CARDIAC ALLOGRAFT VASCULOPATHY IN LARGE ANIMALS

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DESCRIPTION (provided by applicant): Studies conducted during the two previous funding periods using the now well-characterized, MHC inbred miniature swine model of CAV have clearly shown that both the direct and indirect pathways of allorecognition contribute to CAV and demonstrated that the induction of tolerance can abrogate the development of CAV. However, they also revealed that different tolerance protocols were not equally effective in preventing CAV and that tolerance protocols developed for kidney allografts were not necessarily successful with heart allografts. This suggests the vital need for a comprehensive, mechanistic analysis of tolerance protocols aimed specifically at preventing CAV in heart allograft recipients. Our overall goal is to develop a clinically relevant tolerance protocol in large animals that will specifically prolong heart allograft survival and prevent CAV. We plan to examine the efficacy of three very different and novel tolerance induction strategies. For each strategy we will employ mechanistic assays to compare 1) alloreactive T cell clone size (CFSE), 2) evidence for apoptosis (Annexin), 3) changes in cytokine profile (ELISPOT), 4) indirect allorecognition (peptide MLR assays), 5) Vbeta usage (CDR3 length spectratyping) and 6) regulatory T cell function (phenotypic and coculture suppression assays). By evaluating the efficacy of each protocol and elucidating differences in their mechanisms, we hope to achieve a strategy or combination of strategies that will achieve our goal. Our aims are to 1) determine whether treatment with immature host dendritic cells loaded with immunogenic donor MHC peptides will delay or eliminate the development of CAV, 2) determine whether cotransplantation of vascularized donor thymus can induce tolerance and prevent CAV, 3) determine whether achieving a stable state of multilineage mixed chimerism will induce long term tolerance to MHC disparate hearts and prevent CAV.