We seek to develop a clinically applicable strategy to induce """"""""true"""""""" transplantation tolerance treated with allochimeric Class I MHC molecules. Our novel findings in a rat cardiac allograft model provide the foundation of this proposal. We hypothesize: (i) allochimeric-induced tolerance is applicable to genetically diverse allograft recipients; (ii) allochimeric molecules modulate the immune response by indirect presentation; (iii) aIlochimeric molecules generate unique regulatory T cells with distinctive functional and TcR allospecificities. We propose: 1. To Define the Requirements of Different AIIochimeric Molecules to Induce Tolerance. Multiple allochimeric molecules, in different strain combinations, will be constructed 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 their ability to induce """"""""true"""""""" chronic rejection-free tolerance. 2. To Analyze AIIoimmune Modulation by Indirect Presentation of Allochimeric [alpha1h I/u]-RT1.Aa Class I MHC Molecules. Fine mapping of immunogenic/cryptic self-epitopes that are critical for allograft tolerance will be performed. Indirect allochimeric deviation of 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 alpha1h 1/u]-RT1.Aa molecules. AIIochimeric protein labeling and detection will localize the site and cell-type involved in allochimeric processing and presentation. Requirement of 'immature vs mature' hepatic DC for allochimeric tolerance induction will be investigated in vivo by functional stimulation of DC maturity and allostimulatory capacity using FIt3L, a hematopoietic growth factor, to potentially abrogate indirectly induced tolerance following intra-portal allochimeric delivery. 3. To Characterize the Unique Population of Regulatory T Cells Induced by Indirect Allorecognition. We will perform in vivo and ex vivo phenotypic and functional characterization of regulatory T cells. Allochimeric 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. Third, in vivo analysis of chronic rejection coupled with CDR3 spectrotyping, immunoscope and sequence analysis of allochimeric-induced clonally-restricted regulatory T cells will define unique T cell functional specificities and the influence of allochimeric sequence on the allospecific TcR repertoire.
