Organ transplantation is the therapy of choice for end stage organ failure. The major goal in transplantation research is the induction of tolerance to major histocompatibility complex (MHC) antigens. The thymus plays the major role in development of self tolerance, but its role in acquired tolerance to alloantigen is unknown. Several investigators have recently shown that intrathymic injection of donor cells into adult animals can induce a state of long term specific unresponsiveness to tissue and organ allografts. The availability of sequence data for the MHC genes has made it possible to synthesize polymorphic peptides and utilize them to study the mechanisms of allo-recognition. Intrathymic injection of polymorphic MHC allopeptides reproduces the same effects observed with allogeneic cells, suggesting that thymocytes recognize processed MHC molecules. The main focus of this proposal is to use synthetic MHC peptides to study the mechanisms of acquired intrathymic tolerance in the rat model of vascularized organ allografts. Specifically, the plan will be to: 1. Investigate the mechanisms of thymic recognition of MHC allopeptides. This will be accomplished by studying which thymic antigen-presenting cells, bone marrow-derived macrophages/dendritic cells or epithelial cells, bind and present MHC allopeptides to T cells. Methods will include binding studies of biotinylated MHC peptides, determining the requirement for processing, and blocking with specific anti-class I and class II MHC monoclonal antibodies. The response of T cells to specific MHC epitopes after priming by immunization with the peptides, and by an allograft, will be determined. 2. Investigate the role of thymic regulatory cells. The rat renal and cardiac transplant model will be used. The presence of regulatory mechanisms will be investigated by performing thymectomies at different time intervals after transplantation, performing adoptive transfer experiments, and studying graft infiltrating cells and cytokines by immunohistology. 3. Investigate whether thymic recognition of MHC peptides anergizes or deletes activated T cell clones. This will involve induction of immune unresponsiveness to specific MHC epitopes by injecting individual MHC peptides into the thymus, and using in vitro proliferation and precursor frequency assays to study the functional characteristics of T cells from tolerized animals. Anergy will be studied by testing for reversal of T cell unresponsiveness by cytokines. Clonal deletion will be investigated by analyzing T cell receptor Vbeta repertoire. The long term objectives are to develop novel approaches to study the mechanisms of transplantation unresponsiveness, including new strategies to induce specific transplantation tolerance.
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