CARDIAC ALLOGRAFT VASCULOPATHY IN LARGE ANIMALS

描述（由申请人提供）： 在前两个资助期内，使用现已充分表征的MHC近交系小型猪CAV模型进行的研究清楚地表明，同种异体识别的直接和间接途径都有助于CAV，并证明诱导耐受性可以消除CAV的发展。然而，他们也揭示了不同的耐受方案在预防CAV方面并不同样有效，并且为肾脏同种异体移植开发的耐受方案不一定能成功用于心脏同种异体移植。这表明迫切需要一个全面的，专门针对防止心脏移植受者CAV的耐受性协议的机制分析。我们的总体目标是在大型动物中开发一种临床相关的耐受方案，专门延长心脏移植物存活并预防CAV。我们计划研究三种非常不同的新的耐受诱导策略的有效性。对于每种策略，我们将采用机制测定来比较1）同种异体反应性T细胞克隆大小（CFSE），2）细胞凋亡的证据（膜联蛋白），3）细胞因子谱的变化（ELISPOT），4）间接同种异体识别（肽MLR测定），5）V β使用（CDR 3长度谱分析）和6）调节性T细胞功能（表型和共培养抑制测定）。通过评估每种方案的有效性并阐明其机制的差异，我们希望实现一种策略或策略组合，以实现我们的目标。我们的目的是1）确定用负载有免疫原性供体MHC肽的未成熟宿主树突状细胞治疗是否会延迟或消除CAV的发展，2）确定血管化供体胸腺的共移植是否可以诱导耐受并预防CAV，3）确定获得稳定状态的多谱系混合嵌合体是否会诱导对MHC不同心脏的长期耐受并预防CAV。

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.