Mechanisms of Acceptance in Mouse Renal Allografts

描述(由申请人提供)：这项提案针对Foxp3+/T调节细胞在各种手术耐受环境下接受生理上相关的完全MHC不相容器官移植中所扮演的角色提出了明确的证据。在资助的最初一年(ARRA)，MHC不匹配的小鼠肾移植的自发接受被证明非常依赖于使用白喉毒素(DT)的Foxp3细胞来瞬时耗尽B6Foxp3细胞和移植物内的Foxp3细胞。Foxp3DTR小鼠携带维持DBA/2肾移植的生命。移植血管周围有明显的结节状Foxp3细胞和树突状细胞(DC)浸润。Foxp3细胞的缺失导致结节聚集溶解，T细胞广泛侵入皮质、小管和血管，这是急性排斥反应的典型表现，表现为7天后BUN升高。在接受了混合嵌合耐受诱导方案的MHC不匹配肾脏的患者的移植肾中，也出现了类似的Foxp3细胞结节聚集体。具体目标1将把这些观察结果扩展到其他形式的诱导耐受、混合嵌合体和新生儿耐受，其中一些预计是Foxp3依赖的；那些具有缺失机制的应该是Foxp3不依赖的。我们将在移植物中寻找独特的病理特征，表明Treg活性，例如潜伏期相关蛋白(LAP)和Foxp3的共同表达或吲哚胺2，3-双加氧酶(IDO)的DC表达，这些可能被用来区分Foxp3依赖和Foxp3非依赖的移植物接受形式。ELISPOT将测试移植物和淋巴器官中细胞的供体反应性和调节潜力，并比较不同形式的接受和急性排斥反应。对于接受肾移植的受者，移植肾和皮肤移植物中剩余的天然肾脏，将通过移植物肾切除来测试全身耐受性对移植物内浸润物的依赖性。具体目标2将通过比较B6受者的BALB/c(排斥)和DBA/2(接受)肾脏(均为H-2d)，试图确定B6受体BALB/c(排斥)和DBA/2(接受)肾脏在自发移植接受易感性方面存在显著差异的分子和细胞基础。我们将测试DC功能、细胞因子/趋化因子/补体介体、次要组织相容性抗原和产生Foxp3细胞的能力的菌株差异。后续实验试图改变已确定的因素，以促进BALB/c肾脏的接受。具体目的3使用定制的实时共聚焦多光子内窥镜观察Foxp3+细胞与其他T细胞、受者和供者树突状细胞以及肾小管细胞在接受和排斥肾移植过程中的相互作用。该系统允许随着时间的推移观察相同的移植物，实时视频记录，描绘3-4种不同的细胞类型，以及计算机辅助测量细胞的运动性和相互作用。这些研究旨在揭示Foxp3+Treg在小鼠肾移植接受中的意义和体内功能，并为临床肾移植提供潜在的可应用的见解。 与公共卫生相关：小鼠肾脏移植系统提供了与人类病理过程的保真度、丰富的不用药结果谱以及为机制研究操纵和观察免疫反应组件的能力的最佳组合。这些研究将提供对肾移植Treg生理学的见解，阐明它们的治疗相关性，并揭示区分病理和有益的移植物内浸润物的特征，这在临床移植和耐受诱导研究中具有相当重要的意义。

DESCRIPTION (provided by applicant): This proposal addresses the need for definitive evidence on the role of Foxp3+/T regulatory cells in the acceptance of physiologically relevant full MHC incompatible organ grafts in various settings of operational tolerance. In the initial year of funding (ARRA), spontaneous acceptance of MHC-mismatched mouse renal allografts was shown to depend exquisitely on Foxp3 cells using diphtheria toxin (DT) to transiently deplete circulating and intragraft Foxp3 cells in B6.Foxp3DTR mice bearing life sustaining DBA/2 renal allografts. The accepted grafts had distinctive nodular perivascular infiltrates of Foxp3 cells and dendritic cells (DC). Deletion of foxp3 cells caused dissolution of the nodular aggregates and widespread infiltration of the cortex, tubules and blood vessels by T cells, typical of acute rejection, and manifested by a rise in BUN over 7 days. Similar nodular aggregates of Foxp3 cells occur in grafts of patients who have accepted MHC mismatched kidneys on mixed chimerism tolerance induction protocols. Specific aim 1 will extend these observations to other forms of induced tolerance, mixed chimerism and neonatal tolerance, some of which are expected to be Foxp3 dependent; those with deletional mechanisms are expected to be Foxp3 independent. We will seek distinctive pathologic features in the graft, indicating Treg activity, such as the co-expression of latency associated protein (LAP) and Foxp3 or DC expression of indoleamine 2,3-dioxygenase (IDO), that might be used to distinguish foxp3 dependent from foxp3 independent forms of graft acceptance. The donor reactivity and regulatory potential of the cells in grafts and in lymphoid organs will be tested by ELISPOT and compared between the different forms of acceptance and acute rejection. The dependence of systemic tolerance on the intragraft infiltrates will be tested by graft nephrectomy in recipients with accepted kidney and skin grafts that have a remaining native kidney. Specific aim 2 will seek to identify the molecular and cellular basis for the dramatic strain differences in susceptibility to spontaneous graft acceptance, comparing BALB/c (rejected) and DBA/2 (accepted) kidneys (both H-2d) in B6 recipients. We will test for strain differences in DC function, cytokine/chemokine/complement mediators, minor histocompatibility antigens and ability to generate Foxp3 cells. Follow-up experiments seek to alter identified factors to promote acceptance of BALB/c kidneys. Specific aim 3 visualize interaction of Foxp3+ cells with other T cells, recipient and donor dendritic cells and tubular cells in accepting and rejecting renal grafts using a custom real time confocal, multiphoton endomicroscope. This system permits observations of the same graft over time, live video recording, delineation of 3-4 different cell types and computer assisted measurement of cell motility and interactions. These studies are designed to reveal the significance and in vivo function of Foxp3+ Treg in murine renal allograft acceptance and provide insights potentially applicable in clinical renal transplantation. PUBLIC HEALTH RELEVANCE: The mouse kidney transplantation system offers an optimal combination of fidelity to human pathological processes, a rich spectrum of outcomes without drugs, and the ability to manipulate and observe the components of the immune response for mechanistic studies. These studies will provide insights into Treg physiology in renal allografts that will clarify their therapeutic relevance and reveal features that distinguish pathologic from beneficial intragraft infiltrates, of considerable importance in clinical transplantation and tolerance induction studies.