CARDIAC ALLOGRAFT VASCULOPATHY IN LARGE ANIMALS

描述(由申请人提供)： 在前两个资助期，使用现已具有良好特征的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的发生，并证明诱导耐受可以消除CAV的发展。然而，他们也揭示了不同的耐受方案在预防CAV方面并不是同样有效，并且为同种异体肾移植开发的耐受方案在同种异体心脏移植中并不一定成功。这表明，迫切需要对耐受性方案进行全面的、机械性的分析，特别是为了预防心脏移植受者的CAV。我们的总体目标是在大型动物身上开发一种临床相关的耐受方案，以明确延长同种异体心脏移植的存活时间并预防CAV。我们计划检验三种截然不同且新颖的耐受诱导策略的有效性。对于每种策略，我们将使用机械分析来比较1)异基因反应T细胞克隆大小(CFSE)，2)凋亡证据(Annexin)，3)细胞因子谱变化(ELISPOT)，4)间接同种异体识别(多肽MLR分析)，5)Vbeta使用(CDR3长度谱分析)和6)调节性T细胞功能(表型和共培养抑制分析)。通过评估每项议定书的效力并阐明其机制的差异，我们希望实现一项战略或战略组合，以实现我们的目标。我们的目的是1)确定未成熟的宿主树突状细胞负载免疫原性供体MHC多肽治疗是否会延缓或消除CAV的发展；2)确定带血管的供体胸腺共移植是否可以诱导耐受并预防CAV；3)确定达到稳定的多系混合嵌合体状态是否会诱导对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.