EXCEED THE SPACE PROVIDED We seek to develop a clinically applicable strategy to induce a 'true' transplantation tolerance in allograft recipients treated with allochimeric Class I MHC molecules. Our preliminary findings in a rat cardiac allograft model provide the foundation of this proposal. We hypothesize that: (i) allochimeric-induced tolerance, as a function of indirect presentation of donor-type immunogenic epitopes displayed by recipient MHC Class I molecules, is applicable to genetically diverse graft recipients; (ii) indirect presentation of allochimeric MHC Class I molecules induces generation of a unique population of regulatory T cells that maintain and transfer a state of chronic rejection-free 'true' tolerance. We propose the following specific aims: 1. To Define the Requirements of Different AIIochimeric Molecules to Induce Tolerance. Hypothesis: Recipient-type allochimeric class I molecules that exhibit donor-type immunogenic epitopes induce '_true' tolerance to donor-type allografts. Multiple allochimeric molecules, in different strain combinations, will be constructed by PCR gene SOEing, to define critical immunogenic epitopes in the polymorphic regions of class I MHC. AIIochimeric molecules that display donor-dominant epitopes on recipient-class I antigens will be tested for the ability to induce a 'true' chronic rejection-free tolerance. 2. To Analyze AIIoimmune Modulation by Indirect Presentation of AIIochimeric [c%_JU]-RT1.Aa Class I MHC Molecules. Hypothesis: AIIochimeric therapy deviates host immune responses to organ allografts via indirect pathway. Fine mapping of immunogenic/cryptic self-epitopes that are critical for allograft survival will be performed. Indirect allochimeric deviation of nafve or primed T cell responses will be addressed by employing host or donor-type dendritic cells that have been pulsed in vitro with donor wild-type or [cqh_/U]-RTl.A a molecules. AIIochimeric generation of regulatory T cells, a possible key tolerogenic mechanism in this model, will be probed in vitro by studying immature hepatic dendritic cells that indirectly present allochimeric molecules to CD4+ T cells. In vivo stimulation of hepatic dendritic cell allostimulatory capacity/maturity will be investigated by using FIt3L, a heamatopoietic growth factor, to abrogate indirectly induced tolerance after intra-portal allochimeric infusion. 3. To Characterize the Unique Population of Regulatory T Cells Induced by Indirect AIIorecoflnition. Hypothesis: Regulatory T cells that are induced by indirect allorecognition orchestrate the acquisition of tolerance and abrogation of chronic rejection. We will perform in vivo and ex vivo phenotypic and functional characterization of regulatory T cells. Their ability to mediate CD8+ cytotoxic T cells inhibition or directly suppress antigen presentation by dendritic cells will be analyzed in vitro. Transwell experiments in parallel with cytokine supplementation and/or inhibition, will analyze the requirement of cell-cell contact vs. specific cytokine requirement. The unique regulatory T cell orchestrated inhibition of chronic rejection will be investigated by analyzing (i) vascular 'accommodation' induced by deviated host anti-donor alloantibody responses, (ii) upregulation of cytoprotective molecules, and (iii) functional status of graft-infiltrating mononuclear cells. > PERFORMANCE SITE ========================================Section End===========================================
