact, B7-H4 binds to a receptor on activated T cells but not to CTLA-4, ICOS or PD-1 according to FACS analysis of a B7-H4 transfected 293 cell line [114]. B- and T-lymphocyte attenuator (BTLA) was previously suggested as the receptor for B7-H4 [123]. However, recent studies have indicated that BTLA does not bind to B7-H4 directly and that herpes virus entry mediator (HVEM) may be the unique BTLA ligand [124, 125]. 1.4.6.4 Mechanisms of B7-H4 engagement B7-H4 has been established as a novel negative regulator in the B7 family. However, how B7-H4 regulates T cell immunity is still not understood. Studies show that the suppressive activity of Treg is associated with expression of B7-H4 in vitro [126]. B7-H4 expression on APCs is triggered by Tregs through production of IL-10. Blockade of B7-H4 reduces the suppressive activity mediated by Tregs. In addition, Treg exhibits suppressive activity by stimulating B7-H4 expression through IL-10. Furthermore, the high expression levels of B7-H4 in various cancer cells suggests that it may help cancer cells escape immune surveillance [127]. Primary ovarian tumour cells express intracellular B7-H4 whereas only a fraction of tumour macrophages express surface B7-H4. B7-H4+ tumour macrophages, but not primary ovarian tumour cells, suppress tumour-associated antigen-specific T cell immunity. Interleukin 6 (IL-6) and IL-10 are expressed high in the tumour environment and trigger macrophage B7-H4 26  expression [127]. Collectively, these results suggest that a collaborative interaction between B7H4 and Tregs may downregulate T cell immunity. B7-H4 deficient mice display normal responses to several types of airway inflammation and they only show mildly augumented Th1 response, suggesting a redundant role of B7-H4 in immune response [128]. Interestingly, a subsequent study reveals that B7-H4 suppresses neutrophil-mediated immune response, suggesting a dominant role in innate immunity [129].  1.5 TOLERANCE STRATEGIES Islet transplantation is an attractive therapeutic option for diabetes but patients still require lifelong systemic immunosuppression with unwanted side effects, including an increased susceptibility to infection and malignancy. The ultimate goal in islet transplantation is the induction of tolerance that would permit graft survival in the absence of continuous immunosuppressive treatment. Induction of tolerance involves both central and peripheral mechanisms [130].  1.5.1  Central tolerance  Central tolerance occurs during lymphocyte development in the thymus and bone marrow [131]. Complete-, mixed-, and micro-chimerism describes compositions of 100% haematopoietic cells, between 1-100%, and less than 1%, respectively that are comprised of donor cells, respectively [132]. Proof of concept for induction of tolerance in humans through the transplantation of haematopoietic cells was primarily established in clinical case studies [133]. Kidney transplants failed to reject in the absence of immunosuppression when kidney transplantation was accompanied by bone marrow transplant from the same donor. This chimerism approach has been used before bone marrow transplantation, but it requires irradiation 27  of endogenous haematopoietic cells. This is an acceptable risk for patients with haematopoietic malignant disorders. However, it would not be acceptable to irradiate haematopoietic cells of transplantation recipients who possess normal bone marrow function. Recent studies showed that mixed chimerism can lead to long-term donor-specific tolerance following organ transplantation [130, 131, 134–136]. 1.5.2  Peripherial tolerance Unlike central tolerance, peripherial tolerance is developed after T and B cells mature. In  order to promote peripherial tolerance, the alloreactive effector T-cell pool must be minimized. Various strategies have been explored to induce peripheral tolerance such as deletion of effector T cells, inhibition of T cell activation by costimulation blockade, or active regulation of effector T cells by Tregs.  1.5.3  Tregs and tolerance  Treg-mediated tolerance induction has been well established as a non-deletional strategy to modulate alloreactive T cell responses. The key role of Tregs in maintaining self tolerance was described by Sakaguche’s group [137]. They demonstrated that Tregs which originated from the thymus play a critical role in protecting the host from autoimmune diseases. Tregs can be categorized into two main types (naturally occurring Tregs, nTregs, and inducible Tregs, iTregs) based on their origins, modes of action, and mechanisms [138]. nTregs develop in the thymus and constitute approximately 5-10% of mature thymocytes and about 10% of peripheral CD4+ T cells. CD4+CD25+ Tregs are known to suppress effector T cell proliferation in vitro through a cell contact-dependent mechanism. The function of nTregs is cytokine independent [139]. A role for nTregs in the development of transplantation tolerance was first 28  indicated by their ability to suppress mouse GVHD following allogeneic bone marrow transplantation [140]. nTregs arise during T-cell development in the thymus whereas a second population of Treg subsets (iTregs) arise during immune responses in the periphery. iTregs are distinct from nTregs and suppress immune responses through secretion of immunosuppressive cytokines. For example, Th3 and Tr1 cells induce tolerance through secretion of TGF-β and IL-10, respectively. [141, 142]. Tr1 cells tend to migrate toward sites of inflammation while nTregs are predominately found in lymphoid organs, suggesting distinct regulatory mechanisms at different sites [143]. Both nTregs and iTregs regulate immune responses depending on the site (lymphoid organ versus peripheral tissue) and milieu (steady-state homeostasis versus inflammatory conditions). Several strategies have been proposed to maintain tolerance through Tregs. Repetitive stimulation of naïve T cells with immature allogeneic DCs results in the development of a suppressive phenotype by responding T cells [144]. Suppression of conventional T cells is mediated in part by secreted cytokines. TGF-βdeficient or wild type CD4+CD25+ Tregs are equally able to suppress the development of inflammatory bowel disease (IBD) when each population is co-transferred with normal CD4+CD45RBhigh cells into SCID mice, suggesting a dispensable role of TGF-β in Tregmediated suppression [145]. By contrast, IL-10 deficient Tregs are unable to suppress IBD. Transferred mice develop IBD but not systemic autoimmue diseases, suggesting that an IL-10dependent mechanism is important for mucosal immune homeostasis but may be dispensable for systemic self-tolerance. However, IL-10 deficient Tregs are able to suppress autoimmune gastritis, suggesting a dispensable role of IL-10 in Treg-mediated suppression in autoimmune gastritis [146]. The other two properties of immune regulation by Tregs are called linked suppression and infectious tolerance. Linked suppression refers to tolerance generated against a specific 29  antigen (Ag-Y) that leads to tolerance against unrelated or third-party antigen (Ag-X or Ag-Z). The specific (Ag-Y) and unrelated (Ag-X) or third-party (Ag-Z) must be expressed on the surface of the same APC [147, 148]. Such linked suppression can be achieved through cell contact or suppressive cytokines such as TGF-β and IL-10. Infectious tolerance refers to that which can be transferred from one recipient to another. CD4+ T cells from tolerized mice could prevent graft rejection in naïve recipients [149-151]. The generation of tolerance can be achieved through various strategies such as costimulation blockade [149-151]. The detailed mechanisms remain elusive.  1.6 TCR SIGNAL TRANSDUCTION TCR signal transduction is triggered by antigen-specific recognition of a peptide presented on MHC by an APC and various co-signalling molecules. The following series of intracellular signal cascades result in T cell activation, development, acquisition of effector’s functions and apoptosis. The earliest step in intracellular signaling following TCR ligation is the activation of Src (LCK and FYN) protein tyrosine kinases (PTKs), leading to phosphorylation of CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) [152-155]. The CD4 (or CD8) coreceptor is constitutively associated with membrane-bound LCK [156, 157]. The binding of CD4 and MHC II brings the tyrosine kinase LCK into proximity with CD3 ITAMs. This movement allows Lck to phosphorylate the ITAMs of CD3. The binding site for the syk-family kinase, ZAP70, is exposed by the phosphorylation of these ITAMs. ZAP70 can now be phophorylated and therefore activated by LCK. Phosphorylation facilitates the binding of ZAP70 to Lck through a Src homology 2 (SH2) domain. Kinase activity is enhanced by this phosphorylation process and can be further increased when ZAP70 phosphorylates LCK. Thus, interaction between LCK and ZAP70 makes a kinase activation cycle. This initial step allows the TCR, which has no intrinsic enzymatic function, to associate with an active PTK 30  which is able to phosphorylate proximal proteins. The phosphorylated ITAMs also serve as docking sites for interactions with other proteins (Fig. 1.2) [158]. The transmembrane adapter protein linker for the activation of T cells (LAT) is one of the most important targets of the ZAP70 [159]. LAT can be phosphorylated by Lck and/or ZAP70. Phosphorylated LAT can bind to phospholipase C-γ1 (PLC), the p85 subunit of phosphoinositide 3-kinase (PI3K). The binding PLC-γ1 to LAT brings it in proximity to the TCR signal complex. This association promotes phosphorylation and activation. Activated PLC-γ1 can cleave PI4, 5 bisphosphate to generate IP3 and DAG. Intracellular Ca++ levels are increased and protein kinase C (PKC) is activated by this movement. At the same time, phosphorylated LAT can also bind to the adaptor protein growth factor receptor-bound protein 2 (GRB2). GRB2 interacts with the guanine exchange of GDP for GTP. RAS is activated by this exchange. The activation of RAS results in activation of the RAS regulated mitogen-associated protein kinase (MAPK) pathway which leads to extracellular signal-regulated kinase (ERK) and JNK phosphorylation. The activation of MAPK cascade leads to the phosphorylation and activation of transcription factors and results in transcription of IL-2 gene (Fig. 1.2) [160, 161]. The PI3K dependent kinase AKT is activated in response to TCR ligation. AKT phosphorylates many targets and regulates the expression of many genes. For example, activation of AKT promotes cell survival through upregulation of prosurvival genes including BCL-XL. AKT also regulates IL-2 gene expression by optimizing transcription of nuclear factor of activated T-cells (NFAT) (Fig. 1.2) [162].  31  Antigen presenting cell MHC  ZAP70  LCK  GRB2  LAT PLCγ1  B7.2 CD28 CD28  CD4/CD8  B7.1  TCRα TCRβ CD3δ CD3ε CD3ε CD3γ  CD3ζ CD3ζ  Ag  PI3K  T cell  SOS  RAS AKT  DAG RAF1  MEKK1  PKC  NF-κB JNK  ERK  P38  JUN  FOS  NF-κB  Gene expression  Figure 1.2 TCR-mediated signal transduction The engagement of the TCR with antigen presented on the MHC and co-stimulatory molecules trigger a series of biochemical reactions. The earliest step is involved in phosphorylation of various proteins on tyrosine residues. Activation of LCK and ZAP70 induces localization of adaptor proteins to the cytoplasmic membrane. Associations of these adaptor protein with RAS, allows the rapid conversion of RAS from the inactive form (GDP-RAS) to the active form (GTP-RAS).  32  1.7 THESIS OBJECTIVES  Islet transplantation has been acknowledged as a valuable treatment option for type 1 diabetes. However, shortage of donor supply and side effects of current immunosuppressive drugs have impaired this application. Allograft rejection is initiated by the recognition of T-cell receptor (TCR) and peptide presented on MHC molecules. In the presence of signal 2, alloreactive T cells become fully activated. Without signal 2, also known as co-stimulatory molecules, T cells undergo apoptosis or anergy. The requirement of signal 2 for full activation of T cells suggests the promising prospect that allografts could become tolerated if signal 2 could be blocked. Therefore, co-stimulation blockade may represent an efficient means to prevent islet allograft rejection and induce tolerance. B7-H4 was identified in 2003 as a co-inhibitory molecule in the B7 family. B7-H4 (B7S1/B7x), which binds to a receptor, not yet identified on activated T cells, inhibits T-cell function. However, its role in vivo has rarely been reported. There is no published data on the function of B7-H4 is controlling allograft rejection. Therefore, the objective of this project is to study the role of B7-H4 pathway on the prolongation of islet allograft survival and the mechanisms involved. Based on the fact that B7-H4 has been demonstrated to have inhibitory regulation on T cell responses, our hypothesis is that activation of the negative co-signaling pathway by local expression of B7-H4 in islets will inhibit activated T-cell proliferation and protect  cells from damage mediated by alloreactive responses.  33  Three specific aims will be addressed in the project.  1. Effects of local expression of B7-H4 by a recombinant adenovirus on the survival of islet allografts. 2. The prospect and mechanisms of generating tolerance by B7-H4 treatment. 3. The mechanisms of T cell responses which inhibit signal transduction associated with B7-H4 engagement.  Targeted use of B7-H4 to improve transplantation outcomes and generate donor-specific tolerance may help to improve the paradigm of immunosuppression. The understanding of B7H4 signalling transduction pathway will aid us in further investigation of the function of B7-H4, especially on the selection of combination therapy (a combination of other reagents to have a synergistic effect). New classes of T-cell inhibitors may reduce side effects of immunosuppression in islet transplantation or rationalize life-long usage of immunosuppression, thus providing a more physiological setting than other therapies for patients with T1D.  34  CHAPTER 2 METHODS AND MATERIALS  35  2.1 ANIMALS C57BL/6, Balb/c, and CBA/J mice were purchased from Jackson Laboratory and housed in the Jack Bell Research Center. All mice were cared for according to the guidelines of the Canadian Council on Animal Care and regulations of the University of British Columbia.  2.2 ISLET ISOLATION  Donor islets were isolated by ductal collagenase injection from 8–10 week-old female Balb/c mice by a protocol modified from Lacy and Kostianovsky, as described previously [163]. The collected islets were hand-picked and counted, and groups of 400 islets were cultured overnight at 37C with or without viral infection. For ex vivo viral-transduction groups, 5 plaque forming units (pfu) of Ad-B7-H4 or Ad-LacZ were absorbed at 37C for 2 h in minimal volume of medium containing no FBS followed by replacement with RPMI 1640 containing 10% FBS and incubation for another 24 h.  2.3 PRIMARY AND SECONDARY ISLET TRANSPLANTATIONS  Recipients were rendered diabetic by a single intraperitoneal injection of streptozotocin at the dose of 200 mg/kg (Sigma, Oakville, Canada) 3–4 days before transplantation. Animals were considered diabetic after two consecutive days with blood glucose levels >300 mg/dl measured by Glucometer Elite (Bayer, Elkhart IN). A total of 400 donor islets were gently injected into the upper pole of the left kidney of age-matched diabetic C57BL/6 recipient mouse. Graft function in transplanted mice was defined as a primary sustained decrease of random blood glucose concentration to less than 200 mg/dl beyond day 3 after transplantation, and a graft was 36  considered as failed when the blood glucose rebounded to a level of >250 mg/dl after primary graft success. To test for immunologic tolerance, the kidneys bearing the primary islet grafts were removed after long-term function (over 60 days). These nephrectomized mice were retransplanted with the same donor strain (BALB/c) or third-party (CBA/J) islets into the right kidney capsule. No immunosuppressive treatment was provided.  2.4 GENERATION OF RECOMBINANT B7-H4 (Ad-B7-H4)  Briefly, cDNA of full-length mouse B7-H4 was cloned from mB7-H4 pcDNA 3.1– plasmids and was sub-cloned into the pShuttle plasmid, in which B7-H4 is driven by the CMV promoter/enhancer. Ad-B7-H4 was constructed by co-transfection of HEK 293 cells with the B7H4pShuttle plus the purified fragment of PI-SceI-digested DNA from both E1- and E3-deleted adenovirus type 5. Ad-B7-H4 was grown in 293 cells and purified by adenovirus purification kits (Clontech Laboratories, Inc. Mountain View, CA). Recombinant virus was titrated by the endpoint dilution method of tissue culture infective dose (TCID50). Ad-LacZ, which contains the Escherichia coli β-galactosidase gene, was grown and purified as described above and used as a control adenovirus.  2.5 RNA ISOLATION AND REAL-TIME PCR ANALYSIS  Total RNA was extracted from mouse islets using RNeasy Mini Kits (QIAGEN, Mississauga, Canada). All RNA samples were digested with RNase-free DNase I (QIAGEN). First-strand complementary deoxyribonucleic acid (cDNA) was then synthesized from this RNA by Superscript II Reverse Transcriptase (GIBCO) using oligo(dT). The primer for adenovirusderived B7-H4 was designed to distinguish between endogenous B7-H4 expression and viral37  delivered gene expression. The primer pairs were listed in Table 2.1. Mouse GAPDH mRNA was used as an internal control to confirm equivalent loading of the total RNA. Kidney graft tissue was excised with the aid of a dissection microscope for RNA isolation. Quantitative real-time PCR was done in duplicate using 25 ng cDNA with 0.4 µM of each primer in a final reaction volume of 20 µl containing 10 µl of 2 SYBR PCR Master Mix (QIAGEN). PCR parameters were as follows: 50°C for 2 min and 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec, 55°C for 20 sec and 72°C for 30 sec. Relative expression level was expressed as 2-(CTubiquitin-CTgene) (where CT is cycling threshold) with ubiquitin RNA as the endogenous control for normalization.  Table 2.1 PCR primer pairs gene Ad-B7-H4 B7-H4 Foxp3 Gzmb IL-2 IL-4 IL-10 IFN-γ TGF-β1  sense CTC CAT AGA GCT AGC GTT TAA CAG CTG GAA ACA TTG GAG AGG TTC ATG CAT CAG CTC TCC AC TCG ACC CTA CAT GGC CTT AC CCC ACT TCA AGC TCC ACT TC CCT CAC ACT CCA CAC CAA TG CCA AGC CTT ATC GGA AAT GA ACT GGC AAA AGG ATG GTG AC TTG CTT CAG CTC CAC AGA GA  anti-sense TGC GGC CTC TGA ACA TCT CAT TGC GGC CTC TGA ACA TCT CAT CTG GAC ACC CAT TCC AGA CT GAG CAG CAG TCG GCA CAA AG ATC CTG GGG AGT TTC AGG TT AGC CTG GGT AGT TCC TTG GT TTT TCA CAG GGG AGA AAT CG TGA GCT CAT TGA ATG CTT GG TGG TTG TAG AGG GCA AGG AC  2.6 WESTERN BLOT  For B7-H4 detection, 25 μg of each cell lysate from islets were loaded onto a 12% separating Bis-Tris gel. The proteins were transferred to a nitrocellular membrane (BioRad, 38  Mississauga, Canada). The membrane was blocked in PBS containing 5% skim milk for 1 h followed by incubation with the primary antibody goat anti-mouse B7-H4 (R&D, Burlington, Canada) or mouse anti-actin at the concentration of 2.5 µg/ml. Secondary antibodies for B7-H4 and actin are goat anti-rat and rabbit anti-goat conjugated with HRPO, respectively. The blot was developed with Enhanced Chemiluminescence Plus Developer (Pierce, Nepean, Canada). For enzymatic deglycosylation, islet proteins were first extracted and denatured, and the denatured islet protein was digested with N-glycosidase F (also known as PNGase, Biolabs, Pickering, Canada) at 37°C for 1 h. Treated and untreated samples were handled in parallel for Western blot analysis. For the proteins involved in signal transduction, T cells were lysed in lysis buffer (150 mM sodium chloride,1.0% NP-40, 50 mM Tris, pH 8.0) supplemented with a cocktail of protease and phosphatase inhibitors (Roche, Laval, QC, Canada). Lysates were made in 1 SDSPAGE buffer and boiled for 2 min prior to separation by SDS-PAGE and electro-transferred to nitrocellulose membranes (Bio-Rad). The amount of protein was determined by Sigma protein assay to ensure equal protein loading in each lane. Antibodies against phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-AKT (Ser473), phospho-GSK-3/ (Ser21/9, GSK-3α preferred), and phosphor-ZAP70 (Tyr319) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to phospho-LCK (Tyr394) were from Santa Cruz Biotechnology. Secondary antibody horseradish peroxidase conjugated goat against rabbit was purchased from R&D (Minneapolis, MN, USA). The blot was developed with Enhanced Chemiluminescence Plus Developer (Pierce, Nepean, ON, Canada) and autographs were quantified using Bio-Rad Quantity One software (Bio-Rad).  39  2.7 GLUCOSE STIMULATION ASSAY Freshly isolated islets were transduced with Ad-B7-H4 or Ad-LacZ (mock) treated overnight at 37°C and ability of insulin secretion in response to glucose stimulation was tested by static incubation.. Islets were then cultured in Krebs-Ringer’s Buffer (Sigma) at 37°C for 30 min. The supernatant was then replaced with Krebs-Ringer’s Buffer with 5 mmol/l or 20 mmol/l of glucose and cultured for 60 min and stored for insulin measurement. Immunoreactive insulin was measured in the incubation media by a sensitive insulin enzyme-linked immunosorbent assay (Crystal Chem. Chicago, IL). Proteins were detected by the Bradford (Sigma). The ratio of insulin release (u/ml) to protein concentration (g/ml) is obtained for each sample. Stimulation Index is calculated by a percentage of the ratio of each sample to uninfected islets incubated at 5 mmol/l glucose (considered as 100).  2.8 INTRAPERITONEAL GLUCOSE TOLERANCE TEST (IPGTT)  After 5 h fasting, mice were injected with 2 g/kg body weight of glucose intraperitoneally. Tail blood samples were taken at 0, 15, 30, 60, and 120 min after injection and tested for blood glucose level and/or insulin by ELISA assay according to manufacturer’s instructions (Crystal Chem. Inc., Downers Grove, IL).  2.9 MIXED LYMPHOCYTE REACTION ASSAY (MLR)  Responder splenocytes were isolated from C57BL/6 recipients of B7-H4 transduced islets at 60 days post-transplant and were compared with those from non-transplanted controls in MLR assays. Responder cells were seeded at 1×105/well and cultured in RPMI 1640 (GIBCO) medium 40  supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 50 µM 2-ME in triplicate and stimulated with γ-irradiated (3000 rad) Balb/c or CBA/J stimulator splenocytes at various stimulator/responder ratios or at a stimulator/responder ratio of 3/1 in some experiments for 3 days. The wells were pulsed with 1 µCi/well of [3H] methylthymidine (PerkinElmer, Montreal, Canada) and the radio-labelled plate was harvested 18 h later with a multiple Harvester 96 (Tomtec, Hamden, CT) and [3H] incorporation was measured in a liquid scintillation counter (Wallac). Results were calculated as mean counts per minute (cpm). Recombinant mouse IL-2 at 100 u/mL was added in some MLR assays. For some MLRs, CD4+CD25+ T cells were depleted from lymphocytes by microbeads coated with anti-CD25 monoclonal antibody (mAb) (Miltenyi Biotec Inc.). The negatively selected CD25– cells were collected. Purity and viability, as determined by FACS analysis and trypan blue staining respectively, exceeded 95% in all cases.  2.10 HISTOLOGICAL ANALYSIS  One half of the islet graft-bearing kidney was snap-frozen in Tissue –Tek OCT (Sakura Finetek, Torrence, CA) and the other half was fixed in 4% paraformaldehyde and paraffinembedded. For B7-H4 (R&D), CD3 (Abcam, Cambridge, USA), and Foxp3 (eBioscience) stainings in the graft fixed tissue sections of 5 μm, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min at room temperature. HRP-conjugated anti-goat or antirat antibody was used for secondary antibody. Brown color was developed by 3,3'diaminobenzidine substrate (Sigma). Also, sections were stained with guinea pig anti-insulin or rat anti-mouse CD45 (BD Bioscience), followed by secondary Texas Red– or FITC-conjugated  41  antibodies. Staining of CD4 and CD8 was performed on the cryostat section according to the manufacture’s instructions (BD Bioscience). Isletitis grade was assessed by Yoon’s method for grading insulitis (177). Grade: 0, normal islets; 1, mononuclear infiltration, largely in the periphery, in less than 25% of the islet; 2, 25 to 50% of islet showing mononuclear infiltration; 3, over 50% of islet showing mononuclear infiltration; and 4, small, retracted islet with few mononuclear cells.  2.11 FLOW CYTOMETRY Single cells were re-suspended in 50 L ice-cold PBS/1% calf serum buffer and stained with fluorochrome-conjugated mAbs. MAbs against CD4, CD3, CD69, CD25, Foxp3, and control IgG were purchased from eBioscience (San Diego, CA). Intracellular staining of Foxp3 was performed using a commercially available kit (eBioscience). 100,000 or 20,000 live gated events were acquired. Cells were analyzed on FACSCanto (BD Bioscience, Mississauga, Canada) using De Novo software (De Novo Software, Los Angeles, CA).  2.12 T CELL STIMULATION  CD3+, CD4+, or CD8+ T cells were purified from mouse spleens and lymph nodes by using magnetic negative selection (StemCell Inc., Vancouver, BC, Canada). The purity of CD3+, CD4+, or CD8+ T cells was more than 95% or 90% for CD8+ by FACS analysis (FACSCalibur, Becton Dickinson). CD3+, CD4+, or CD8+ T cells were cultured in tissue culture plates that were pre-coated with anti-CD3 mAb (145-2C11, BD Bioscience) at indicated concentrations with soluble anti-CD28 (clone 37.51, BD Bioscience). Pre-activated CD3+ T cells were first stimulated with anti-CD3 mAb at 0.3 g/mL. These pre-activated T cells were rested for 30 h 42  before being re-activated with the indicated amounts of plate-bound anti-CD3 mAb and soluble anti-CD28 (2 g/mL).  2.13 GENERATION AND PURIFICATION OF B7-H4.Ig FUSION PROTEIN  B7-H4.Ig was harvested from the supernatant of 293 cells with transient transfection of B7-H4 cDNA in which the extracellular domain was in-frame fused with human Ig G1 Fc fragment. This B7-H4-containing supernatant was purified by Protein A affinity chromatography (Bio-Rad, Mississauga, ON, Canada). The purified B7-H4.Ig was quantitated by SDS-PAGE electrophoresis with bovine serum albumin (BSA) as a standard control and further confirmed by direct ELISA. Control human IgG1.Ig protein was from Cedarlane Laboratories (Cedarlane, Burlington, ON, Canada). 2.14 [3H]-THYMIDINE INCORPORATON ASSAY B7-H4.Ig or control human IgG1 Fc.Ig (Fc.Ig) was incubated in 96-well flat-bottom tissue culture plates that were pre-coated with anti-CD3 antibody for 2 to 4 h at 37C. Purified T cells (3×104 cells per well) were cultured in RPMI-1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 u/mL penicillin, 100 g/mL streptomycin and 50 M 2-mercaptoethanol for 72 h, followed by pulse with addition of 1 Ci [3H]-thymidine (PerkinElmer, Montreal, QC, Canada) in the last 18 h. [3H]-thymidine incorporation was measured by liquid scintillation counting.  43  2.15 CYTOKINE DETECTION  The production of IL-2 protein in the cell supernatants was measured by sandwich ELISA with a pair of anti-IL-2 antibodies (eBioscience, San Diego, CA, USA). Briefly, capture antibodies (clone JES6-IA12) were coated at 2.5 g/mL on NUNC ELISA plates at 4C overnight. After three washes, the coated plates were blocked with PBS/3% BSA for 1 h at 37C. The collected cell supernatants and serial dilutions of recombinant mouse IL-2 standard protein (eBioscience) were added and incubated for 2 h at 37C. Biotinylated detection antibodies (clone JES6-5H4) were added to capture the IL-2 protein, followed by Streptavidin-HRPO incubation. TMB (Sigma, Oakville, ON, Canada) was used as developing reagent. Absorbance was determined at 405 nm. Triplicate absorbance values of supernatant samples were calculated according to standard recombinant IL-2 concentration in ng/mL.  2.16 STATISTICAL ANALYSIS  Glucose-stimulated insulin release, glucose tolerance, and MLR data were analyzed by two-tailed Student’s T tests or post-hoc ANOVA for multiple comparisons (Turkey’s range test). Graft survival curves were created by Prism 4.03 software using Kaplan-Meier life-table analysis, and the two sets of survival curves were compared by the log-rank test. Student’s t test (two-tailed and paired analysis) was used for comparison of means between experimental groups examined by MLR, real-time PCR, and FACS assays. Differences were considered significant if p<0.05, unless stated otherwise.  44  CHAPTER 3 THE ROLE OF B7-H4 SIGNALLING IN ISLET ALLOGRAFT REJECTION  45  3.1 BACKGROUND Studies in experimental models and recent clinical trials have demonstrated that islet transplantation is a potential therapeutic treatment for diabetes patients [7–12]. However, loss of insulin independence by 5 years in the majority of recipients remains of concern [11]. Many factors play a role in allograft failure, including islet injury during isolation, hypoxia, recurrence of β-cell autoimmunity, insufficient revascularization, and islet graft destruction by alloreactive T cells. Among these factors, immunological rejection contributes the most to graft failure [15, 19]. Allograft rejection depends on the activation of alloreactive T cells controlled by the generation of two signals. Signal 1 results from engagement of TCR by its cognate antigen– specific MHC. Signal 2 invloves co-stimulatory/co-inhibitory molecules and results in either promoting or inhibiting T-cell activation. Given the central role of T cells in transplant rejection, the goal of therapies to prevent graft rejection is to block destructive T-cell responses. Many studies have established the effectiveness in controlling graft rejection by co-stimulatory/coinhibitory molecules [68, 164, 165]. B7-H4 (also called B7S1 or B7x) is a more recently identified co-inhibitory molecule of the B7-CD28 family [114–116]. B7-H4 mRNA can be found in both lymphoid and nonlymphoid tissues, indicating that the B7-H4 pathway may have more profound role in the periphery than in the secondary lymphoid tissue [114]. Various studies indicate that B7-H4 inhibits T-cell proliferation and cytotoxicity of antigenic peptide-specific T cells, and it reduces their secretion of IL-2, IL-4, IL-10 and IFN-γ in response to anti-CD3 antibody [114–116]. Little is known about the role of B7-H4 in regulating islet graft rejection. mRNAs of B7H4 have been shown to be expressed in mouse islets but not the B7-H4 protein. In this study, we developed a recombinant B7-H4 adenovirus (Ad-B7-H4) for expression of B7-H4 protein in islet  46  grafts. We use this construct to determine the effect of local expression of B7-H4 on inhibiting alloreactive T cells in islet transplantation.  3.2 RESULTS  3.2.1 B7-H4 expression in Ad-B7-H4–transduced primary islets  Ad-B7-H4 transduction efficiency at various times and concentrations was initially tested in the 293 cell line and in primary islets by flow cytometric analysis, and dose- and timedependent increases of B7-H4 expression were observed. To avoid possible cytotoxicity associated with adenovirus transduction and to optimize the balance between transduction efficiency and islet cell function, an optimal Ad-B7-H4 concentration of 5 pfu was chosen for use in the following transduction experiments, based on our preliminary results. Endogenous B7H4 mRNA was expressed in primary islets, as well as in recombinant adenovirus-transduced islets. Ad-derived B7-H4 mRNA was only expressed in Ad-B7-H4–transduced islets but not in control Ad-LacZ–infected or uninfected islets (Fig. 3.1A). To further define the nature of B7-H4 protein derived by adenoviral transduction, Western blots were performed. As expected, mouse islets did not express B7-H4 protein (Fig. 3.1B). An approximately 70 kD band was detected in Ad-B7-H4–transduced islets, but not Ad-LacZ–transduced islets (Fig. 3.1B). However, the predicted size based on amino acid sequence of the B7-H4 protein is around 28 kD, significantly smaller than that of the derived B7-H4 protein observed [117, 122]. There are seven potential Nlinked glycosylation sites mainly in exons 2, 3, and 6 of the B7-H4 gene [117].  47  A.  B.  Figure 3.1 B7-H4 expression in primary mouse islets transduced by Ad-B7-H4 Ad-derived and endogenous B7-H4 mRNA expression in primary islets with various ex vivo treatments (untreated, Ad-B7-H4–infected or Ad-LacZ–infected at 5 pfu for 24 h) were detected in RT-PCR assay (A). Expression of Ad-derived B7-H4 protein in primary islets after various ex vivo treatments (untreated, Ad-B7-H4–infected or Ad-LacZ–infected at 5 pfu for 48 h) (B. left three channels) and expression of undigested and PNGase-digested Ad-derived B7-H4 protein in Ad-B7-H4–treated islets (B. right two channels) were detected in Western blot assays. GAPDH and actin are islet housekeeping mRNA and protein controls, respectively.  48  To determine if the glycosylation sites are responsible for the increased size of B7-H4 protein expressed in Ad-B7-H4–transduced islets, the protein was digested with PNGase, which can remove N-linked carbohydrates. The size of the B7-H4 protein was reduced from 70 kD to 28 kD upon PNGase treatment, suggesting that the additional carbohydrates were added to the backbone of B7-H4 by post-translational modification (Fig. 3.1B). Therefore, the protein expressed by adenoviral vector ex vivo is a biological active mature protein. To define the influence of virus infection on insulin and glucagon expression, insulin and glucagon were stained in islet tissue sections. Insulin distributed similarly in the cytoplasmic compartments of both viral-infected and uninfected islets. Glucagon staining showed a typical ring-like structure and was distributed in the periphery of the islets in both infected and uninfected islets (Fig. 3.2).  49  Figure 3.2 Expression of B7-H4 on isolated islets after recombinant Ad-B7-H4 transduction Isolated islets from BALB/c mice were transduced with Ad-B7-H4 (left panel) or Ad-LacZ (right panel) at 5 pfu for 24 h. H&E, insulin (in red) plus B7-H4 (in green), or glucagon (in red) plus B7-H4 (in green) staining of islets from these two groups was shown from the top to the bottom. Scale bar= 100 m  50  3.2.2 B7-H4 recombinant adenovirus transduction does not affect β cell function  To determine whether there is any adverse affect of adenovirus-transduction on insulin secretion from freshly isolated islets, glucose-stimulated insulin secretion (GSIS) was examined after adenoviral transduction. The results showed that insulin levels increased approximately 3fold in response to high glucose stimulation in both infected and uninfected islets (Fig. 3.3). The insulin secretory function from Ad-B7-H4–transduced islets was similar to non-transduced islets (p=0.9), indicating that recombinant B7-H4 adenovirus exhibited no negative influence on islet function at the transfection condition. uninfected  Ad-B7-H4  GSIS (% badal)  350 *  300  *  250 200 150 100 50 0 5  20  5  20  Glucose Concentration (mM)  Figure 3.3 Influence of Ad-B7-H4 transduction in glucose-stimulated insulin secretion in islets Mouse islets were infected with Ad-B7-H4 at 5 pfu for 24 h or untreated as controls. Mouse insulin secretion (u/ml) was then measured and normalized with its protein content (g/ml) and expressed as glucose stimulated-insulin secretion index (GSIS), shown on the Y-axis. The data are represented as 100% GSIS by uninfected islet corresponding to 5 mM glucose. Results are performed in triplicate and represented three independent experiments.  51  In addition, we used syngeneic islet transplantation to confirm our in vitro results. Blood glucose in diabetic Balb/c recipients induced by STZ decreased <200 mg/dl upon transplantation in three groups of Balb/c donor islets without treatment or those infected with adenovirus (AdLacZ or Ad-B7-H4, n=6 each). Furthermore, these islet grafts survived indefinitely in all groups (Fig. 3.4). These results confirmed that adenoviral transduction did not induce deleterious effects on islet β cell function.  Blood Glucose (mg/dL)  600  untreated  500  Ad-LacZ  400  Ad-B7-H4  300 200 100 0 1  5  9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69  Transplantation  Days Post-Transplant  nephrectomy  Figure 3.4 Blood glucose of recipients transplanted with syngeneic donor islets which were treated with adenovirus Blood glucose level was shown in diabetic BALB/c recipients transplanted with either untreated (shown in black lines), Ad-LacZ- (shown in red lines), or Ad-B7H4-infected (shown in green lines) BALB/c donor islets. Blood glucose returned to a normal level after islet transplantation and rebounded to a high level upon removal of donor engrafted left kidney in three groups. 6 mice were included in each group.  52  3.2.3 Local B7-H4 expression in islets prolongs allograft survival  The role of B7-H4 expression in islet grafts was evaluated in a mouse islet allotransplant model. As shown in Fig. 3.5A, transplantation of 400 Ad-B7-H4 or Ad-LacZ–transduced islets under the kidney capsule promptly reduced blood glucose in streptozotocin-induced diabetic C57BL/6 recipients. Mice transplanted with Ad-LacZ–transduced islets rejected the allografts around 2 weeks post-transplant (mean 14.5±3.3 days, n=12) after establishing primary islet function. In contrast, mice transplanted with Ad-B7-H4–transduced islets demonstrated prolonged graft function (mean 56.5±9.4 days, n=12) with some extending to 60 days post-transplantation, the designated experimental endpoint. These data suggest that local B7-H4 expression in islets by Ad-B7-H4 transduction significantly prolongs allograft β-cell survival in vivo (p=0.0001) (Fig. 3.5B). When the kidneys bearing the donor islet grafts that had survived for more than 60 days were removed, blood glucose values rapidly returned to pre-transplant levels, confirming that the Ad-B7-H4–transduced donor islets were responsible for the reversal of high blood glucose levels in diabetic recipient mice rather than reversion/regeneration of endogenous pancreatic β cell function (Fig. 4.5A, p71). To further define the function of the transplanted Ad-B7-H4– transduced islets in the graft, we performed intraperitoneal glucose tolerance tests on day 30 post-transplant. After a 5-h fast, glucose level increased in response to glucose injection at 15 and 30 min and dropped at 60 and 90 min to the basal level at 120 min in both transplanted and non-transplanted mice (Fig. 3.5C). Serum insulin levels increased from 0.22  0.05 ng/ml at baseline to 2.3 ± 0.3 15 min post-injection in the transplanted group (n=6) vs. from 0.21  0.04 to 2.1 ± 0.5 in the non- transplanted group (n=6), confirming that islet function in transplanted mice responded similarly to nontransplanted control subjects.  53  Blood Glucose (mg/dL)  A.  700  Ad-LacZ  600  Ad-B 7 -H4  500 400 300 200 100 0 -2  8  18  28  38  48  58  68  D ays Post-Tran spl an t  B. Islet Graft Survival  100  Ad-LacZ Ad-B7-H4  75 50 25 0 0  10  20  30  40  50  60  70  Days Post-Transplant  Blood Glucose (mg/dl)  C.  600  Control  500  Ad-B7-H4  400 300 200 100 0 0  30  60  90  120  Minutes After Glucose Injection  Figure 3.5 Prolongation of allograft survival in streptozotocin-induced diabetic recipient C57BL/6 mice transplanted with Ad-B7-H4–transduced Balb/c mouse islets Significant prolongation of graft function in diabetic C57BL/6 mice transplanted with Ad-B7H4–transduced islets (black lines) was shown in (A), compared with that in mice with Ad-LacZ– treated islets (grey lines) (n=12, mean 56.5±9.4 vs. 14.5±3.3 days, respectively). Kaplan-Meier graft survival curve is derived from blood glucose data (B), with P=0.001 by the log-rank test. Intraperitoneal glucose tolerance test results in C57BL/6 recipients transplanted with Ad-B7-H4– treated islets (n=6) 30 days post transplant (black square) were compared to wild type control mice without transplant (n=6) (open triangle). Data are expressed as means ± SE, and there in no significant difference found between two groups (C).  54  3.2.4 B7-H4 is expressed transiently in the islet grafts  According to our results, most of the C57BL/6 recipients transplanted with Ad-B7-H4– transduced islets survived for more than 60 days (Fig. 3.5B). The vector we used is a replicationdeficient adenovirus derived from human serotype 5, lacking the E1 gene that are required for viral replication in the host. Thus, the foreign gene transduced by Ad-B7-H4 is not constitutively expressed in the islet grafts. To address the question whether or not expression of B7-H4 in the graft is essential for controlling graft survival, we tested B7-H4 expression at both mRNA and protein levels in the early and the late stages of transplantation. As shown in Fig. 3.4, RNA expression in the graft quantitated by real-time RT-PCR was dramatically reduced to very minimal levels at day 60, compared with day 10 post-transplant (Fig. 3.6A). In agreement with mRNA expression levels, B7-H4 protein can be easily detected at day 10 but not at day 60 in the grafts of paraformaldehyde-fixed, paraffin-embedded sections (Fig. 3.6B). These results demonstrate that sustained expression of B7-H4 in the graft is not associated with allograft function during the late stage post-transplant, suggesting that expression is not essential for function.  55  A.  B.  D60  D10  D10  0  50  Relative B7-H4 Expression  100  D60  Figure 3.6 B7-H4 expression in the transplanted islet grafts transduced with Ad-B7-H4 (A). Relative B7-H4 mRNA expression of excised grafts with Ad-B7-H4–transduced islets at 10 days and 60 days post-transplant was tested by real-time PCR. (B). B7-H4 protein expression (in brown) in tissue sections of these grafts was detected by antimouse B7-H4 antibody plus HRP-conjugated secondary anti-goat antibody with 3,3'diaminobenzidine as substrate in immunohistochemistry staining. Magnification is 200.  56  3.2.5 Immune responses and β-cell function in the islet grafts To further investigate the mechanism of protective effects of B7-H4 on allograft survival of the islets, graft-bearing kidneys transplanted with Ad-B7-H4 and Ad-LacZ islets were removed from the mice and double stained for the activated leukocyte marker CD45 and insulin by specific antibodies for monitoring engrafted islet lymphoid infiltration and β cell insulin content. As shown in Fig. 3.7A, in the early stage 10 days post-transplant, infiltration of CD45+ leukocytes is found surrounding the Ad-LacZ–transduced grafts and the Ad-B7-H4–transduced grafts (90% and 80% respectively), but no infiltration is found in the kidney. At day 60 posttransplant, CD45+ leukocyte infiltration became much less (20%), compared with the early stage (Fig. 3.7A). Insulin-positive cells remained strong 60 days post-transplant in the Ad-B7-H4– transduced graft (Fig. 3.7A). In order to distinguish the phenotypes of CD45+ cells, CD4, CD8, and Foxp3 staining was performed (Fig. 3.7A). The number of CD8+ cells was lower in the AdB7-H4-treated groups at both day 10 and day 60 (123 and 185 cells per field respectively) compared with the Ad-LacZ-infected group (425 cells per field, p=0.001). CD4+ expressing cells were similar and high in all three groups. However, expression of Foxp3+ at day 60 (Fig. 3.7A, low right panel) was elevated significantly compared with the early stage groups (82±6 vs 29±7, and 17±4 cells per field respectively, three randomly selected fields were counted, p=0.001 compared with two groups of early stage). Isletitis was assessed according to Yoon’s method (177). The lowest frequency of isletitis scores was found in the Ad-B7-H4-treated grafts at day 10 post transplant (Fig. 3.7B)  57  A.  CD45 + Insulin  CD4  CD8  Foxp3  Figure 3.7 Inflammatory infiltration and residual β cells in the islet grafts transplanted with Ad-LacZ– or Ad-B7-H4–transduced islets (A). Tissue sections of transplanted grafts of Ad-LacZ–tranduced islets 10 days post-transplant , Ad-B7-H4–transduced islets 10 days or 60 days post-transplant were double stained by secondary Texas Red– and FITC-conjugated antibodies for detecting insulin (in red) and CD45 (in green) positive cells or stained by rat anti-mouse CD4, CD8 and Foxp3 antibodies respectively, followed by HRP-conjugated secondary antibody with 3,3'-diaminobenzidine as substrate for identifying the subsets of the infiltrating cells in immunohistochemistry assays. Magnification is 200. (B). Isletitis grade was blindly assessed by Yoon’s method. Grade 0, normal islets; 1, mononuclear infiltration, largely in the periphery, in less than 25% of the islet; 2, 25 to 50% of islet showing mononuclear infiltration; 3, over 50% of islet showing mononuclear infiltration; and 4, small, retracted islet with few mononuclear cells.  58  3.2.6 Lymphocyte responses to alloantigen stimulation in B7-H4 recipients To confirm the hypothesis of acceptance of allograft in the C57BL/6 recipients of Ad-B7H4–treated islets, MLR assays with the splenocyte mixtures at various stimulator (Balb/c)/responder ratios of wild type C57BL/6 (group 1) and of C57BL/6 recipients transplanted with Ad-B7-H4–transduced islets (group 2) were performed. Lymphocytes proliferated vigorously after alloantigen stimulation (Fig. 3.8). Significantly lower C57BL/6 mixed lymphocyte responses to alloantigens of Balb/c mice were observed in group 2 vs. those in group 1 (p=0.005, p=0.006, and p=0.01, at stimulators/responders ratios 5:1, 2:1, and 1:1, respectively) (Fig. 3.8).  [3H]-thymidine incorporation  18000  Non-Tx CTRL  16000  D60 Post-Tx  14000 12000 10000 8000 6000 4000 2000 0 5:1  2:1  1:1  1:2  Stimulators: Responders ratios  Figure 3.8 Allogeneic mixed-lymphocyte reaction of transplanted mice with Ad-B7-H4– treated islets in comparison with that of non-transplanted wild type mice The responses of the responder splenocytes from C57BL/6 recipients transplanted with Ad-B7H4–transduced islets 60 days post-transplant (D60 post-Tx, black square) and those of responders from wild type mice without transplant (Non-Tx CTRL, open square) to stimulator γirradiated-splenocytes (at 2500 rad) from Balb/c mice were tested in 3-day [3H] methylthymidine proliferation assays. The results are representative of three independent experiments. *Significant differences between the Ad-B7-H4 treated group and the control group at stimulators/responder ratios 5:1, 2:1, and 1:1.  59  3.3 SUMMARY OF FINDINGS AND DISCUSSION This study is the first report to show that local expression of B7-H4 can prolong mouse islet allotransplant survival. The functional role of B7-H4 provides the fundamental understanding of potential new immunosuppressive drugs development. The mechanism whereby B7-H4 prolongs islet graft survival remains unclear. Sica et al. showed that B7-H4.Ig injection significantly reduced the activity of alloreactive cytotoxic T cells and extended the survival of mice developing GVHD [114]. Consistent with their results, we found that the transplantation of Ad-B7-H4–transduced islet grafts induced hyporesponsiveness of the recipients’ lymphocytes to alloantigenic stimulation in MLR assays. Sica et al.’s study suggests that the inhibitory effect of B7-H4 on T-cell proliferation in mouse MLR occurs through cell-cycle arrest [114]. Our previous study also indicates that B7-H4.Ig protein arrests cell-cycle progression of activated human CD4+ T cells in G0/G1 phase and in addition induces apoptosis of both activated CD4+ and CD8+ T cells from human T1D patients [166]; therefore, the observed alloreactive cell hyporesponsiveness in this study could be induced by anergy and/or apoptosis of responder cells. The study of Prasad et al. indicated that mice treated with anti-B7-H4 (B7S1) blocking antibody consistently developed greatly accelerated and much more robust EAE, a mouse model of autoimmune disease (multiple sclerosis), resulting in greater CD4 and CD8 cell infiltration in the brain compared with controls [115]. Similar to their results, our study showed that fewer CD45+ immune response cells were infiltrated into the transplanted islet grafts treated by Ad-B7H4, suggesting the inhibition of immunologically active infiltrating cells by B7-H4 blockade. Moreover, the CD8+ T subset was reduced in grafts from Ad-B7-H4-treated group (Fig. 3.5A), suggesting CD8 population may be associated with graft rejection [167]. A recent study indicated that epithelial cells expressing B7-H4 showed a 40% decrease in apoptosis [168]; this suggests that the anti-apoptotic effect of B7-H4 could be an additional mode 60  of action. The potential anti-apoptotic action of B7-H4 on islet cells raises the potential of malignant transformation induced by ectopic over-expression of B7-H4. B7-H4 has been observed to be over-expressed in various cancers, such as breast, ovarian and pancreatic cancers, suggesting that B7-H4 over-expression might associate with epithelial cell transformation [169, 170]. Although B7-H4 gene transfer may on one hand enhance β cell survival, it may also result in transformation (e.g. β-cell tumor, insulinoma). In this study, we chose to transiently express B7-H4 using a replication-competent adenovirus which would help prevent tumor formation due to constitutive B7-H4 expression. No transformation or tumorigenesis in the Ad-B7-H4-treated islets was detected for both syngeneic and allogeneic islet transplants. Potential tumor formation will be an important issue to consider in further clinical development of B7-H4 transduction strategies for islet protection. In summary, transient expression of the negative co-stimulatory molecule B7-H4 by adenovirus vector in islet inhibits graft loss in transplantation, while insulin-secretory function is preserved. The results from this finding demonstrate that local B7-H4 expression in transplanted islet grafts is able to inhibit alloreactive lymphocyte response and prolong mouse islet allotransplant survival.  61  CHAPTER 4 THE ROLE OF B7-H4 IN GENERATING TOLERANCE  62  4.1 BACKGROUND The success of islet transplantation is largely reliant on effective immunosuppressive therapy. Current immunosuppressive drugs are able to control acute rejection efficiently, in particular changes to the immunosuppressive regimen introduced by the Edmonton Protocol improved graft survival at one year. However, toxic effects of these drugs on β cells have been implicated as a major factor in the long-term dysfunction of islet allografts [7, 11, 35, 171, 172]. In order to improve β-cell function, rationalization of immunosuppressive therapy is needed using a new generation of drugs with minimal side effects. Greater immunoselectivity is desirable with the overall objective of tolerance induction. Immune–mediated rejection is mainly controlled by an alloreactive T-cell response to transplanted grafts. The specificity for alloreactivity is determined by the recognition of the peptide presented on MHC and TCR. However, the antigen-specific signal generated by the TCR alone is insufficient for optimal T-cell activation [173]. Full T-cell activation requires a second positive co-signal, such as is provided by CD28 stimulation [54, 173]. In the presence of a negative co-signaling molecule, T-cell proliferation is terminated and likely results in a state of anergy, which is defined as inability to respond when re-exposed to the same antigen [55, 173]. The ability of negative co-signals to generate anergy provides us with an attractive strategy to inhibit allograft rejection and to induce tolerance. Generation of tolerance with co-signaling blockade has been reported to occur through various mechanisms including anergy, clonal deletion, and immune regulation/suppression [51, 98, 140, 174, 175]. For example, blocking both CD28–B7 and CD40–CD40 ligand binding results in apoptosis of proliferating T cells and permanent engraftment of cardiac allografts [176]. In another study, combined CTLA-4.Ig and anti-CD40L treatment results in tolerance to alloantigen in vivo through induction of T regulatory cells (Tregs) within the local graft environment and/or peripheral tissues [140, 174].  63  Several studies have demonstrated that it is feasible to withdraw continuing immunosuppressive therapy after blockade of co-signaling molecules [51, 98, 140, 174, 175]. B7-H4 functions as a negative co-signaling molecule in vitro [114–116] . Local expression of B7-H4 by a recombinant adenovirus (Ad-B7-H4) prolongs murine islet allograft survival in a strongly immunogenic combination, from BALB/c to C57BL/6 [177]. These promising data prompted us to explore whether alloantigen tolerance arises in mice receiving B7-H4 transduced islets.  4.2 RESULTS  4.2.1 A regulatory phenotype develops in B7-H4 treated grafts and periphery Expression of genes associated with regulatory T cells was sought in both the graft and the periphery. Foxp3, IL-10, and TGFβ1 mRNA expression in islet grafts and spleens of longterm surviving and syngeneic recipients were analyzed by quantitative real-time PCR (Q-PCR) on days 2, 10, and 60 (n=8 in each group). Tissues from syngeneic (C57BL/6  C57BL/6) recipients were used for comparison. Fig. 4.1A shows that Foxp3 expression in both islet graft and spleens from D60_Tx recipients was higher than that of the syngeneic transplant controls, but statistical significance was achieved only on the day 60 after first transplant (Fig. 4.1A). TGFβ1 was expressed at a similar level to syngeneic controls in all tissue analyzed (data not shown). In contrast, IL-10 expression was significantly increased in the spleen at day 60 (p=0.005, n=8) (Fig. 4.1B). Foxp3+, CD4+CD25+Foxp3+ Tregs, and CD4+CD25–Foxp3– T effectors (Teffs) in the draining renal lymph nodes and spleens of long-term surviving graft recipients were quantified by FACS. Fig. 4.1C shows that increased Foxp3+ Tregs were detected in the renal lymph nodes at day 2 post-transplant (p=0.004, n=8). However, there was no significant difference in absolute 64  number (6.05  104 ± 1.82 versus 9.03  104 ± 2.97, p=0.06). There was no difference in the quantity of CD4+CD25–Foxp3– Teffs in either renal lymph nodes or spleens of the two groups of recipients (data not shown). At day 60, a significantly higher number of Foxp3+ Tregs was detected in the spleen of the mice that had B7-H4–transduced islets compared with the syngeneic recipients (Fig. 4.1D, p=0.04, n=8). There was also a significant increase in absolute number (7.57  105 ± 2.91 versus 1.06  106 ± 1.06, p=0.05).  Foxp3 mRNA  10  Syngeneic B7-H4  8  10  *p=0.03  6  4  4  2  2  0  0 10  Syngeneic B7-H4  8  6  2  C.  Spleen  Graft  *p=0.05  6 4  2  60  D. 10  Syngeneic B7-H4  8 6  6  4  4  2  2  0  0 2  10  Syngeneic B7-H4  8  60  Days post-transplant  *p=0.005  10  60  Spleen  10  % Foxp3  10  Syngeneic B7-H4  *p=0.004  0  10  B. IL-10 mRNA  8  Lymph node  2  2  60  10  % Foxp3  A.  Syngeneic B7-H4  8 6  *p=0.04  4 2 0  2  10  60  Days post-transplant  2  10  60  Days post-transplant  Figure 4.1 High levels of Foxp3, Tregs, and IL-10 were found in the grafts and periphery Results from syngeneic islet transplant recipients and D60_Tx recipients are shown in white and grey bars, respectively. 18s mRNA was used as loading control. (A). mRNA expression level in the grafts and spleens. There was a significantly increased expression of Foxp3 in grafts of D60_Tx recipients, compared with that of the syngeneic recipients (p=0.03). (B). mRNA expression level in the grafts and spleens. IL-10 expression level was significantly higher in the D60_Tx mice, compared with syngeneic recipients at day 60 (p=0.005). Kinetics of Foxp3+ cells in the local renal lymph nodes (C) and spleens (D). Percentages of renal lymph node Foxp3+ cells in the D60_Tx mice were significantly higher than that in the syngeneic mice at day 2 posttransplant (p=0.004). Frequencies of spleen Foxp3+ Tregs in the D60_Tx mice were significantly higher than that in the syngeneic mice at day 60 post-transplant (p=0.04). Each group consisted of 8 recipients.  65  4.2.2 MLR demonstrates attenuated proliferation in B7-H4 treated recipients  We examined the proliferation of splenic leukocyte cells (SLCs) from either naïve C57BL/6 mice (B6 mice) or C57BL/6 mice bearing 60–day surviving B7-H4–transduced BALB/c islet grafts (D60_Tx mice) in response to BALB/c stimulator cells. Fig. 4.2A shows that SLCs from D60_Tx mice demonstrated attenuated proliferation [(5.3±1.2) ×103 cpm] compared with cells from naïve B6 mice [(10.2±1.3) ×103 cpm] (p=0.003). The response of D60_Tx SLCs to other alloantigen was evaluated by challenging them with third-party CBA/J (H-2k) stimulator cells. SLCs from both naïve B6 and transplanted D60_Tx mice demonstrated similar responses to CBA/J cells (Fig. 4.2A, p=0.14 for D60_Tx cells stimulated with BALB/c vs. CBA/J, n=8). Hence, SLCs from D60_Tx recipients are anergic to primary donor alloantigen but mount a normal proliferative response to other alloantigens. Fig. 4.2B demonstrates an experiment with a 1:1 mixture of SLCs from naïve B6 and D60_Tx transplanted mice. Cell proliferation was [(6.2±2.4) ×103 cpm] in response to BALB/c (H-2d) donor antigen stimulation compared with [(10.2±1.3) ×103 cpm] for naïve B6 cells (p=0.04), demonstrating that D60_Tx SLCs caused suppression of naïve SLC proliferation in response to donor alloantigen stimulation.  4.2.3 The presence of CD4+CD25+ T cells is required for hyporesponsiveness  To investigate the mechanism of donor-specific hyporesponsiveness observed in vitro, the role of CD4+CD25+ T cells was examined. CD4+CD25+ Tregs were depleted from SLCs isolated from D60_Tx mice and were subjected to MLR with BALB/c stimulators. As shown in Fig. 4.2C, depletion of CD4+CD25+ T cells restored the response of D60_Tx cells to alloantigen  66  stimulation, suggesting that CD4+CD25+ T cells plays a role in B7-H4–induced islet allograft tolerance. We next tested whether or not exogenously added IL-2 could reverse the donor-specific alloantigen hyporesponsiveness. The addition of IL-2 to the MLR co-culture resulted in increased proliferation compared with co-cultures without IL-2 [(11.9±2.1) ×103 vs. (5.3±1.2) ×103 cpm] (p=0.001) (Fig. 4.2D). The results revealed that exogenous provision of IL-2 to the MLR co-culture ablated the suppressive response mediated by CD4+CD25+. Therefore, the hyporesponsive state in the D60_Tx cells is dependent on IL-2. Production of IL-2 in the MLR co-cultures was also detected. At both mRNA and protein levels, transcription (Fig. 4.2E) and secretion (data not shown) was lower in the co-culture of D60-Tx than in that of B6, in response to BALB/c stimulation. The reduced amount of IL-2 in the former co-culture could be due to either a declined in the number of cells able to secrete IL-2, or to impaired function of IL-2 secretion from each cell. To test these two possibilities, ELISPOT was performed to test IL-2 secretion at a single cell level. It showed a significant decrease in the number of spots in the former co-culture, suggesting that the low production of IL-2 was in part through the reduced numbers of cells able to secrete proliferative cytokine IL-2 (Fig. 4.2F, p=0.05, n=5)  67  B.  B  6+  6+ B  5  5  0  0  B  6+ B AL D 60 +B IL AL -2 +B 6+ IL B -2 AL +D 60 +B AL  10  B 6+ B AL D 60 +B D ep AL _D 60 +B AL  10  200  *p=0.05  150 100 50 0  AL  15 No treatment  *p=0.03  AL  F. Spots per 106 cells  15 *p=0.003  +IL-2  D 60 +B  B 20  60 +B  L  0.0  D.  20  0.2  B A  0  0.4  D  0  0.6  AL  5  0.8  6+ B  5  AL D 60 +B D AL 60 +B 6+ B AL  10  cpm  103  *p=0.003  15  10  1.0  *p=0.04  IL-2 mRNA  p=0.14  C.  E.  20  B 6+ B AL D 60 +B AL B 6+ C B A D 60 +C B A  cpm  103  15 *p=0.003  *p=0.03  B  A. 20  Figure 4.2 Mixed lymphocyte reaction for lymphocytes from long-term surviving recipients with Ad-B7-H4–transduced islets showed hyporesponsiveness (A). Splenic lymphocytes (SLCs) harvested from spleens of naïve C57BL/6 (B6) mice or mice transplanted with Ad-B7-H4–treated BALB/c islets at 60 days (D60) were co-cultured with γirradiated (at 3000 rad) stimulators from either donor-specific BALB/c (BAL) (H-2d) or thirdparty CBA/J at a responder/stimulator (R/S) ratio of 1:3 for 3 days in a MLR assay. Cell proliferation was calculated as mean counts per minute (cpm ± SE) of triplicate culture wells. Response of SLCs from D60 recipients was significantly lower than those from naïve B6 mice (p=0.003). Proliferation of D60 cells to donor-specific and third-party controls were significantly different (p=0.03). There was no significant difference between naïve B6 mice and D60_Tx mice in response to CBA/J stimulation (p=0.14). (B). SLCs from naïve B6 mice showed hyporesponsiveness in the presence of D60 SLCs. A 1:1 mixture of naïve B6 and D60 responder cells was co-cultured with BAL at R/S ratio 1:3. Proliferation in this MLR assay was significantly lower than that of the control B6 without responder from D60 mice (p=0.04). (C). The presence of CD4+CD25+ is required for the anergic state of SLCs from D60 mice. CD4+CD25+ were depleted from D60 SLCs mice were co-cultured with BAL SLCs. The ability of D60 cells to respond to antigen stimulation was significantly increased after CD25 depletion (P=0.03). (D). IL-2 addition could reverse the suppressive state in D60 cells. Proliferation was significantly higher after exogenous IL-2 (100 U/mL) was added to the MLR culture (p=0.001). (E). Relative expression level of IL-2 in the co-culture of B6 or D60_Tx plus BAL was measured by real-time PCR. (F). Frequency of IL-2 secretion in the MLR culture was detected by ELISPOT. 106 of purified CD3+ T cells from B6 or D60_Tx were co-cultured with irradiated BALB/c splenocytes. The numbers of spot in the co-culture of D60+BAL were significantly lower than that of B6+BAL co-culture (p=0.05). Results were generated from 3 independent experiments. Each group consisted of 5 to 8 recipients.  68  4.2.4 B7-H4 inhibits expression of pro-inflammatory cytokines in the grafts It has been shown previously that polarization of the Th1 response results in graft failure, whereas a Th2 response is associated with graft acceptance. In order to test whether or not the protective role of B7-H4 in alloimmune response is due to the shift of Th1/Th2 balance, IL-2 (for monitoring T cell proliferation), IFN-γ (Th1), IL-4 (Th2), and granzyme B (Gzmb, for monitoring destructive CTL response) was quantitiated by real-time PCR 10 days after first transplantation. Transcription of IL-2 was lower in Ad-B7-H4-treated mice compared with AdLaz-treated mice, but this was not significant (Fig. 4.3). In contrast, both IFN-γ and Gzmb were significantly decreased in Ad-B7-H4-treated mice (Fig. 4.3, p=0.007, p=0.04 for IFN-γ and Gzmb, respectively). Expression of IL-4 was similar in the two groups, suggesting that B7-H4 preferentially inhibits Th1 and CTL responses locally.  Relative expression of RNA  12  Control ns  B7-H4  *p=0.007  9  *p=0.04 6  ns 3  0 IL-2  IFNg  IL-4  Gzmb  Figure 4.3 Cytokine RNA expression in the grafts Relative expression of mRNA of IL-2, IFN-γ, IL-4, and Granzyme B (Gzmb) was measured by real-time PCR at 10 day post first transplant in Ad-Lacz (shown in square, labeled as “control”) or Ad-B7-H4 (shown in triangle, labeled as “B7-H4”) recipients. The syngeneic transplantation of B6 to B6 were used for comparison and considered as expression level of 1. Significant reduction of IFN-γ and Gzmb was detected (p=0.007 and p=0.04, respectively, n=5).  69  4.2.5 Second-set islet allografts survive after removal of long-term surviving Ad-B7-H4– transduced islet grafts  Fig. 4.4A demonstrates the results of random blood glucose levels following transplantation of B7-H4–transduced islets, subsequent graft nephrectomy at day 60, then secondary transplantation from the same donor haplotype (BALB/c) without any further immunosuppressive treatment. Blood glucose rose promptly in all recipients after graft nephrectomy, thus confirming that the grafts were functioning. A second non-transduced BALB/c graft restored blood glucose control in all five recipients. Fig. 4.4B compares the survival of secondary transplants implanted with BALB/c donor specific islets versus third-party CBA/J islets. Three of five BALB/c donor specific allografts survived indefinitely (>100 days), and the other two failed with delayed kinetics on days 38 and 58, respectively. Overall survival of grafts in this group of recipients compared favorably with third-party CBA/J islet graft survival which was 21±6 days (group 1 versus 2, p=0.001) .  70  B. 100  700 600 500 400 300 200 100 -3  17  60 100 120 140 160 Days Post-Transplant  Percent survival  Blood Glucose (mg/dL)  A.  75 50  B7-H4 Third Party  25 0  Tx  T2x  0  50  100  150  200  250  Days Post-Transplant  Nephrectomy  Figure 4.4 B7-H4 induces unresponsiveness to second-set donor islet allografts, but not third party islets (A). Blood glucose levels in the long-term surviving recipients with Ad-B7-H4–transduced BALB/c islets. After removal of the primary islet-engrafted left kidney, D60_Tx mice became hyperglycemic, then were re-transplanted with a second donor-specific BALB/c islet allograft (n=5) without any immune treatment. (B). Comparison of survival of islet grafts after removal of the primary islet-engrafted left kidney, D60_Tx mice are re-transplantation with a second donor-specific BALB/c islet allograft (black line, n=5) or with third-party control CBA/J islets (grey line, n=5) without any further treatment. Donor-specific, second-transplanted recipients survived significantly longer than that of third-party control (p=0.0017, n=5, mean 79±46 vs. 21±6 days, respectively).  71  4.2.6 Inflammation, beta cell function, and immune infiltrates are distinct in the secondary transplantation recipients  In order to analyze the immune responses of accepted or rejected recipients in second set transplantation, grafts were harvested from secondary transplanted mice and stained for hematoxylin and eosin (H&E), insulin, CD45, CD3, and Foxp3. Massive infiltrates of CD45 leukocytes and residual insulin positive cells were observed in two failed grafts that rejected at day 38 and 58 after secondary transplant. In contrast, three long term survivors showed well preserved islets with a minimal amount of infiltration (Fig. 4.5A, B). Further identification of the infiltration in failed grafts revealed that majority are CD3+ (33029 per field) and very few Foxp3+ T cells (178 per field) (Fig. 4.5C, D). Moreover, the percentage of Foxp3 versus CD3 positive cells in the surviving allografts exceeded that in the failed allografts (Fig. 4.5E, 39.8% vs. 5.1%), suggesting that alloreactive T cells were responsible for the failure of two allografts upon secondary transplantation. Fig. 4.6 shows the percentage of Foxp3+, Treg, and Teff cells in the renal lymph node and spleen of failed or surviving recipients after secondary transplant. No significant differences were observed among the different subsets, suggesting that other factors (for example, cytokine enviroment or cell activation state) which we did not examine might play a role in tolerance maintenance.  72  A.  B. % Islets  100  0-Normal islets 1-Less than 25% isletitis 2-25% to 50% isletitis 3-Invasive isletitis 4-Retracted islets  80 60 40 20 0  Failed  Surviving  73  C.  E.  D.  Positive  400  Failed  Failed  Surviving  300  Surviving  200 100 0  0 +  CD3  +  20  40 +  FoxP3  60  80  100  +  FoxP3 : CD3 ratio  Figure 4.5 Histology of rejected and surviving allografts after secondary transplant shows distinct patterns (A). Rejected and surviving grafts were stained with H&E, anti-insulin (red), and CD45 (green). Magnification is 200. (B). Grafts from two failed and three surviving recipients were harvested and stained with H&E. The slides were scored for isletitis according to Yoon’s methods. (C). Rejected and surviving grafts were stained with CD3 and Foxp3 (brown). Magnification is 200. (D). CD3+ and Foxp3+ cells were quantified and shown as numbers per field. (E). Ratio of Foxp3+ cells in the CD3+ population. Foxp3+ and CD3+ cells were randomly selected and blindly counted. Data represent two rejected and three long term surviving recipients after secondary transplantation with donor specific BALB/c islets. 74  A. Spleen  LN  SSC  26 21 44  19 66 08  Gate 1  B.  G a te 1  13 10 72  65 53 6  0  65536  131072  196608  26214  30  0 0  65 53 6  13 10 7 2  1 96 60 8  2 62 14  Gate 2  10  10  10  10 -10  2  0  2  3  -10 10 10  4  10  G ate 2  5  4  3  2 2  2  5  10  Percentage  CD25  FSC  0  -10 10 10  10  2  3  10  10  4  10  5  Foxp3  CD4 10  10  10  20  Failed Survived  10  5  4  3  0 Foxp3  1  10 2 - 10 2  -10 10  2  3  10  10  4  2  5  10  - 10 10  2  3  10  10  4  10  CD25 64  49  48  33  32  Count  65  Foxp3  Treg  Treg  Teff  Teff  5  Spleen  LN  Spleen  LN  Spleen  LN  16  16  0  0 2  0  2  -10 10 10  10  3  4  10  10  5  2  0  -10 10 10  2  10  3  10  4  10  5  Foxp3  Figure 4.6 FACS analysis of Foxp3+, Tregs, and Teffs in the spleens and renal lymph nodes in 2 failed and 3 surviving recipients after secondary transplantation (A). Representative plots of FSC versus SSC, CD4+ versus CD25+, CD25+ versus Foxp3+, and histogram of Foxp3+ T cells. (B). Percentage of Foxp3+ Tregs, and Teffs in the spleen and lymph node after secondary transplant. There are no significant differences between the two groups. 100,000 events were collected.  75  4.3 SUMMARY OF FINDINGS AND DISCUSSION  B7-H4 has been recognized as a potent negative co-stimulatory regulator of T-cell responses and function [114-116]. In chapter 3, we showed that local expression of B7-H4 on donor islets improved islet transplantation outcomes [177]. Reduced numbers of alloreactive CD8+ cells were detected in the allografts transduced with Ad-B7-H4 compared with untreated allografts at day 10 post-transplant [177]. This observation suggests that long-term surviving mice could be due to reduced immune responses controlled by local B7-H4 expression. Therefore, B7-H4-treated recipients are unable to destroy a well-established allograft that recruits only a small number of effector cells to the graft site by B7-H4–mediated negative cosignalling. Furthermore, T cells isolated from recipient mice with long-term surviving BALB/c allografts responded to donor-specific alloantigen (BALB/c) significantly less than to third-party alloantigen (CBA/J) in MLR co-culture. This result was further confirmed in vivo by the enhanced survival of BALB/c over CBA/J secondary allografts transplanted into mice with longterm surviving BALB/c primary allografts. Three out of five recipients had islet survival times of >100 days upon re-transplantation with the first-donor (BALB/c) islets without any further immune treatment. More importantly, these mice rejected islets from CBA/J mice shortly after second transplant, excluding the possibility of non-specific immune suppression or immune ignorance. Our data show that acceptance of a second graft without ongoing immune treatment is not a consequence of reduced immunogenicity against donor antigen. Instead, B7-H4 induces donor– specific tolerance in our model. This result agrees with other studies which confirm that donor– specific tolerance, rather than ignorance, is achievable by co-signaling blockade [51, 140, 174, 98, 175].  76  Tregs play important roles in the development of type 1 diabetes and mediation of allograft tolerance [178-183]. Studies have shown that B7-H4 plays an important role in mediating the immunosuppressive effects of Tregs and B7-H4+ tumor macrophages suppress tumor-associated antigen-specific T-cell immunity in cancer [126, 127]. The role of CD4+CD25+ T cells as suppressive cells is well established in allograft protection and tolerance induction. Some argue that the presence of CD4+CD25+ T cells is not a unique feature of allograft acceptance [178]. In our model, we established that the presence of CD4+CD25+ T cells is required for hyporesponsiveness to alloantigen stimulation in recipients. This finding is consistent with the notion that the level of CD4+CD25+Foxp3+ Tregs is directly associated with allograft acceptance [184]. Tregs can be divided into two distinct groups of nTregs and iTregs based on their origins, specificity, and mechanism of action [185]. CD4+CD25+ nTregs are generated in thymus during normal T-cell development and enter the periphery as functionally mature T cells. nTregs suppress effector T-cell proliferation in vitro through a cell contact–dependent manner [185]. nTregs depend on IL-2 for survival in combination with TCR engagement [186, 187]. Consistent with this, the present study has established that the hyporesponsiveness- mediated by CD4+CD25+ T cells in the long-term surviving mice is IL-2–dependent. The contribution of nTregs in inducing tolerance is best illustrated by the spontaneous development of autoimmune disease in normal mice when CD4+CD25+ T cells are depleted [137]. nTregs were first identified by their ability to suppress murine allogeneic bone marrow transplantation. Co-transfer of purified CD4+CD25+ Tregs with naïve T cells significantly delayed graft versus host disease. Depletion of CD4+CD25+ T cells completely abrogated tolerance generated by CD40/CD40L or CTLA-4 in vitro and in vivo [142]. Similar protection was reported in solid transplantation [140]. The data presented in our current study support the idea that tolerance induction by B7-H4 is associated with Treg-  77  mediated suppression. Despite the ability of nTreg to maintain tolerance, they cannot fully protect grafts from destruction [141, 182, 186]. A second group of Tregs called inducible Tregs (iTregs) can be induced in the periphery and include both Foxp3+ Th3 and Tr1 cells. Th3 cells have been shown to elicit immune tolerance through the secretion of TGF-β1 [143]. Tr1 cells, on the other hand, act through and secrete large amounts of IL-10 [183]. Tr1 cells tend to migrate toward sites of inflammation, while nTregs are predominately found in lymphoid organs [137]. The relative role of each of the different types of Tregs in allograft survival is not well characterized and may depend on many factors, including the type of and site of the allograft. Some studies have demonstrated that high levels of Tregs in the graft and/or periphery are associated with graft survival [140, 174, 137, 184]. In this study, Foxp3+ T cells were upregulated in the local allografts and spleens of the long term surviving receipients treated with Ad-B7-H4 compared with syngeneic islet recipient mice (Figs. 3.5, 4.1, and 4.2), indicating Tregs involvement in B7-H4 associated prolongation of allograft. We have detected a significant increase in IL-10 expression at the same time point when Tregs were examined, implying that IL-10–induced iTregs might play a role in tolerance induction. Furthermore, although TGF-β plays a critical role in the induction of Foxp3+ Treg cells, and exerts suppressive actions on many immune cell types, its importance is not observed in all systems [178, 179]. Consistent with other studies showing that production of TGF-β by Tregs is not essential for suppression in vivo, our data showed that TGF-β mRNA expression did not change significantly in the local graft or the spleen of the recipients with long-term surviving allografts, compared with mice receiving syngeneic grafts, indicating that it might play a minimal role in B7-H4–mediated tolerance induction [180, 181]. The contribution of TGF-β to the induction of iTregs from several studies remains unclear, but it may relate to its ability to induce IL-10 expression [182, 183]. Kryczek demonstrated that one mechanism by which Treg 78  cells activate APC is through IL-10–induced B7-H4 expression on these cells [126]. The importance of IL-10 is also indicated by the fact that in the tumor environment, IL-10 is responsible for upregulation of B7-H4 expression on APCs, resulting in B7-H4–dependent suppressive APCs in the tumor microenvironment [127, 189]. Our current data also suggest a role for IL-10–dependent mechanisms in B7-H4–mediated inhibition of allotransplant rejection in our model. Several mechanisms have been proposed for tolerance induction. In this study, the data indicate that both nTregs and iTreg are involved in tolerance induction mediated by B7-H4. However, the action of Tregs is reversible. B7-H4 is able to induce Tregs in the local graft site and periphery. However, induction of Tregs seems to be independent of persistent B7-H4 expression. We showed previously that expression of B7-H4 remained only 5% at day 60 post transplant due to the replication-defective adenoviral vector used in our model. The reason, in part, may be due to the Treg-mediated hyporesponsiveness in the spleen since hyporesponsiveness is restored upon depletion of CD4+CD25+. In the B7-H4 treated mice, three second set transplant recipients without any immune treatment survived more than 100 days, but the other two failed at 38 and 58 days, respectively. The survival time of two failed grafts was longer compared with those transplanted with donor non-specific third party CBA/J islets (day 38 and 58 on the fromer vs. 21±4 days in the latter). The failure is associated with massive alloreactive CD3+ T cell infiltrates into the graft. A similar expression of Tregs in the periphery of two failed and three surviving recipients after second-set transplants suggests that factors other than Tregs may contribute to tolerance maintenance, such as inefficient inhibition of memory T cells generated in second-set transplants. Because of the small numbers of second-set transplants, we cannot conclude on the relative contribution of Tregs and Teffs in determining the fate of secondary transplant. The importance of Tregs and Teffs in generating and maintaining transplantation tolerance has been described in many other studies [190-192]. Our 79  data suggest that Tregs are involved in tolerance induction but play a minimal role in tolerance maintenance. An important finding in this study is that protection of islet allograft by B7-H4 is mediated through inhibition of the Th1 response. Loss of function of B7-H4 in vivo results in a mounted Th1 response (128), suggesting that B7-H4 preferentially inhibits Th1 polarization. This may be due to a high level of expression of B7-H4 receptors on the Th1 subset (123) . We did not observe an influence on the Th2 response after B7-H4 treatment, as was shown in Yuan’s paper (220). The reason for this may be due to the different system we used. In their study, diabetic C57BL/6 mice were injected intraperitoneally with B7-H4-transfected NIT cells. In our study, diabetic C57BL/6 mice were transplanted with Ad-B7-H4-transduced BALB/c islets under the kidney capsule. In fact, we observed local effects in the early stage, whereas Yuan et al. detected systemic changes in general. Although different systems were used in our study and theirs, both groups observed a systemic effect on Treg involvement. We did detect a high expression of Foxp3 in local draining lymph node. This result may reflect a robust alloreactive response in the renal lymph node, when immature APCs encounter alloantigen from the graft and migrate to the lymph node and proliferate there. Alternatively, B7-H4 limited priming T cells inefficiently at early time point, as transcription of IL-2 in the allograft was lower, but not significantly reduced. Consistent with this notion, we also stained a massive infiltrate in the early stage (177), suggesting inefficient suppression of alloreactive T cell priming by B7-H4. At an early stage, B7-H4 was able to control local inflammatory cytokine production, such as IFN-γ and granzyme B. At a later stage, B7-H4-mediated induction of Tregs in the periphery facilitates a reduced response to alloantigen stimulation.  80  CHAPTER 5 B7-H4 SIGNAL PATHWAY  81  5.1 BACKGROUND B7-H4 is an inhibitory member of the B7 family of co-regulatory molecules which is expressed on antigen-presenting cells as well as on non-immune cells and which interacts with an as yet unidentified receptor(s) on activated T cells to inhibit T-cell proliferation and IL-2 production [114-116, 166]. The importance of B7-H4 in regulating immune responses in vivo has been shown through many studies. Administration of a B7-H4 mAb that blocks B7-H4 action, in an experimental autoimmune encephalomyelitis (EAE) mouse model promoted T-cell responses and exacerbated disease [115]. Adenoviral-mediated transduction of islets with B7-H4, on the other hand, protected them from rejection when transplanted into allogeneic mice [177]. Studies into the mechanisms by which B7-H4 engagement prevents T-cell proliferation have shown that cells are arrested at the G0/G1 phase of the cell cycle [114]. Addition of exogenous IL-2 can partially reverse B7-H4–induced suppression of T-cell proliferation, suggesting that inhibition of IL-2 production is an important component of B7-H4 action on T cells. Ligation of the T-cell receptor (TCR) in conjunction with co-stimulatory receptors initiates a cascade of signal transduction events that result in IL-2 production and T-cell clonal expansion and differentiation [193]. The tyrosine kinase LCK is the first signaling molecule to be activated downstream of the TCR [194]. Activated LCK phosphorylates ITAM motifs in the cytoplasmic domain of the TCR gamma, epsilon and zeta chains [152]. ZAP70, another tyrosine kinase, is recruited to the phosphorylated zeta chain and is activated by phosphorylation of LCK. ZAP70 then phosphorylates a number of downstream signaling molecules [195], activating a signaling cascade which includes ERK and JNK kinases, which leads to stimulation of IL-2 transcription [196]. However, these signaling pathways downstream of the TCR work in conjunction with signaling pathways downstream of the co-stimulatory receptors. One of the key co-stimulatory receptors is CD28, a positive signaling member of the B7 co-regulatory family. CD28 interacts with its cognate ligands (B7.1/B7.2) on antigen-presenting cells and leads to 82  activation of phosphatidylinositol 3-kinase (PI3K). PI3K catalyzes the production of phosphoinositol-3,4,5-triphosphate (PIP3) which functions to activate PH domain–containing proteins such as the protein kinase AKT. AKT is a master regulator involved in protein synthesis, anti-apoptosis, cell survival/proliferation, and glucose metabolism. Activation of PI3K/AKT pathway is a fundamental requirement for cell-cycle progression and T-cell proliferation [193, 194]. TCR activation in the absence of CD28 stimulation results in impaired or altered T-cell responses ranging from decreased proliferation/IL-2 production to anergy (non-responsiveness to antigen) or apoptosis. The requirement for co-stimulatory receptor signaling has been used to regulate T-cell responses. CTLA-4 is another member of the B7 family, but it functions to inhibit T-cell activation. CTLA-4 is a surface protein that can be expressed on activated T cells and competes with CD28 for binding to B7.1/B7.2. CTLA-4 binds to B7.1/B7.2 and does not result in PI3K activation. Soluble versions of CTLA-4 have been used clinically to interfere with T-cell responses in autoimmunity and organ transplantation. Studies of the signaling pathways which are altered in T cells exposed to soluble CTLA-4 have confirmed interference with PI3Kdependent events and also revealed an inhibitory effect on ERK and JNK activation [80]. The signaling pathways by which B7-H4 alters T-cell responses, have not been well characterized. Based on our knowledge of how the other inhibitory B7 family members interfere with T-cell activation, we anticipate that B7-H4–mediated signaling may inhibit MAPK and AKT kinases. We examine this possibility and whether B7-H4 signaling also alters LCK or ZAP70 activation. Characterization of the molecular mechanism by which B7-H4 functions will provide important information guiding rational use of B7-H4 therapy for cancer, autoimmune diseases and transplantation.  83  5.2 RESULTS 5.2.1 B7-H4 engagement inhibits T cell We and others have shown previously that administration of a soluble immunoglobulin fusion protein of B7-H4, B7-H4.Ig, inhibited CD4+ and CD8+ T-cell proliferation upon TCR/CD28 ligation [114-116, 166]. However a question that has not been addressed is whether the inhibitory effect of B7-H4.Ig administration affects naïve and pre-activated T cells equally. We have thus compared the inhibitory effect of B7-H4.Ig on naïve splenic CD3+ T cells versus the same cells which have been stimulated with CD3 for 16 h and rested for 30 h prior to use. Naïve or pre-activated cells were cultured with a range of concentrations of anti-CD3 agonistic antibody, in the presence of a constant concentration of an agonistic CD28 antibody, and with B7-H4.Ig or a control human IgG1 Fc.Ig protein (Fc.Ig). As shown in Fig. 5.1A and 5.1C, although the calculated half maximal effective concentration (EC50) for CD3 was similar in both the naïve and pre-activated cells ( 0.15 g/mL), maximum thymidine incorporation was different, plateauing at 40  103 cpm for pre-activated cells vs. 27  103 cpm for the naïve cells. Thus the pre-activated cells proliferate much more vigorously than the naïve cells. However, the ability of B7-H4.Ig to inhibit thymidine incorporation appeared to be similar in both naïve and pre-activated cells. The addition of B7-H4.Ig to the culture reduced thymidine incorporation by almost 50% in the presence of 0.3 g/mL anti-CD3 in both cell types. We also tested the sensitivity of naïve vs. pre-activated cells to B7-H4.Ig. In Fig. 5.1B and 1D, the indicated concentration of B7-H4.Ig was added to cultures in the presence of 0.3 g/mL anti-CD3 and 1 g/mL anti-CD28. As seen in Fig. 5.1B and 5.1D, the naïve cells were more sensitive to B7-H4.Ig inhibition. At 10 g/mL B7-H4.Ig, thymidine incorporation was inhibited by 70% in naïve cells (p=0.005). In contrast, the same concentration of B7-H4.Ig  84  inhibited thymidine incorporation in pre-activated cells by only 40% (p=0.012). For the remainder of the studies in this paper, we chose to use pre-activated cells because these cells represent a more stringent system (i.e. cells are more resistant to B7-H4.Ig) with which to examine the mechanism by which B7-H4.Ig interferes with T-cell activation. In order to determine the sensitivity of inhibition by B7-H4 on different T-cell subsets, CD3+, CD4+, or CD8+ T cells were purified by magnetic beads. As shown in Fig. 5.1E, B7-H4.Ig protein inhibited thymidine incorporation in each subset to a similar degree. The proliferation of total pre-activated CD3+, CD4+, and CD8+ was reduced by 50.3%, 57.9%, and 58.5%, respectively. There were no significant differences in the ability of B7-H4 to inhibit three T-cell subsets (p=0.6331).  85  anti-CD3/CD28  CPM  A. 35000 30000 25000 20000 15000 10000 5000 0  B.  anti-CD3/CD28+B7-H4  B7-H4.Ig (2)  p=0.003*  B7-H4.Ig (5)  Fc.Ig  p=0.001*  no Fc.Ig 0.1  0.3  0.9  1.2  0  anti-CD3 (μg/ml)  10000  15000 20000  D.  50000  anti-CD3/CD28+B7-H4  40000  p=0.003*  B7-H4 (5) B7-H4 (10)  p=0.005*  30000  5000  CPM  anti-CD3/CD28  C.  CPM  *p=0. 005  B7-H4.Ig (10)  p=0.008*  0  *p=0.01 *p=0.008  B7-H4 (30)  20000 10000  * p=0.01  Fc.Ig  p=0.001*  no Fc.Ig  0 0  0.1  0.3  0.9  1.2  0  anti-CD3 (μg/ml)  E.  CPM  20000  30000  40000  CPM Control B7-H4  50000 40000  10000  *p=0.008 *p=0.005 *p=0.002  30000 20000 10000 0 CD3  CD4  CD8  Figure 5.1 B7-H4 inhibits T-cell proliferation Naïve (A and B) and pre-activated (C, D, and E) CD3+ T cells were stimulated with various concentrations of plate-bound anti-CD3 and soluble anti-CD28 (1 g/mL) for 72 h. 18 h before harvest, cultures were pulsed with 1 Ci of [3H]-thymidine. B7-H4.Ig or Fc.Ig was added at indicated concentrations (B and D). 10 g/ml and 30 g/ml of B7-H4.Ig or Fc.Ig was added for naïve and pre-activated T cells, respectively. Preactivated CD3+, CD4+, or CD8+ T-cell subsets were stimulated with 0.3 g/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 g/ml). Triplicate wells were harvested and counted. Data represent at least 3 independent experiments from 6 mice in each group.  86  We also examined the expression of the B7-H4 receptor on pre-activated CD4+ vs. CD8+ T-cell subsets (Fig. 5.2). The B7-H4 receptor has not yet been identified, but it can be detected through its binding of B7-H4.g [116]. Briefly, cells were incubated with 10 µg/mL of B7-H4.Ig or control Fc.Ig for 30 min, prior to washing and detection of bound B7-H4.Ig using a PE conjugated goat anti-human Ig Ab. B7-H4 receptor was detectable on pre-activated CD3+, CD4+, or CD8+ T cells starting at 3 days and peaking at 4 to 5 days after stimulation (Fig. 5.2A). The mean fluorescence intensity (MFI) of B7-H4.Ig staining was similar in all three different subsets (Fig. 5.2B, p=0.55), indicating similar expression of the B7-H4 receptor on pre-activated CD3+, CD4+, or CD8+ T cells. The inability to detect B7-H4.Ig binding prior to 3 days likely reflects the limitation of sensitivity of staining method dependent on B7-H4.Ig protein binding followed by detection of the bound B7-H4.Ig using an anti-Ig Ab. Our proliferation data above indicates that cells are inhibited by B7-H4.Ig even at day 0 (naïve cells) after CD3/CD28 activation, at which  B7-H4 receptor (MFI)  time B7-H4.Ig protein binding can’t be detected using current methods. B. A. 50 40 30 20 10 0  CD3  CD4  CD8  Figure 5.2 Expression of B7-H4 receptor on activated T-cell subsets Naïve CD3+, CD4+, or CD8+ T-cell subsets were stimulated with 0.3 g/ml of plate-bound antiCD3 and soluble anti-CD28 (1 g/ml) for 16 h and then rested for 30 h prior to re-activation. Activated CD3+, CD4+, or CD8+ T-cell subsets were stimulated with plate-bound anti-CD3 (5 g/ml) and soluble anti-CD28 (1 g/ml) for 4 d. Expression of putative B7-H4 receptor was detected as follows. Cells were stained with a control human IgG1 (filled) or with a B7H4.hIgG1 (open), followed by goat anti-human IgG-PE. (A). Representative histograms of expression of B7-H4 receptor on CD3+, CD4+, or CD8+ T-cell subsets. (B). Mean fluorescence intensity (MFI) of B7-H4 receptor was plotted. There is no significant difference in the expression of B7-H4 receptor among three different T-cell subsets. Data represent 3 independent experiments from 5 mice in each group.  87  We also compared the responsiveness of CD3+, CD4+, or CD8+ to B7-H4.Ig inhibition using readout that is more immediate than proliferation assays. CD3+, CD4+, or CD8+ were stimulated with anti-CD3 for 16 h and rested for 30 h prior to use. Cells were then stimulated with 5 g/mL anti-CD3 and 2 g/mL anti-CD28 in the presence of 30 g/mL Fc.Ig or B7-H4.Ig for the indicated length of time. Cell lysates were prepared and subjected to Western blot analysis to determine the levels of CD3/CD28 induced AKT phosphorylation on serine 473 using an AKT-phospho-AKT-ser-473 Ab. AKT is a serine/threonine protein kinase that is essential for multiple cellular processes including the progression of cell cycle and T-cell proliferation and phosphorylation on Ser-473 is a marker of its activation state (201). As shown in Fig (Fig. 5.3), anti-CD3/CD28 stimulation could induce AKT-Ser-473 phosphorylation within 10 min. In the presence of B7-H4.Ig, the level of p-AKT-Ser-473 was reduced by ~70% compared to the Fc.Ig control in all three different T-cell subsets (Fig. 5.3B). Our data show that CD3+, CD4+, or CD8+ T cells all express similar levels of the B7H4.Ig receptor (Fig. 5.2) and respond to similarly with respect to inhibition of AKT phosphorylation (Fig. 5.3). We therefore used total CD3+ T cells in the following experiments.  88  Figure 5.3 B7-H4 inhibits phosphorylation of AKT on different T cell subsets similarly Naïve CD3+, CD4+, or CD8+ T-cell subsets were stimulated with 0.3 g/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 g/ml) for 16 h and then rested for 30 h prior to reactivation. Activated CD3+, CD4+, or CD8+ T-cell subsets were stimulated with plate-bound anti-CD3 (5 g/ml) and soluble anti-CD28 (1 g/ml) for 10 min. protein extracts from CD3+, CD4+, or CD8+ T-cell subsets were detected by phosphorylated AKT Ser473 (A) and quantitated by using Bio-Rad Quantity One program. The y-axis was normalized for the loading control. B7-H4 treatment significantly inhibited AKT phosphorylation at 10 min to a similar degree on different T-cell subsets (B). Data represent 3 independent experiments from 9 mice in each group.  89  5.2.2 B7-H4 engagement inhibits IL-2 production and CD25 expression  We next determined the effect of B7-H4.Ig treatment on anti-CD3/CD28–stimulated Tcell production of IL-2. Pre-activated T cells were treated with 5 g/mL anti-CD3 and 2 g/mL anti-CD28 in the presence of 30 g/mL Fc.Ig or B7-H4.Ig for the indicated length of time (Fig. 5.4A). RNAs were isolated and the level of IL-2 mRNA was quantitated by Q-PCR. In the antiCD3/CD28 + Fc.Ig cells, IL-2 mRNA levels peaked at 4 h and declined over the course of 24 h to basal levels at 48 h (Fig. 5.4A). The presence of B7-H4.Ig dramatically reduced the amount IL-2 mRNA detected at 4 h (p=0.00003), but interestingly, IL-2 mRNA levels rise at 24 h before declining to basal levels at 48 h. We also collected the cell culture supernatant and measured IL2 protein levels by ELISA. As shown in Fig. 5.4B, at 24 h, B7-H4.Ig also significantly decreased IL-2 protein detected in the culture supernatant (p=0.002). The difference in IL-2 protein levels between B7-H4.Ig– and Fc.Ig–treated cells was not as great as the difference in IL-2 mRNA. There was also no difference in IL-2 protein levels between treated and untreated cells in 48-h supernatants. Part of the reason for the discrepancy between the protein and mRNA data may be the fact that the IL-2 is an autocrine growth factor for T cells and is thus depleted from the culture through binding to T cells. The anti-CD3/CD28 + Fc.Ig cultures had more cells than the B7-H4.Ig–treated cultures. Thus the amount of IL-2 detected in the supernatants of the antiCD3/CD28 + Fc.Ig cells was an underestimate of the total IL-2 that was made. Activated T cells up-regulate the IL-2 receptor alpha chain CD25 needed for high affinity binding of IL-2. Therefore, we investigated whether the addition of B7-H4.Ig would interfere with expression of CD25. Naïve cells were stimulated and rested as described above. CD25 increased from 6% to 40% within 16 h, but they decreased back to 15% during the 30-h rest period. Cells were then treated with anti-CD3/CD28 with Fc.Ig or B7-H4.Ig as before, and the percent of cells expressing CD25 were determined by flow cytometry. As shown in Fig. 5.4C 90  and 5.4D, at 4 h after stimulation, the percent of cells which have upregulated was 37% in Fc.Ig– treated cells vs. 26% in B7-H4.Ig–treated cells (p=0.003). Thus B7-H4.Ig also inhibited expression of IL-2 receptor alpha chain CD25.  91  A.  B.  anti-CD3/CD28 anti-CD3/CD28+B7-H4  8  300 250  IL-2 (ng/ml)  IL-2 mRNA  350  200 150 100 50 0 0  0.5  1  2  4  24  Control B7-H4  6 4 2 0  48  4  Hours after stimulation  24  48  Hours after stimulation  C. 1  2  10 2 -10  3  10  2  10  2  -10 3  10  4  10  APC-A  5  2  10  5  PerCP-Cy5-5-A  10  4  10  3  10  2  10  2  -10  2  -10 10  3  10  4  10  APC-A  3  10  4  10  APC-A  5  10  4  10  3  10  2  10  5  -10  10  2 2  -10 10  3  10  4  10  APC-A  4  10  3  10  2  10 2 -10  5  2  2 2  -10 10  5  10  2  10  5  10  2  -10 10  PerCP-Cy5-5-A  2 2  -1010  PerCP-Cy5-5-A  4  10  PerCP-Cy5-5-A  3  10  5  10  4  3  10  4  10  APC-A  5 4  10  3  10  2  10 2 -10 2  2  3  10  4  10  APC-A  4  10  Fc.Ig  3  10  2  10 2 -10  5  10  -10 10  5  10  2 2  10  -10 10  PerCP-Cy5-5-A  4  10  2 PerCP-Cy5-5-A  5  10  PerCP-Cy5-5-A  PerCP-Cy5-5-A  0.5  CD3  CD3  Time (h)  5  10  3  10  4  10  APC-A  5  10  5  10  4  10  B7-H4  3  10  2  10 2 -10 2  2  -10 10  3  10  4  10  APC-A  5  10  CD25 D. CD25 expression (%)  50  anti-CD3/CD28 anti-CD3/CD28+B7-H4  40 30 20 10 0.5  1  2  4  Hours after stimulation  Figure 5.4 B7-H4 inhibits IL-2 and IL-2 receptor  chain (CD25) expression Pre-activated CD3+ T cells were stimulated with anti-CD3 (5 g/mL) and anti-CD28 (2 g/mL) in the presence of B7-H4.Ig or Fc.Ig. Cells were collected for IL-2 mRNA detection (A) or stained with anti-CD25 at indicated time (C). The expression of CD25 was quantitated and plotted in C and D. Living cells were analyzed by flow cytometry. Culture supernatant was collected and tested by ELISA (B).  92  5.2.3 B7-H4 engagement inhibits expression of CD69  CD69 is another cell-surface molecule, the expression of which is transiently induced on T cells early during the activation process [197]. We also monitored CD69 expression in the cells used for the CD25 expression studies above. In the pre-activation period, the percent of cells expressing CD69 increased from 7% to 65% at 16 h, decreasing almost to basal levels after a 30h rest. Upon re-stimulation with anti-CD3/CD28 (Fig. 5.5A, 5.5B), the percent of cells expressing CD69 increased to 71% in anti-CD3/CD28 + Fc.Ig cells compared to 39% in antiCD3/CD28 + B7-H4.Ig at 4 h (p=0.0004).  93  A. Time (h)  2  4 43  32  32  22  22  22  11  11  11  0  0  0  2  2  3  4  10  10  PE-A  2  5  2  -10 10  10  Count  43  32  Count  43  32  Count  43  -10 10  3  4  10  10  PE-A  5  22 11 0  2 1  10  -1010  3  4  10  10  PE-A  5  2 1  10  43  43  43  32  32  32  32  Count  Count  22 11  11  11  0  0  0  2 1  -1010  3  10  4  10  PE-A  5  10  2  2  -10 10  3  10  4  10  PE-A  5  10  10  2 1  10  4  10  4  10  10  PE-A  5  22  22  22  3  -1010  Count  43  Count  B7-H4  1  Count  Fc.Ig  0.5  11 0 2 1  -1010  3  10  4  10  PE-A  5  10  -1010  3  10  PE-A  5  CD69  CD69 expression (%)  B. anti-CD3/CD28  70  anti-CD3/CD28+B7-H4  60 50 40 30 20 10 0 0.5  1  2  4  Hours after stimulation  Figure 5.5 B7-H4 inhibits CD69 expression Pre-activated CD3+ T cells were stimulated as above. Cells were collected and stained with anti-CD69 or rat IgG (A). This experiment represents the mean of three individual trials. The average expression of CD69 was shown in (B).  94  5.2.4 ERK activity is reduced upon B7-H4 engagement  One of the key pathways in transcription and expression of IL-2 involves the ERK1/2 kinases [198, 199]. Therefore, we investigated whether or not ERK1/2 activation would be affected by B7-H4 ligation. Cells were pre-activated and rested as before, and then stimulated with anti-CD3/CD28 and Fc.Ig or B7-H4.Ig protein. Cells lysates were prepared and subjected immunoblot analysis with antibodies to the phosphorylated, active form of ERK [phosphop44/42 MARK (ERK1/2) (Thr202/Tyr204)]. Phosphorylated ERK1/2 (p-ERK1/2) could be detected as early as 5 min, reaching maximal expression at 10 min, and decreasing to minimal levels at 30 min (Fig. 5.6A, 5.6E). After 60 min, the p-ERK expression rebounded to a high level, likely due to the action of autocrine IL-2. The expression of p-ERK1/2 was reduced with B7-H4.Ig engagement at all time points (Fig. 5.6A, 5.6E). For example, at 10 min, p-ERK1/2 reached peak levels. B7-H4.Ig treatment reduced ERK1/2 phosphorylation to 7% of the level seen in the Fc.Ig treated samples. 5.2.5 JNK activity is reduced upon B7-H4 engagement  JNK kinase is also involved in T-cell activation and IL-2 production [200, 201]. We therefore also examined the effect of B7-H4.Ig treatment on JNK phosphorylation as a measure of its activation state. The samples from the ERK studies described above were subjected to analyses with antibodies to phospho-SAPK/JNK (Thr183/Tyr185). We found that phosphorylation of JNK could indeed be detected as early as 1 min in response to antiCD3/CD28 stimulation, reaching a maximum at 10 min, and decreasing to a constant level thereafter (Fig. 5.6B, 5.6F). B7-H4 clearly reduced phosphorylation of JNK at all time points  95  (Fig. 5.6B, 5.6F), with 60% of JNK activity remaining at 10 min, demonstrating that B7-H4 also interferes with JNK activity.  5.2.6 AKT activity is reduced upon B7-H4 engagement  CD28 co-receptor activation of the PI3K/AKT pathway is essential for proliferation, IL-2 production and prevention of development of anergy or tolerance. We probed the lysates prepared above with antibodies to the phosphorylated active form of AKT (phospho-AKTSer473). Phosphorylated AKT (p-AKT-Ser473) could be seen as early as 1 min, increasing to a plateau level at 60 min, which remained to 120 min in the presence of anti-CD3/CD28 (Fig. 5.6C, 5.6G). At time points up to and including 60 min, B7-H4.Ig treatment significantly inhibited phosphorylation of p-AKT-Ser473 to 30% of the level in the Fc.Ig samples (Fig. 5.6C, 5.6G). Interestingly, inhibition at the 120-min time point was only 50%. We next examined the effect of B7-H4.Ig treatment on the phosphorylation state of GSK3, an endogenous substrate of AKT kinase, as a separate marker of in situ AKT kinase activity [202]. Immunoblots prepared as above were probed with antibodies to phosphorylated GSK-3 (phospho-GSK-3/) (Ser21/9). The kinetics of activation of GSK-3 phosphorylation in response to anti-CD3/CD28 was similar to that of AKT-Ser473 phosphorylation, suggesting that p-AKT-Ser473 was a good surrogate marker of kinase activity (Fig. 5.6D, 5.6H). More importantly, B7-H4.Ig treatment inhibited p-GSK-3 in the same way it did p-AKT-Ser473, demonstrating that B7-H4.Ig also inhibited phosphorylation and activation of AKT enzymatic activity.  96  Time (mins) 0  1  5  10  20  CD3/CD28 - + + + + + + + + B7-H4 - - + - + - + - +  30  60  + + + + - + - +  A.  120 + + - + p-ERK1/2 Actin  B.  p-JNK Actin  C.  p-AKT Actin  D.  pGSK-3 Actin  97  E.  F. Control B7-H4  10  4  JNK  ERK  Control B7-H4  5  15  5  3 2 1  0  0  1  5  10 20 30 60 120  0  0  1  G.  10 20 30 60 120  H. 2.0  2.5  1.5 1.0  B7-H4  1.0 0.5  0.5 0.0  Control  1.5  GSK-3  Control B7-H4  2.0  AKT473  5  Minutes after stimulation  Minutes after stimulation  0  1  5  10 20 30 60 120  0.0  0  1  5  10 20 30 60 120  Minutes after stimulation  Minutes after stimulation  Figure 5.6 B7-H4 suppresses activation of ERK, JNK, and AKT Western blot analyses of protein extracts from CD3+ T cells stimulated with immobilized antiCD3 and soluble anti-CD28. Fc.Ig or B7-H4.Ig was added after plate-bound anti-CD3 incubation. A. detection of phosphorylated ERK1/2 Thr202/Tyr204 B. detection of phosphorylated JNK Thr183/Tyr185 C. detection of phosphorylated AKT Ser473 D. detection of phosphorylated GSK-3/ Ser21/9 ERK (E), JNK (F), AKT (G), and GSK-3α/β (H) activity were quantitated by using Bio-Rad Quantity One program. The y-axis was normalized for the loading control.  98  5.2.7 B7-H4 does not affect early parameters of TCR triggering  The signaling molecules we examined above are those that CD28 co-receptor signaling regulates (either directly, AKT, GSK-3 or indirectly, ERK, JNK). We next examined the effect of B7-H4.Ig on two signaling molecules which the TCR directly regulates with minimal input from co-receptor action. LCK and ZAP70 tyrosine kinases are directly associated with the TCR complex, and their activation state can be monitored by their phosphorylation state. Phosphorylation of LCK and ZAP70 could be seen as early as 1 min upon anti-CD3/CD28 stimulation, and this was maintained up to 60 min (Fig. 5.7A, 5.7B). B7-H4.Ig treatment did not alter the phosphorylation of either LCK or ZAP-70.  99  Time (mins)  0  1  5  CD3/CD28 B7-H4  -  + + - +  + -  10 + +  20  + + - +  + -  30  + +  + -  60 + +  + + - + LCK  A. Actin ZAP70 B. Actin  C.  D. Control B7-H4  1.0  ZAP70  LCK  0.8 0.6 0.4 0.2 0.0  0  1  5  10  20  30  60  0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00  Control B7-H4  0  1  5  10  20  30  60  Minutes after stimulation  Minutes after stimulation  Figure 5.7 B7-H4 does not affect phosphorylation of LCK or ZAP70 Western blot analyses of protein extracts from CD3+ T cells stimulated with immobilized antiCD3 and soluble anti-CD28 in the presence of control Ig or B7-H4.Ig. A. detection of phosphorylated LCK B. detection of phosphorylated ZAP70 LCK (C) and ZAP70 (D) were quantitated by using Bio-Rad Quantity One program. The y-axis was normalized for the loading control.  100  5.3 SUMMARY OF FINDINGS AND DISCUSSION The B7-H4 negative regulation of T cell responses has been established. However, the signaling pathways in T cells altered by B7-H4 ligation have never been reported. Production of IL-2 is a hallmark of T-cell activation. The transcription and secretion of IL-2 by activated T cells promotes T cell cycle progression, clonal expansion and effector function. IL-2 mRNA expression increased as early as 30 min in pre-activated T cells upon TCR/CD28 stimulation (Fig. 5.2). The IL-2R is composed of three subunits. Of these three, the CD25  chain is required for high affinity IL-2 binding, while the  and  chains are responsible for the transduction of IL-2–generated signals. IL-2R is upregulated by CD3/CD28 ligation through autocrine IL-2–dependent and –independent mechanisms [203]. This study revealed that B7-H4.Ig inhibition of both IL-2 and CD25 expression occurred with similar kinetics, suggesting that IL-2 and CD25 expression are regulated through autocrine IL-2–independent mechanisms. IL-2 gene transcription occurs in activated T cells through the consequence of various signaling pathways. Both the ERK and JNK kinase pathways have been recognized as important regulators for IL-2 gene transcription. In this study, the data showed that both ERK and JNK activity was stimulated upon anti-CD3/CD28 ligation and reduced in the presence of B7-H4. CTLA-4, another potent negative regulator of the B7 family, has also been reported to inhibit both TCR-induced ERK and JNK activation [80]. It is interesting that B7-H4.Ig inhibited ERK phosphorylation to a greater degree, with only 7% of ERK activity remaining at 10 min (Fig. 4A, 4E), than JNK phosphorylation. This apparent preferential effect on ERK and JNK and the question of whether B7-H4.Ig treatment leads to inhibition of ERK and JNK kinases directly or via a molecule upstream of these kinases is the subject of future investigation. The inhibition of IL-2 mRNA levels by B7-H4.Ig appears to reverse at 24 h. This may be the result of the action of  101  autocrine IL-2 accumulating in the culture, since the addition of exogenous IL-2 is known to be able to overcome the inhibitory effect of B7-H4 ligation [114–116]. CD28 co-receptor signaling is essential for productive T-cell activation, especially of naïve or resting T cells. Blocking CD28 ligation impairs IL-2 production and can lead to T-cell anergy and apoptosis [53, 204]. Inhibition of CD28 co-receptor signaling has been reported to alleviate T cell–dependent diseases [205, 206]. Administration of anti-B7-H4 exacerbated EAE disease in mice [115]. We have also shown that B7-H4.Ig addition induces apoptosis of activated CD8+ T cells from patients with T1D [166] and that in vivo administration to allogeneic islet graft recipient mice is associated with decreased CD8+ T-cell levels in the graft and with prolonged survival [177]. Consistent with the well described central role of the PI3K/AKT pathway in CD28 signaling, we found that B7-H4.Ig treatment significantly reduced AKT phosphorylation and kinase activity (as shown by the phosphorylation state of GSK-3). Phosphorylation and activation of AKT is induced directly by CD28 ligation [207–209] and also by the action of cytokines such as IL-2 [210–212]. Our observation that B7-H4.Ig treatment interferes with AKT activation has a number of important corollaries. First, the observation that exogenously added IL-2 can reverse the effect of B7-H4 action [114] may be due to the fact IL-2 can activate PI3K/AKT via binding to the IL-2 receptor [211, 212] and in this way compensate for the B7-H4.Ig inhibition of CD28-induced AKT activation. Second, the greater sensitivity of naïve vs. pre-activated cells to B7-H4.Ig protein may be related to greater dependence of naïve cells on CD28 signaling [53, 204]. In contrast to the inhibitory effect of B7-H4.Ig treatment on ERK, JNK and AKT activation by anti-CD3/CD28, B7-H4.Ig did not alter the phosphorylation state of LCK or ZAP70 tyrosine kinases. The activation and phosphorylation of these kinases are regulated directly by ligation of TCR itself. LCK is constitutively associated with the cytoplasmic domain of the co-receptor molecules CD4 and CD8 [156] and is the first downstream signaling molecule 102  activated upon TCR ligation [194, 213]. Upon TCR stimulation, LCK (constitutively associated with the CD4/8 chains) autophosphorylates at tyrosine 394 in the activation loop [214], resulting in its activation and the phosphorylation of numerous substrates, including the intracellular domains of the TCR chains. ZAP70 rapidly associates with the phosphorylated CD3, which brings it in proximity to LCK. LCK phosphorylates ZAP70 tyrosine 493 in its activation loop, also resulting in its activation [215]. The phosphorylation state of these tyrosine residues in their activation loops is a strong indicator of their enzymatic activity. We did not observe any effect of B7-H4.Ig on the phosphorylation state of these residues, and it is possible that B7-H4.Ig can alter the ability of these kinases to access certain substrates. This remains to be determined, however, we note that the molecules for which we did observe an effect of B7-H4.Ig are either entirely dependent on CD28 signaling (AKT), or they integrate signals from CD28 (ERK and JNK). Our data show for the first time that B7-H4.Ig interferes with T-cell activation, at least in part through antagonizing signaling pathways downstream of CD28. In summary, our current data have provided insight into how B7-H4.Ig protein alters T-cell signaling and function that will be useful in developing B7-H4.Ig for clinical therapy. For example, monitoring CD25/69 expression could be a good biomarker for B7-H4.Ig action and B7-H4.Ig might best be used in combination with agents which target the TCR directly, or which target co-receptor signaling in ways different than B7-H4.Ig.  103  CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS  104  Alloimmunity is an intrinsic host response to foreign antigen stimulation. T cell-mediated alloimmune responses play a key role in determining the fate of islet transplantation. The requirement of a positive signal 2 for full T cell activation allows modulation of the immune response. Alloreactive T cell responses can be either activated or inhibited depending on whether positive or negative signaling predominates. B7-H4, first discovered in 2003, was recognized as a negative regulator in T cell responses in both in vitro assays and mouse experimental autoimmune encephalitis models [114, 115]. In this thesis, we have examined the potential protective role of B7-H4 ligation on inhibiting alloimmune responses. We show novel data that B7-H4 ligation can prolong islet graft survival. This likely occurs through inhibition of effector T cell function and through a Treg dependent mechanism, and that B7-H4 receptor signaling involves inhibition of TCR-stimulated AKT, JNK and ERK activation. As described in the introduction, alloimmune responses involve multiple steps. Antigen presenting cells take up graft alloantigens, migrate to regional lymph nodes where they prime naïve T cells. Primed T cells differentiate, proliferate and migrate to the periphery where they carry out their effector functions. We found that local expression of B7-H4 in the graft reduced the numbers of graft infiltrating CD8 effector cells at an early stage and substantially increased the numbers of FoxP3+ Treg cells at a later stage. Furthermore, expression of IL-2, a hallmark of T cell proliferation, is inhibited, but not significantly reduced in the local graft, suggesting an inefficient suppression of T cell proliferation in the local graft. If local B7-H4 expression inhibits T cell priming, it does so very inefficiently, since effector CD4 and CD8 cells are still found in the graft (albeit in lower numbers). However, B7-H4 is able to suppress expression of Th1associated IFN-γ and CTL-related granzyme B in the islet allograft, demonstrating a strong inhibition on pro-inflammatory cytokines in the early stage. Graft survival in B7-H4 treated primary recipients appears to be associated with Treg-dependent tolerance and that the presence of Treg cells interferes with effector T cell function in the long term (Fig. 6.1). 105  Figure 6.1 The effects of B7-H4 on alloreactive responses in transplantation Local expression of B7-H4 on donor islets prolongs islet allograft survival. Interaction between B7-H4 and an unknown receptor on activated T cells leads to protection of allograft from rejection through deletional and non-deletional mechanisms. B7-H4 inhibits alloreactive CD8+ proliferation, Th1-associated IFNγ and cytotoxic-related granzyme B response which may facilitate generation of suppressive Foxp3+ Tregs (shown in green). The combination of controlling activated T-cell proliferation and promoting regulatory T-cell subsets results in enhanced allograft survival and induction of donor-specific tolerance.  106  An important conclusion of our studies is that B7-H4 treatment allows long term graft survival in the absence of immunosuppressive drugs. Although allogeneic islet grafts eventually all fail, autologous islet transplants survive long term [216]. This has been attributed to the cytotoxic effect of the currently used immunosuppressive drugs on beta cells. All the drugs available for transplantation are cytotoxic to beta cells to a varying degree [8, 9, 13, 35, 217, 218, 219] so islet allograft function is better maintained without ongoing immunosupppression. Induction of allograft tolerance, as an alternative to global immunsuppression, is preferred. Our data suggest that B7-H4 therapy can result in such a tolerant state. In terms of the mechanism of B7-H4-mediated tolerance generation, our data suggest that Tregs are important in tolerance induction in the primary graft recipients, but whether they are sufficient for graft survival in the secondary graft recipients remains to be determined. We also investigated the intracellular signal transduction mechanism by which B7-H4 ligation interferes with T cell activation. Such molecular studies could give insight into better design of tolerance inducing protocols, and also into potential development of small molecule drugs that mimic the action of B7-H4. For instance, CTLA-4 directly interferes with CD28 signaling by competing with B7.1/B7.2 (activating ligands) for binding to CD28 [55, 57, 69, 70, 71]. This knowledge suggests that CTLA-4-Ig needs to be administered prior to graft implantation for maximum efficacy. In contrast, we found that B7-H4 does not globally interfere with all signal transduction events downstream of CD28 (Fig. 6.2). Instead B7-H4 treatment selectively interferes with AKT, JNK and ERK kinase activation. More work is required to fully understand how signaling from the B7-H4 receptor inhibits these kinases. This suggests that these two agents have great synergistic potential in preventing graft rejection.  107  Figure 6.2 The role of B7-H4 in islet transplantation B7-H4-mediated signal transduction pathway. The engagement of the B7-H4 with an unknown receptor on activated T cells inhibits the activation of JNK, ERK, and AKT (shown in red) but allows the proximal LCK and ZAP70 phosphorylation (shown in green). This pattern of B7-H4 action results in inhibition of IL-2 transcription/production and T cell proliferation.  108  In summary, we are the first to report that B7-H4 treatment prolongs graft survival in an islet model and show that this is associated with donor specific tolerance. The regulatory effects of B7-H4 in alloimmune responses were subsequently tested in studies using cell (expressed B7H4 in an insulinoma cell line derived from NOD) and solid transplantation models (heart), respectively [220, 221]. This provides pre-clinical proof of principle data to pursue the development of B7-H4 based therapy for induction of tolerance in human transplant recipients. Our data have also raised a number of interesting basic research questions to be investigated in future studies. Current data support the hypothesis that tolerance induction mediated by B7-H4 may be mediated through both nTregs in the graft and IL-10–expressing iTreg modulation in the periphery. We have not determined the relative contribution of nTregs versus iTregs, and of Tregs in the local graft versus peripheral tissues. These questions can be addressed through adoptive transfer experiments of each of the cell types in question. Determining the exact nature of Tregs involved in eliciting graft tolerance is important in understanding the relationship between B7-H4 and regulation of Treg function and/or development. The recurrence of autoimmune responses in islet transplantation was not investigated in current study. Published data show that administration of B7-H4 monoclonal antibody that blocks endogenous B7-H4 accelerates mouse experimental autoimmune encephalitis [115]. This suggests that B7-H4 ligation will conversely be beneficial in interfering with established autoimmunity, a condition which is extremely important when transplanting islet cells into a T1D recipient. The very enticing possibility that B7-H4 can interfere with autoimmunity can be addressed with studies using the NOD mouse model of autoimmune diabetes. Finally, future studies related to the signalling mechanisms used by B7-H4 to inhibit T cell activation include analysis of the state of the regulators of AKT, JNK and ERK kinases. 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