Mechanisms of acceptance of mouse renal allografts

这项建议解决了对确定性证据的巨大需求，以测试流行的 Foxp 3 +/T调节细胞对于接受生理学上的 相关的MHC不相容的器官移植。MHC不匹配的小鼠肾移植物 在某些菌株组合中，通常在没有特定治疗的情况下被接受， 接受来自同一捐赠者的其他组织。心脏移植没有这个 属性，表明肾脏的器官特异性特征。尽管示威游行 这种现象在30多年前，肾脏促进的机制 对其他组织的耐受性尚未确定。该项目将测试的作用， Foxp 3 + T调节细胞和吲哚胺2，3-双加氧酶（IDO）在原发性肝癌发病机制中的作用 接受小鼠肾移植，使用完全MHC不匹配的组合， 已知接受33-80%的移植物。将特别注意以下方面的相互作用： Foxp 3细胞与肾小管，因为Foxp 3细胞集中在这个网站。foxp 3+细胞 将被删除系统和在移植物中使用白喉毒素受体 (DTR)具有由foxp 3启动子表达的DTR的转基因小鼠 关于GFP再移植实验将测试移植物内T细胞是否足以 一旦移植物内Foxp 3细胞被去除，就排斥移植物。Foxp 3+细胞与 肾移植物中的其它T细胞、树突细胞和肾小管细胞将用免疫组织化学方法进行评估。 真实的时间共焦，多光子显微内镜在我们的实验室开发使用 在这些细胞中表达GFP或其它荧光标记蛋白的转基因小鼠。这 系统允许随着时间的推移重复观察同一移植物，实时视频记录， 三种不同细胞类型的描绘和细胞运动性的计算机分析， 交互.将分析肾同种异体移植物的活组织检查的细胞含量（Foxp 3+， Tbet+，IDO表达），以及在人中进行的标准Banff评分， 使用全载玻片成像确定哪些特征与后期移植物接受相关 技术和形态计量免疫组织化学。移植物内TGF的作用β 在Treg的产生中，活化、IL-6和IDO的产生以及CD 103的表达将 进行评估。浸润细胞的功能研究将与 脾脏对供体的ELISPOT直接和间接反应性以及Foxp 3+的抑制 细胞

This proposal addresses the great need for definitive evidence to test the prevalent dogma that Foxp3+/Tregulatory cells are essential for the acceptance of physiologically relevant MHC incompatible organ grafts. MHC-mismatched mouse renal allografts are often accepted without specific therapy in certain strain combinations, and can promote acceptance of other tissues from the same donor. Heart allografts do not have this property, suggesting an organ specific feature of the kidney. Despite demonstration of this phenomenon over 30 years ago, the mechanisms by which kidneys promote tolerance to other tissues have not been established. This project will test the role of Foxp3+ T regulatory cells and indoleamine 2,3-dioxygenase (IDO) in the pathogenesis of acceptance of mouse renal allografts, using full MHC mismatched combinations that are known to accept 33-80% of the grafts. Particular attention will be paid to interactions of Foxp3 cells with renal tubules, since Foxp3 cells concentrate in this site. Foxp3+ cells will be deleted systemically and in the graft with using the diphtheria toxin receptor (DTR) transgenic mouse with DTR expressed from the foxp3 promoter in conjunction with GFP. Retransplant experiments will test whether intragraft T cells are sufficient to reject the graft once intragraft Foxp3 cells are removed. Interaction of Foxp3+ cells with other T cells, dendritic cells and tubular cells in the renal grafts will be assessed with a real time confocal, multiphoton endomicroscope developed in our laboratory using transgenic mice expressing GFP or other fluorescent marker proteins in these cells. This system permits repeated observations of the same graft over time, live video recording, delineation of three different cell types and computer analysis of cell motility and interactions. Biopsies of renal allografts will be analyzed for cellular content (Foxp3+, Tbet+, IDO expression) in addition to standard Banff scoring as done in the human to determine which features correlate with later graft acceptance, using whole slide imaging technology and morphometric immunohistochemistry. The role of intragraft TGFβ activation, IL-6 and IDO production and CD103 expression in the generation of Treg will be assessed. Functional studies of infiltrating cells will be compared with those in the spleen for ELISPOT direct and indirect reactivity to the donor and inhibition by Foxp3+ cells.