Cell Costimulatory Pathways Research Articles

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

Targeting Co-Stimulatory Pathways in Gene Therapy

Gene therapy with recombinant viral vectors such as adenovirus and adenovirus-associated virus holds great promise in treating a wide range of diseases because of the high efficiency with which the viruses transfer their genomes into host cells in vivo. However, the activation of the host immune responses remains a major hurdle to successful gene therapy. Studies in the past two decades have elucidated the important role co-stimulation plays in the activation of both T and B cells. This review summarizes our current understanding of T cell co-stimulatory pathways, and strategies targeting these co-stimulatory pathways in gene therapy applications as well as potential future directions.

Topical inhibition of T cell costimulatory pathways in draining lymph nodes may suppress allograft rejection

Topical immune suppression is an attractive and practical therapeutic option to prolong survival time of allografts, before the appearance of new agent with higher immunosuppressive efficacy and lower undesirable side effects. The initiation of rejection and outcome of allografts is principally mediated by alloantigen reactive T cells. The activation of T cells requires at least two signals, first is T-cell receptor signal and second is costimulatory signal. T cells that encounter antigen without the appropriate costimulatory signal become anergy or tolerance. Migration of alloantigen-bearing dendritic cells into the T-cell zone of secondary lymphoid tissues, which are essential for primary alloimmune responses, effectively induces T-cell activation and expansion with the presence of two signals. Draining lymph nodes are the promising targets for topical immune suppression, as disrupting lymphatic drainage from the transplanted graft to lymph nodes prevented rejection of skin allografts and lymphadenectomy prolong the survival time of skin and corneal allografts in experimental animals. Therefore, we hypothesize that inhibition of T cell costimulatory pathways in draining lymph nodes could impair the alloantigen-specific immune response and reduce systemic immunosuppressive drugs dose for allografts survival. Further investigations are required to identify most efficient way for draining lymph nodes transfer of costimulatory molecule gene or topical drug administration of costimulatory inhibitors to draining lymph nodes.

Targeting dendritic cells to treat multiple sclerosis

Multiple sclerosis (MS) is considered to be a predominantly T-cell-mediated disease, and emerging evidence indicates that dendritic cells have a critical role in the initiation and progression of this debilitating condition. Dendritic cells are specialized antigen-presenting cells that can prime naive T cells and modulate adaptive immune responses. Their powerful biological functions indicate that these cells can be exploited by immunotherapeutic approaches. Therapies that inhibit the immunogenic actions of dendritic cells through the blockade of proinflammatory cytokine production and T cell co-stimulatory pathways are currently being pursued. Furthermore, novel strategies that can regulate dendritic cell development and differentiation and harness the tolerogenic capacity of these cells are also being developed. Here, we evaluate the prospects of these future therapeutic strategies, which focus on dendritic cells and dendritic cell-related targets to treat MS.

Structural immunology of costimualtory and coinhibitory molecules

The T cell costimulatory pathways are central to regulating immune responses, and targeting these pathways represents one of the most promising approaches for achieving immunotherapy. The molecular structures of costimulation revealed invaluable mechanistic insights underlying costimulatory receptor/ligand specificity, affinity, oligomeric state, and valency, which provided the bases for better manipulation of these signaling pathways. The incredible growth of this field led to identification of new members and unexpected interactions, revealing a complicated regulatory network of immune responses. The advances in structural biology of costimulation will promise unprecedented opportunities for furthering our understanding and therapeutic application of T cell costimulatory pathways.

A Novel Function of CD26-Mediated Costimulation in the Cytotoxic Activity of Human CD8+ T Cells in Xenogeneic Chronic Graft-Versus-Host Disease (cGVHD) and GVL Mice Model.

Abstract 3548Poster Board III-485CD26 is a 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain, capable of cleaving amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. CD26 activity is dependent on cell type and the microenvironment, which influence its multiple biological roles. CD26 plays an important role in immunology, autoimmunity, diabetes and cancer. The role of CD26 in immune regulation has been extensively characterized, with our recent findings elucidating its linkage with signaling pathways and structures involved in T cells activation as well as antigen presenting cell (APC)-T-cell interaction. CD26 is preferentially expressed on human CD4+ memory T cells and CD4+CD45RO+CD26+ T cells exhibit response maximally to the recall antigen tetanus toxoid. Human T-helper 1 (Th1) cells display a higher expression of CD26 and are much more sensitive to CD26-mediated costimulation than human Th2 cells. In contrast, the role of CD26 in human CD8+ T cells still remains to be elucidated, while CD8+ T cells express CD26 as well as CD4+ T cells. In this study, we studied the effector function of CD26-mediated costimulation of CD8+ T cells. In comparison with CD28-medaited costimulation, which is one of representative T cell costimulatory pathway, CD26-mediated costimulation in CD8+ T cells showed delayed proliferation than that of CD28 stimuatlion, but finally expanded to a similar extent. The secretion of inflammatory cytokines, TNF-alpha and IFN-gamma, was strongly induced after CD26-mediated costimulation. In contrast, the secretion of IL-2 and IL-5 was significantly less as compared with CD28-mediated costimulation. Finally, we showed that the expression of Granzyme B, one of the major effector molecules in the cytotoxic activity of CD8+ T cells, was markedly enhanced by CD26-mediated costimulation in dose dependent manner of anti-CD26 monoclonal antibody stimulation. Moreover, with CD26-mediated costimulation, CD8+ T cells were observed to kill target cells in a Granzyme B-dependent manner. Next, we examined CD26-dependent organ injury by human T cells using xenogeneic chronic GVHD (x-cGVHD) mice model. About 6 weeks after transplantation of human peripheral blood mononuclear cells (PBMC) into NOG-Scid mice, mice develop loss pf hair, loss of weight, and human CD8+ lymphocytes infiltration into liver, lung, and salivary glands, which exhibit pathological changes of cGVHD. Administration of anti-human CD26 mAb decreased onset of x-cGVHD, while human lymphocytes were grafted successfully. Finally, we examined whether GVL effect is exerted via CD26-mediated costimulation, using x-cGVHD mice model in which P388 leukemic cells were grafted subcutaneously. In this tumor-graft model, P388 cells were not grafted in mice with onset of x-cGVHD, which indicated that GVL effect was exerted by human lymphocytes. On the other hand, P388 cells were also diminished in mice with decreased x-cGVHD by administration of anti-CD26 mAb. Taken together, our data strongly suggest that CD26-mediated costimulation in CD8+ T cells deeply involves in the pathology of cGVHD. The present study offers a novel notion that CD26 may become one of therapeutic targets of GVHD without impairment of GVL effect. Disclosures:Yamada:Y's Therapeutics: Membership on an entity's Board of Directors or advisory committees. Dang:Y's Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Morimoto:Y's Therapeutics: Patents & Royalties, a board member.

Accelerated Course of Experimental Autoimmune Encephalomyelitis in PD-1-Deficient Central Nervous System Myelin Mutants

It is assumed that the onset and course of autoimmune inflammatory central nervous system (CNS) disorders (eg, multiple sclerosis) are influenced by factors that afflict immune regulation as well as CNS vulnerability. We challenged this concept experimentally by investigating how genetic alterations that affect myelin (primary oligodendrocyte damage in PLPtg mice) and/or T-cell regulation (deficiency of PD-1) influence both the onset and course of an experimental autoimmune CNS inflammatory disease [MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE)]. We observed that double pathology was associated with a significantly earlier onset of disease, a slight increase in the neurological score, an increase in the number of infiltrating cells, and enhanced axonal degeneration compared with wild-type mice and the respective, single mutant controls. Double-mutant PLPtg/PD-1(-/-) mice showed an increased production of interferon-gamma by CNS immune cells at the peak of disease. Neither PD-1 deficiency nor oligodendropathy led to detectable spread of antigenic MHC class I- or class II-restricted epitopes during EAE. However, absence of PD-1 clearly increased the propensity of T lymphocytes to expand, and the number of clonal expansions reliably reflected the severity of the EAE disease course. Our data show that the interplay between immune dysregulation and myelinopathy results in a stable exacerbation of actively induced autoimmune CNS inflammation, suggesting that the combination of several pathological issues contributes significantly to disease susceptibility or relapses in human disease.

Prevention of Acute GvHD during Haplo-BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model

Introduction: While bone marrow transplantation (BMT) offers curative therapy for many malignant and non-malignant hematologic diseases, it remains plagued by high morbidity and mortality, most prominently through the complications of graft-versus-host disease (GvHD). The field of GvHD research has been limited by a small number of clinically relevant model systems, which, until now, have focused on murine and canine models. However, many new human therapeutics, including those directed at T cell costimulation pathways, do not cross-react with targets in these model systems, limiting their utility to fully evaluate the new generation biologic anti-GvHD therapies.Methods: We have developed a fully pedigreed and MHC-typed rhesus macaque bone marrow transplant system, with which to model GvHD and its prevention. This model relies upon microsatellite typing of both autosomal chromosomes to create genetically-verified family trees and microsatellite-and allele-specific PCR-based MHC typing, with which to identify MHC haplotypes within each family group. By this methodology, full and half-sibling pairs can be identified with either full MHC matching, one MHC haplotype matching or full MHC disparity. This represents a significant advance compared to previous primate haplo-studies, which relied upon parent-offspring transplants, as it optimizes animal availability and colony stability as well as permiting the rigorous evaluation of the degree of MHC disparity on the non-shared chromosome 6. For the current study, we have concentrated on MHC haplo-identical BMT. Our preparative regimen consists of TBI (8.00 Gy with lung shielding to 6.00 Gy), which is performed on the day of transplant with hematopoietic stem cells. After transplant, donor engraftment is measured by microsatellite analysis, from both whole blood and from flow cytometrically sorted T and B cell populations. GvHD is graded using standard clinical grading scales. The immune phenotype after transplant is also measured by cell- and serum-based flow cytometric analyses.Results: Three animals have so far been studied. The first animal served as a control for TBI-based preparation. This animal exhibited both neutropenia (nadir = 0.015 × 103/uL at 12 days) and thrombocytopenia (nadir = 2 × 103/uL at 12 days) and prolonged anemia (hgb=5.8 g/dL on the day of sacrifice). This animal was sacrificed at day 29 due to prolonged pancytopenia. The second animal was transplanted with haploidentical hematopoietic stem cells (4.47 × 108 total nucleated cells/kg and 1.10 × 108 CD3+ T cells/kg) and was treated with only rapamycin for immunosuppression. He exhibited profuse diarrhea and necrotic skin changes within 8 days of transplant, coincident with early engraftment and was sacrificed at day 14. A full histologic analysis to determine the cause of these lesions (and the histopathologic extent of GvHD) is currently underway. The third animal was transplanted with haploidentical donor bone marrow (1.1 × 109 total nucleated cells/kg and 6.9 × 107 CD3+ T cells/kg), but was given T cell costimulation blockade with abatacept (targeting the CD28/B7 pathway) and an anti-CD40 antibody (targeting the CD40/CD154 pathway) in addition to rapamycin. Abatacept is currently clinically approved for treatment of rheumatoid arthritis, but has not been added to clinical GvHD regimens, largely due to the paucity of translational efficacy data. This animal is currently >43 days post-transplant, with full donor chimerism (including 100% T and B cell chimerism), without signs of GvHD: he has no rash, no diarrhea and is stable clinically. Additional animals are now being added to the study, to confirm these initial results.Conclusions:We have established a robust non-human primate model of GvHD using pedigreed and MHC-typed rhesus macaques.We have used this model to begin to test the efficacy of a novel agent combination, capable of preventing the onset and complications of acute GvHD.Preliminary results suggest that CD28- and CD40-directed costimulation blockade may be active agents for the prevention of GvHD. A large scale analysis of their efficacy and immune consequences is currently underway.

Constitutive Expression of B7-1 on B Cells Uncovers Autoimmunity toward the B Cell Compartment in the Nonobese Diabetic Mouse

The NOD mouse is an invaluable model for the study of autoimmune diabetes. Furthermore, although less appreciated, NOD mice are susceptible to other autoimmune diseases that can be differentially manifested by altering the balance of T cell costimulatory pathways. In this study, we show that constitutively expressing B7-1 on B cells (NOD-B7-1B-transgenic mice) resulted in reduced insulitis and completely protected NOD mice from developing diabetes. Furthermore, B7-1 expression led to a dramatic reduction of the B cell compartment due to a selective deletion of follicular B cells in the spleen, whereas marginal zone B cells were largely unaffected. B cell depletion was dependent on B cell specificity, mediated by CD8(+) T cells, and occurred exclusively in the autoimmune-prone NOD background. Our results suggest that B cell deletion was a consequence of the specific activation of autoreactive T cells directed at peripheral self Ags presented by maturing B cells that expressed B7-1 costimulatory molecules. This study underscores the importance of B7 costimulatory molecules in controlling the amplitude and target of autoimmunity in genetically prone individuals and has important implications in the use of costimulatory pathway antagonists in the treatment of human autoimmune diseases.

T Cell Dysfunction by Hepatitis C Virus Core Protein Involves PD-1/PDL-1 Signaling

Reports have shown that a negative T cell costimulatory pathway mediated by PD-1 (programmed death-1) and PDL-1 (programmed death ligand-1) is associated with T cell exhaustion and persistent viral infection. Persistent hepatitis C virus (HCV) infection in humans is also characterized by impaired T lymphocyte function, but the role of the PD-1 and PDL-1 pathway in HCV infection is unknown. Here we report that T cells isolated from chronically HCV-infected patients express significantly higher levels of PD-1 when compared with healthy donors. In addition, PD-1 and PDL-1 expression is upregulated on healthy donor T cells exposed to HCV core, a nucleocapsid protein that is immunosuppressive; upregulation of PD-1 is mediated through interaction of HCV core with the complement receptor, gC1qR. Importantly, T cell functions that are dysregulated by HCV core, including T cell activation, proliferation, and apoptosis, can be restored by blocking PD-1 and PDL-1 engagement. Our results indicate that HCV core can upregulate a key negative T cell signaling pathway associated with viral persistence and highly expressed on the T cells of persistently infected individuals. This upregulation of the PD-1 and PDL-1 pathway in humans represents a novel and perhaps common mechanism by which a virus usurps host machinery to facilitate persistence.

T Cell Costimulatory and Inhibitory Receptors as Therapeutic Targets for Inducing Anti-Tumor Immunity

Central to the normal function of the immune system is its ability to distinguish between self and non-self since failure to do so could provoke the onset of autoimmune disease. To avoid this possibility, the immune system employs several processes that include, negative selection, peripheral tolerance, and limiting DC antigen priming of naïve T cells to the lymph nodes. Naïve T cells must receive two independent signals from these antigen-presenting cells (APC) that other cells cannot provide if they are to become productively activated. The first is antigen-specific and occurs when T cell antigen receptors encounter the appropriate antigen-MHC complex on the APC--Signal 1. A second, antigen-independent signal is delivered through a T cell costimulatory molecule that engages its APC-expressed ligands--Signal 2. In the absence of a costimulatory signal T cells typically enter a state of anergy. Furthermore, the extent to which T cell activation occurs can be held in check through specific inhibitory receptors expressed on T cells. Understanding the basic mechanisms of how T cell activation is regulated has led to the development of therapeutic approaches for targeting T cell costimulatory and inhibitory pathways for turning on, or preventing the turning off immune responses in subjects with cancer. In this review we will discuss several T cell costimulatory and inhibitory pathways known to influence the development of anti-tumor immunity and how experimental manipulation of these signaling pathways has led to the generation of protective, or curative anti-tumor immunity in mice and humans.

T cell costimulatory pathways in allograft rejection and tolerance: what's new?

The induction or maintenance of allograft tolerance remains an ongoing challenge. One approach to the development of tolerogenic strategies involves targeting T-cell costimulatory signals. The two most widely studied costimulatory pathways are the CD28/B7 and CD40/CD154 pathways, and blocking of both, either alone or in combination, has been shown to prolong allograft survival in rodents and primates. Recent work revealed that CD28-independent 'novel costimulatory' pathways exist, which can mediate allograft rejection. This review highlights new studies on the role of these pathways in allograft rejection and tolerance. NK cells, CD8 T cells, and memory-effector responses appear to be less dependent on CD28 and/or CD154 costimulation, and utilize these novel costimulatory pathways for activation. The novel signals differ in their ability to enhance or inhibit T-cell activation, in their temporal and spatial expression patterns, and in their relative importance within the hierarchy of costimulatory signals. Emerging data suggest that costimulatory molecules are expressed on parenchymal cells. A strategy to induce tolerance might involve targeting novel costimulatory signals particularly at the time point of maximal expression, and delivering negative signals, while inhibiting the positive signals that drive T-cell alloresponses.

Stable Skin-specific Overexpression of Human CTLA4-Ig in Transgenic Mice through Seven Generations

Skin graft rejection is a typical cellular immune response, mainly mediated by T cells. Cytotoxic T lymphocyte associated antigen 4-immunoglobin (CTLA4-Ig) extends graft survival by blocking the T cell co-stimulation pathway and inhibiting T cell activation. To investigate the efficacy of CTLA4-Ig in prolonging skin graft survival, human CTLA4-Ig (hCTLA4-Ig) was engineered to overexpress in mouse skin by transgenesis using the K14 promoter. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay indicated that the expression of CTLA4-Ig remained skin-specific and relatively constant compared to the internal control protein, AKT, through seven generations. The presence and concentration of the hCTLA4-Ig protein in transgenic mouse sera was determined by enzyme-linked immunosorbent assay (ELISA), and the results indicated that the serum CTLA4-Ig concentration also remained constant through generations. Survival of transgenic mouse skins grafted onto rat wounds was remarkably prolonged compared to that of wild-type skins from the same mouse strain, and remained comparable among all seven generations. This suggested that the bioactive hCTLA4-Ig protein was stably expressed in transgenical mice through at least seven generations, which was consistent with the stable skin-specific CTLA4-Ig expression. The results demonstrated that the transgenic expression of hCTLA4-Ig in skin driven by the K14 promoter remained constant through generations, and a transgenic line can be established to provide transgenic skin with extended survival reproducibly.

Immunotherapy for Prostate Cancer

The absence of curative therapies for advanced or recurrent forms of prostate cancer has prompted a vigorous search for novel treatment strategies. Immunotherapy encompasses one particularly promising systemic approach to treat prostate cancer. Immune-based strategies to treat prostate cancer have recently been facilitated by the identification of a number of prostate tissue/tumor antigens that can be targeted, either by antibody or T cells, to promote prostate tumor cell injury or death. These same prostate antigens can also be used for the construction of vaccines to induce prostate-specific T cell-mediated immunity. Greater insight into specific mechanisms that govern antigen-specific T cell activation has brought with it a number of innovative methods to induce and enhance T cell-mediated responses against prostate tumors. For instance, autologous dendritic cells loaded with prostate antigens have proved useful to induce prostate-specific T cell activation. Similarly, in vivo manipulations of the T cell costimulatory pathway receptors can greatly facilitate tumor-specific T cell activation and potentiate T cell-mediated responses against a number of malignancies, including prostate cancer. Therefore, in this review we summarize recent advances pertaining to immunotherapeutic approaches to treat prostate cancer.

Vascular Endothelial Growth Factor Antagonist Modulates Leukocyte Trafficking and Protects Mouse Livers against Ischemia/Reperfusion Injury

Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.

Enhanced immunosuppression induced by targeted mutation of cytotoxic T lymphocyte antigen 4-immunoglobulin

Purpose of review Blockade of T cell costimulatory pathways has enormous potential as an immunosuppressive strategy to prolong the survival of solid organ transplants. Discovery of the CD28 costimulatory pathway led to the development of both monoclonal antibodies and fusion proteins that interrupt CD28 signaling. Subsequent studies in both animal models and humans demonstrated that although these molecules had promise as immunosuppressive agents for solid organ transplantation, they lacked the desired clinical efficacy. Recent advances in the field of immunobiologics allowed for the development of rationally modified agents, such as LEA29Y (belatacept), that more effectively target costimulatory molecules. This review summarizes the current understanding of the role of CD28 costimulatory blockade as an immunosuppressive strategy in organ transplantation. Recent findings This review highlights the strategy used to engineer a fusion protein (LEA29Y, belatacept) from its parent compound (cytotoxic T lymphocyte 4-immunoglobulin) that has higher avidity for CD28 ligands and more potent immunosuppressive effects. Summary Before the development of cyclosporine in the early 1980s, the success of transplantation was limited by high rates of rejection and consequent organ loss. Unfortunately, cyclosporine and most subsequently developed immunosuppressive drugs target ubiquitously expressed molecules, causing a myriad of significant side effects unrelated to their primary immunosuppressive effect. Early studies demonstrated complete T cell activation to be critically dependent on signals provided by costimulatory molecules such as CD28 and its ligands B7-1 and B7-2. This provided the impetus for the investigation of agents that block or inhibit these signals with the goal of developing compounds that have less deleterious side effects.

Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.

CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.

CD40L AND CD28 BLOCKADE INDUCES REGULATORY MECHANISM AND INHIBITS CHRONIC ALLOGRAFT REJECTION

P1037 Aims: Blockade of individual T cell costimulatory pathways may result in long-term allograft survival but tolerance is not obtained since chronic rejection is observed. To obtain transplantation tolerance, blockade of more than one costimulatory pathway is needed but the importance of different pathways and the optimal reagents to be used have not been fully defined. Methods: Heart transplantation was performed between MHC mismatched rats. CD40-CD154 interactions were blocked by gene transfer of CD40Ig. We interfered with CD28 costimulatory signals by administering an anti-CD28 Mab, which in contrast to CTLA4Ig, preserves T cell inhibitory signals through CTLA4. Results: CD40Ig treatment resulted in long-term (>120d, n=19) heart allograft survival and development of chronic rejection in all grafts whereas acute rejection occurred in the non-coding adenovirus-treated (7.6±1.6d, n=9) group. Treatment with anti-CD28 Mab resulted in a moderate but significant prolongation of allograft survival (17.4±1.7d, n=10). Cotreatment with both CD40Ig and anti-CD28Mab resulted in long term allograft survival (>120d, n=7) and a significant reduction in the percentage and degree of vessels with chronic rejection lesions, with 3/7 grafts completely free of lesions and therefore tolerant. Splenocytes from cotreated tolerant recipients, but not those with chronic rejection (CD40Ig or cotreated), showed completely suppressed MLR responses against 1st but not 3rd party donor cells. T cells purified from splenocytes of tolerant cotreated recipients showed normal MLR responses indicating that among total splenocytes, non T cells inhibited the proliferation of T cells. Splenocytes from tolerant recipients but not those with chronic rejection lesions inhibited naïve MLR, indicating the presence of active tolerogenic mechanisms. Transfer of splenocytes from CD40Ig-treated recipients to naïve sublethaly irradiated rats conferred long term allograft survival (>120d, n=4 vs. 17 d, n=2 in controls). In contrast, transfer of splenocytes from CD40Ig+anti-CD28 cotreated rats, either tolerant or with chronic rejection lesions, did not prolong allograft survival (17d, n=3). Conclusions: The association of CD40Ig with anti-CD28 Mab resulted in decreased chronic rejection lesions and tolerogenic mechanisms different to those seen with CD40Ig.

NEW INSIGHTS INTO ICOS-B7h PATHWAY IN ALLOIMMUNE RESPONSE

O456* Aims: Recent data indicate that the new T cell costimulatory pathway ICOS-B7h plays an important role in alloimmune responses in vivo. Although its role has been investigated in acute allograft rejection, relatively little is known of its functions in the process of chronic rejection. In this study, we investigated the role of ICOS-B7h pathway in development of chronic rejection of vascularized cardiac allografts. Methods: We utilized a MHC class II disparate cardiac transplantation model (Bm12 into C57BL/6). In this model, Bm12 cardiac allografts survive long-term but spontaneously develop moderately severe chronic vasculopathy, cellular infiltration and fibrosis by 6-8 weeks post-transplant. Results: We treated mice with a blocking anti-B7h mAb (HK5.3) either in the initiation phase (n=6, 0.25mg, every other day, 0-20 days after transplantation) or in the progression phase (n=6, 21-41 days) of chronic rejection. At 6 weeks, histopathological examination of grafts was performed by a blinded observer and scored in terms of vasculopathy (score 0-4), fibrosis (grade 0-3) and cellular infiltration (grade 0-3). Early blockade of ICOS-B7h pathway didn’t show a protective effect on chronic rejection (vasculopathy; 3.5±0.4, fibrosis; 2.2±0.3, cellular infiltration; 2.3±0.3: mean±SEM) compared to untreated controls (n=6, vasculopathy; 3.6±0.1, fibrosis; 2.7±0.2, cellular infiltration; 2.7±0.2). In contrast, delayed blockade of B7h significantly inhibited the development of chronic vasculopathy (1.8±0.4 vs. control, P=0.043), fibrosis (1.5±0.2, P=0.004) and cellular infiltration (1.7±0.3, P=0.03). To address the underlying mechanisms, we performed quantative real-time PCR analysis. There was significant decrease in local intragraft expression of cytokines, such as TNF-α, IFN-γ and IL-4, and several key chemokines, such as RANTES and IP-10, as well as adhesion molecules such as E-selectin in recipients with delayed blockade of B7h compared to controls. Moreover, there was significantly less infiltration of CD4 and CD8 T cells (p=0.007 and p=0.042 vs. control). Thus, delayed blockade of ICOS-B7h pathway downregulated local immune activation, thereby resulting in the less infiltration of alloreactive cells into cardiac grafts. Furthermore, to rule out the possibility that early blockade simply was not sufficient enough to display the protective effect on chronic rejection in this model, we treated mice with mAb in the entire period (n=6, 0-41 days, every other day). Interestingly, these mice demonstrated severe chronic rejection (vasculopathy; 3.3±0.4, fibrosis; 2.7±0.2, cellular infiltration; 2.8±0.2). Taken together, ICOS pathway may be critical in the progression phase of chronic rejection. In addition, its blockade in the initiation phase may have a detrimental effect on the prevention of chronic rejection. Conclusions: These data highlight the importance of ICOS-B7h costimulatory pathway in the pathogenesis of progression of chronic allograft rejection and provide the rationale to explore novel blockade strategy to treat this important clinical problem.