The PD-1:PDL pathway plays an important role in regulating alloimmune responses but its role in transplantation tolerance is unknown. We investigated the role of PD-1:PDL costimulatory pathway in peripheral and a well established model of central transplantation tolerance. Early as well as delayed blockade of PDL1 but not PDL2 abrogated tolerance induced by CTLA4Ig in a fully MHC-mismatched cardiac allograft model. Accelerated rejection was associated with a significant increase in the frequency of IFN-γ-producing alloreactive T cells and expansion of effector CD8+ T cells in the periphery, and a decline in the percentage of Foxp3+ graft infiltrating cells. Similarly, studies using PDL1/L2-deficient recipients confirmed the results with Ab blockade. Interestingly, while PDL1-deficient donor allografts were accepted by wild-type recipients treated with CTLA4Ig, the grafts developed severe chronic rejection and vasculopathy when compared with wild-type grafts. Finally, in a model of central tolerance induced by mixed allogeneic chimerism, engraftment was not abrogated by PDL1/L2 blockade. These novel data demonstrate the critical role of PDL1 for induction and maintenance of peripheral transplantation tolerance by its ability to alter the balance between pathogenic and regulatory T cells. Expression of PDL1 in donor tissue is critical for prevention of in situ graft pathology and chronic rejection.

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We studied T-cell clones generated from grafts of rejecting and tolerant animals and investigated the regulatory function of Th2 clones in vitro and in vivo. To prevent allograft rejection, we treated LEW strain recipient rats of WF strain kidney grafts with CTLA4Ig to block CD28-B7 costimulation. We then isolated epitope-specific T-cell clones from the engrafted tissue, using a donor-derived immunodominant class II MHC allopeptide presented by recipient antigen-presenting cells. Acutely rejected tissue from untreated animals yielded self-restricted, allopeptide-specific T-cell clones that produced IFN-γ, whereas clones from tolerant animals produced IL-4 and IL-10. Adoptive transfer into naive recipients of Th1 clones, but not Th2 clones, induced alloantigen-specific delayed-type hypersensitivity (DTH) responses. In addition, Th2 clones suppressed DTH responses mediated by Th1 clones in vivo and blocked Th1 cell proliferation and IFN-γ production in vitro. A pilot human study showed that HLA-DR allopeptide-specific T-cell clones generated from patients with chronic rejection secrete Th1 cytokines, whereas those from patients with stable graft function produce Th2 cytokines in response to donor-specific HLA-DR allopeptides. We suggest that self-restricted alloantigen-specific Th2 clones may regulate the alloimmune responses and promote long-term allograft survival and tolerance. See related Commentary on pages 797-798rats undergoing acute vascularized allograft rejection express a restricted TCR Vβ repertoire and transfer DTH responses in vivo (16). In this study, we compared for the first time, to our knowledge, the functions of self-restricted alloreactive T-cell clones generated from grafts of rejecting and tolerant animals and analyzed the putative regulatory functions of Th2 clones in vitro and in vivo. Results of a pilot study in kidney transplant recipients with chronic rejection or stable graft function establish the biological relevance of our animal studies in humans. Our results confirm the regulatory functions of alloreactive Th2 clones and provide an invaluable tool for the study of the functions of Th1 and Th2 clones in acute/chronic allograft rejection and tolerance in vivo. MethodsAnimals. Inbred 200-250 g male Lewis (LEW; RT1 l ) rats were used as recipients and Wistar Furth (WF; RT1 u ) rats served as donors. They were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, Indiana, USA).Rat kidney transplantation. LEW rats underwent bilateral nephrectomies and received heterotopic MHCincompatible WF renal allografts. For the purpose of this study, we used two groups of animals. The first group was unmodified and the rejecting graft was harvested on day 7. The second group was treated with a single injection of human cytotoxic lymphocyte activation factor 4 (CTLA4Ig; Bristol Myers Squibb Co., Princeton, New Jersey, USA) on day 2 after transplant and the graft was harvested after 100 days. This protocol of CTLA4Ig administration has previously been shown by Sayegh's group to induce long-term a...

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MethodsClass II MHC peptides. We synthesized four 15-to 16-mer peptides, two derived from human class II MHC: HLA-DQA1 (residues 62-77 of the α chain of DQA*0101) and HLA-DQB1 (corresponding residues of the β chain of DQB1*0501) chain, and two derived from rat class II MHC: RT1.D u α (residues 61-75) and RT1.B u α (residues 62-78). The peptides were synthesized by Chiron Mimotopes (Victoria, Australia) using an automated peptide synthesizer. Peptides were purified by reverse-phase HPLC and shown to be >95% homogenous by analytical reverse HPLC and mass spectroscopy. RT1.D u β2 (residues 20-44), a Wistar-Furth (WF) class II MHC peptide, was synthesized in the Protein/Nucleic Acid Laboratory, Brigham and Women's Hospital, Department of Medicine (7,8). Before use, the peptides were dissolved in sterile PBS at a concentration of 1 mg/ml. Peptide sequences are shown in Table 1.Animals. Male Lewis (LEW), Wistar-Furth (WF), and Brown Norway (BN) rats, 8-12 weeks old, were obtained from Harlan-Sprague-Dawley (Indianapolis, Indiana, USA). Adult male CBL/6j and DBA mice, 4-6 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Rat mixed lymphocyte response. Cervical lymph nodes were har- Received for publication November 6, 1998, and accepted in revised form January 26, 1999. Inhibition of allorecognition by a human class II MHC-derived peptide through the induction of apoptosisThe interaction of the T-cell receptor with the major histocomatibility complex (MHC)-peptide complex is central to T-cell activation. Variation in the nature of the peptide bound within the groove of the MHC molecule may result in an altered T-cell response. Because some naturally processed peptides bound within the groove of the class II MHC molecule are derived from the MHC molecules themselves, we studied the inhibitory effects of synthetic class II MHC peptides on alloimmune responses in vitro. Three peptides derived from a highly conserved region of the class II MHC α chains inhibited the rat mixed lymphocyte response (MLR) in a dose-dependent manner, with the human HLA-DQA1 peptide also inhibiting the human and mouse MLR. No effect was seen on mitogen-induced T-cell proliferation. HLA-DQA1 inhibited cytolytic T lymphocyte (CTL) generation in a dose-response fashion, with no reduction in preformed CTL killing, suggesting that the inhibitory effect is targeted at CD4 + T-cell function. Cell-cycle analysis by flow cytometry showed that restimulation of primed T cells in the presence of HLA-DQA1 resulted in increased apoptosis, whereas unstimulated cells were not affected. These data demonstrate that synthetic peptides derived from highly conserved regions of the class II MHC α chain can alter CD4 + T-lymphocyte alloimmune responses in vitro, and this effect is mediated by the induction of apoptosis in activated T cells. vested from naive LEW, WF, and BN rats, and the lymphocytes were isolated as described previously (9). The cells were then washed twice and resuspended in RPMI-1640 medium (BioWhittaker Inc., Walkersvi...

Abstract. Donor brain death has been considered a significant risk factor for both early and late organ allograft dysfunction. This central injury not only evokes an upsurge of catecholamines with resultant peripheral tissue vasoconstriction and ischemia but also promotes release of hormones and inflammatory mediators that may also affect the organs directly. One of the resultant influences of these events is the rapid upregulation of the acute-phase adhesion molecules, the selectins. These initiate leukocyte adhesion to vascular endothelium and trigger subsequent cellular and molecular changes in the compromised tissues. An established F344 3 LEW rat model of chronic rejection was used to examine (1) whether the initial inflammatory events that develop within kidney allografts from brain-dead donors could be normalized using a recombinant soluble form of P-selectin glycoprotein ligand and (2) whether amelioration of these early changes would alter the inexorable progression of chronic allograft rejection. Untreated living donor controls experienced unrelenting chronic rejection over time. This complex process was accelerated in brain-dead donor kidneys. Treatment with P-selectin glycoprotein ligand prevented the early inflammatory changes in the transplanted organs and their subsequent (200 d) functional and morphologic manifestations, particularly when the soluble ligand was administered both to the donor before organ removal and to the recipient after engraftment. This strategy of using a naturally occurring selectin ligand to prevent donor-associated chronic graft dysfunction may be of special clinical interest in cadaver donor transplantation.The clinical findings that kidneys from living unrelated donors perform as well over the short and long term as those from living related sources and that grafts from both donor groups are invariably superior to those from cadavers have emphasized that antigen-independent events, which include increased donor age and intercurrent disease, the state of brain death, and the period of ischemia, influence the quality of solid organs after transplantation and are important risk factors for their eventual outcome (1,2). Brain death, a central catastrophe unique to the cadaver organ donor, produces profound physiologic and structural derangements in the peripheral tissues of experimental animals both before and after placement in the recipient (3,4). These include massive upregulation of major histocompatibility antigens, adhesion molecules, cytokines, and other acute-phase proteins. Ischemia with systemic vasoconstriction is an important facet of brain death, secondary to the initial burst of catecholamines released into the circulation (5). The early injury and the associated reperfusion after transplantation evoke nonspecific inflammatory changes in the affected organs. However, additional factors may contribute to the peripheral effects of brain injury. Important changes in the dynamics of a series of hormones have been identified (6,7). Because brain death may also influ...

Of interest is our finding that IT injection of a short segment of WAG-derived MHC class I peptide induces active acquired tolerance similar to results obtained with the use of pure WF-derived peptide u-5 in the WF-to-ACI rat combination. It is noteworthy that we could not confirm the T helper (Th)1/Th2 paradigm in this model by initial cytokine analysis. Whether induction of tolerance by IT injection of allo-MHC peptides will have clinical usefulness must await results of similar studies in large animals. However, of major interest is the finding that a short segment of RT1.AU represents the tolerogenic

There is increasing evidence that ongoing T-cell recognition of alloantigen and activation are key mediators of chronic allograft rejection. The CD28-B7 pathway is unique among costimulatory pathways in that two alternate ligands for B7 exist: CD28 and CTLA4. Recently, it has been suggested that CTLA4 negative signaling may be required for induction of acquired tolerance in vivo. A strategy by which the T cell is targeted at the CD28 receptor rather than its ligands would theoretically allow the inhibitory functions of the CTLA4-B7-1/2 axis to remain intact. Using a rat-specific monoclonal antibody, we investigated the effect of targeting CD28 in a model of chronic rejection without the confounding variable of immunosuppression. We also used an acute cardiac allograft rejection model to investigate CD28 stimulation-based strategies to induce donor-specific tolerance. We demonstrated that anti-CD28 monoclonal antibody was as effective as CTLA4 immunoglobulin in protecting against chronic allograft vasculopathy. In addition, a short course of cyclosporine therapy synergized with either anti-CD28 monoclonal antibody or CTLA4 immunoglobulin, suggesting that it may be clinically relevant to combine low-dose calcineurin inhibitors with CTLA4 immunoglobulin or anti-B7 antibodies. Finally, we report on the potential mechanisms of action of targeting CD28 in vivo.