Preclinical Studies of Living Donor Islet-Kidney Allograft Tolerance

项目摘要：项目2是专门为制定耐受性诱导策略而设计的 活体供体复合胰岛肾治疗终末期糖尿病肾病 移植(TX)。许多有肾功能衰竭的糖尿病患者，特别是儿童，有潜在的捐赠者愿意 同时提供肾脏和胰岛供移植。不幸的是，通过部分获得的胰岛数量 从活体供体切除胰腺往往不足以在孤立的胰岛后实现胰岛素独立 TX.我们以前已经证明，移植预血管化胰岛的策略是 在大型动物模型中，复合IKs实现胰岛素独立所需的胰岛数量比FREE的Tx少得多， 非血管化的胰岛。通过完全同种异体肾移植，肾和胰岛功能均得到恢复 使用临床相关的免疫抑制方案对糖尿病狒狒进行肾切除。最近，我们 报道了使用一种新的、降低强度的方法成功地诱导恒河猴对iKs的耐受， “亲子”组合中的造血细胞TX方案。这些数据证明了 同种异体胰岛肾耐受诱导方法的原则。然而，尽管同种异体移植 达到了耐受性，嵌合体是短暂的，需要早期补充胰岛素 移植。我们假设预适应方案的成分和/或供体细胞来源 可能对胰岛功能有早期的负面影响。我们还假设，诱导耐受性 通过持久的混合嵌合体可能比瞬时嵌合体更有效地逆转 自身免疫与T1D相关，最近在NOD小鼠模型中证明了这一点。我们 因此，在此建议：1)以下列方式优化条件方案和动员细胞产品 预期在耐受诱导阶段(目标1)改善胰岛功能的保存，包括 用雷帕霉素和抗CD40单抗取代CyA。如果需要，使用纯化的HSC；2)添加体外 将供者特异性Tregs扩大到诱导方案，以促进建立持久的混合 嵌合体(目标2)。通过使用在世的亲属捐赠者，这种方法是可能的，人们可以从他们那里获得 移植前的供者特异性Tregs；以及3)研究效应T细胞和调节性T细胞在移植前的命运 IKs的接受者(目标3)，以确定耐受机制和确定潜在的生物标志物 预测耐受性与排斥性。这些研究将涉及对效应器的删除或扩展的评估 和使用在Core B中开发的高通量TCR测序方法的调节性T细胞。 由核心A提供，并与项目1合作，两个核心将通过核心C进行协调。

Project Summary: Project 2 is specifically designed toward the development of a tolerance induction strategy for curative treatment of end-stage diabetic nephropathy, using living donor composite Islet-Kidney (IK) transplantation (Tx). Many diabetic patients in renal failure, especially children, have potential donors willing to provide both a kidney and islets for transplantation. Unfortunately, the quantity of islets obtained through partial pancreatectomy from living donors is often insufficient to achieve insulin independence following isolated islet Tx. We have previously demonstrated that the strategy of transplanting pre-vascularized islets as part of composite IKs in large animal models requires far fewer islets to achieve insulin independence than Tx of free, non-vascularized islets. Both renal and islet function were restored by IK tx across fully allogeneic barriers in nephrectomized diabetic baboons using a clinically relevant immunosuppression protocol. More recently, we reported the successful induction of tolerance of IKs in rhesus monkeys using a novel, reduced intensity, hematopoietic cell Tx protocol in a “parent-to-offspring” combination. These data demonstrated “proof of principle” for the approach of induction of tolerance of allogeneic islet-kidneys. However, although allograft tolerance was achieved, chimerism was transient and insulin supplementation was required early post transplantation. We hypothesize that components of the conditioning regimen and/or donor cell source may have had an early negative impact on islet function. We also hypothesize that induction of tolerance through durable mixed chimerism may be more effective than transient chimerism in reversing autoimmunity associated with T1D, as has been demonstrated recently in an NOD mouse model. We therefore propose here to: 1) optimize the conditioning protocol and mobilized cell product in ways that are expected to improve preservation of islet function during the tolerance induction phase (Aim 1), including replacement of CyA with Rapamycin and anti-CD40 mAb and. If needed, use of purifed HSC; 2) add ex-vivo expanded donor-specific Tregs to the induction regimen, in order to facilitate the establishment of durable mixed chimerism (Aim 2). This approach is possible with the use of living-related donors, from whom one can generate donor-specific Tregs in advance of the transplant; and 3) study the fate of both effector and regulatory T cells in recipients of IKs (Aim 3), in order to determine the mechanism of tolerance and identify potential biomarkers predicting tolerance vs rejection. These studies will involve assessment of the deletion or expansion of effector and regulatory T cells using a high-throughput TCR sequencing approach developed in Core B. Islets will be provided by Core A and collaboration with Project 1 and both cores will be coordinated through Core C.