TRANSPLANT IMMUNOMODULATION BY ALLOCHIMERIC MOLECULES

我们寻求开发一种临床上可应用的策略，以在用同种异体嵌合I类MHC分子治疗的同种异体移植受者中诱导“真正的”移植耐受。我们在大鼠心脏同种异体移植模型中的初步发现为这一建议提供了基础。我们假设：（i）异源嵌合诱导的耐受性，作为由受体MHC I类分子展示的供体型免疫原性表位的间接呈递的函数，适用于遗传多样性的移植物受体;（ii）异源嵌合MHC I类分子的间接呈递诱导独特的调节性T细胞群的产生，所述调节性T细胞群维持并转移无慢性排斥的“真正”耐受性的状态。我们提出以下具体目标：1。确定诱导耐受的不同全嵌合体分子的要求。假设：显示供体型免疫原性表位的受体型异源嵌合I类分子诱导对供体型同种异体移植物的"真正”耐受。将通过PCR基因SOEing构建不同菌株组合中的多个异源嵌合分子，以确定I类MHC多态性区域中的关键免疫原性表位。将测试在免疫I类抗原上展示供体显性表位的全嵌合分子诱导“真正的”慢性无排斥耐受的能力。 2.通过间接呈递全嵌合[c%_JU]-RT1.Aa I类MHC分子来分析全免疫调节。假设：全嵌合体治疗通过间接途径偏离宿主对器官移植物的免疫应答。将进行对同种异体移植物存活至关重要的免疫原性/隐蔽自身表位的精细定位。通过采用已经用供体野生型或[cqh/U]-RT1.A a分子体外脉冲的宿主或供体型树突细胞，将解决nafve或致敏T细胞应答的间接同种异体嵌合偏离。调节性T细胞的全嵌合生成，在该模型中可能的关键致耐受性机制，将通过研究间接向CD 4 + T细胞呈递异源嵌合分子的未成熟肝树突状细胞在体外进行探索。将通过使用FIt 3L（一种造血生长因子）研究肝树突状细胞同种异体刺激能力/成熟度的体内刺激，以消除门静脉内同种异体嵌合体输注后间接诱导的耐受。 3.间接免疫反应诱导的独特调节性T细胞群的特征假设：由间接同种异体识别诱导的调节性T细胞协调耐受的获得和慢性排斥的消除。我们将进行调节性T细胞的体内和体外表型和功能表征。将在体外分析它们介导CD 8+细胞毒性T细胞抑制或直接抑制树突细胞的抗原呈递的能力。与细胞因子补充和/或抑制平行的Transwell实验将分析细胞-细胞接触的要求与特定细胞因子的要求。将通过分析（i）由偏离的宿主抗供体同种抗体反应诱导的血管“调节”，（ii）细胞保护分子的上调，和（iii）移植物浸润单核细胞的功能状态，研究慢性排斥反应的独特调节性T细胞协调抑制。>表演现场=

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===========================================