Mechanisms of Acceptance in Mouse Renal Allografts

描述（由申请方提供）：本提案解决了对Foxp 3 +/T调节细胞在各种手术耐受性环境下接受生理相关完全MHC不相容器官移植物中作用的明确证据的需求。在资助的最初一年（ARRA），MHC-不匹配的小鼠肾同种异体移植物的自发接受被证明精确地依赖于Foxp 3细胞，其使用白喉毒素（DT）短暂地消耗携带维持生命的DBA/2肾同种异体移植物的B6.Foxp3DTR小鼠中的循环和移植物内Foxp 3细胞。接受的移植物具有独特的Foxp 3细胞和树突状细胞（DC）的结节状血管周围浸润。foxp 3细胞的缺失引起结节聚集体的溶解和T细胞对皮质、小管和血管的广泛浸润，这是急性排斥反应的典型表现，并表现为7天内BUN的升高。在接受混合嵌合耐受诱导方案的MHC不匹配肾脏的患者移植物中也出现了类似的Foxp 3细胞结节聚集体。具体目标1将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，其中一些预期是Foxp 3依赖性的;具有缺失机制的那些预期是Foxp 3独立的。我们将在移植物中寻找不同的病理学特征，表明Treg活性，例如潜伏相关蛋白（LTP）和Foxp 3的共表达或吲哚胺2，3-双加氧酶（IDO）的DC表达，这可能用于区分移植物接受的foxp 3依赖性和foxp 3非依赖性形式。将通过ELISPOT测试移植物和淋巴器官中细胞的供体反应性和调节潜力，并在不同形式的接受和急性排斥之间进行比较。将通过移植肾切除术在接受了肾脏和皮肤移植物的受者中测试系统耐受性对移植物内浸润的依赖性，所述接受者具有剩余的天然肾脏。具体目标2将寻求确定自发移植物接受敏感性的巨大菌株差异的分子和细胞基础，比较B6受体中BALB/c（拒绝）和DBA/2（接受）肾脏（均为H-2d）。我们将测试DC功能、细胞因子/趋化因子/补体介质、次要组织相容性抗原和产生Foxp 3细胞的能力的菌株差异。后续实验试图改变确定的因素，以促进BALB/c肾的接受。具体目标3使用定制的真实的时间共聚焦、多光子显微内镜可视化Foxp 3+细胞与其他T细胞、受体和供体树突状细胞以及肾小管细胞在接受和排斥肾移植物中的相互作用。该系统允许随时间观察相同移植物、实时视频记录、描绘3-4种不同细胞类型和计算机辅助测量细胞运动性和相互作用。这些研究旨在揭示Foxp 3 + Treg在小鼠肾移植物接受中的意义和体内功能，并提供可能适用于临床肾移植的见解。 公共卫生相关性：小鼠肾移植系统提供了对人类病理过程的保真度、无需药物的丰富结果谱以及操纵和观察免疫应答组分以进行机制研究的能力的最佳组合。这些研究将提供对肾移植物中Treg生理学的见解，这将阐明其治疗相关性，并揭示区分病理性和有益的移植物内浸润的特征，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.