Preclinical Studies of Living Donor Islet-Kidney Allograft Tolerance

项目概要：项目2是专门为开发耐受性诱导策略而设计的 使用活体供体复合胰岛-肾（IK）治疗终末期糖尿病肾病 移植（Tx）。许多患有肾衰竭的糖尿病患者，尤其是儿童，都有潜在的捐赠者愿意为他们的肾脏提供血液。 为移植提供肾脏和胰岛。不幸的是，通过局部移植获得的胰岛数量 活体供者的胰腺切除术通常不足以在分离胰岛后实现胰岛素依赖性， TX.我们以前已经证明，移植预血管化胰岛作为治疗的一部分， 大型动物模型中的复合IK比游离的Tx需要少得多的胰岛来实现胰岛素非依赖性， 非血管化胰岛。IK tx通过完全同种异体的屏障恢复了肾脏和胰岛功能， 肾切除糖尿病狒狒使用临床相关的免疫抑制方案。最近，我们 报道了使用一种新的，降低强度的， 造血细胞Tx方案在"亲本到后代"组合中的应用。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，尽管同种异体移植物 达到耐受，嵌合体是短暂的，需要在术后早期补充胰岛素。 移植我们假设预处理方案和/或供体细胞来源的成分 可能对胰岛功能有早期的负面影响。我们还假设耐受的诱导 通过持久的混合嵌合体可能比短暂的嵌合体更有效地逆转 与T1D相关的自身免疫，如最近在NOD小鼠模型中所证明的。我们 因此，在此建议：1）以如下方式优化调节方案和动员的细胞产物： 预期在耐受诱导期（目标1）改善胰岛功能的保护，包括 用雷帕霉素和抗CD40 mAb替代CyA，以及.如果需要，使用纯化的HSC; 2）添加离体HSC 将供体特异性THBE扩大到诱导方案，以促进建立持久的混合 嵌合体（目的2）。这种方法是可能的，与使用生活相关的捐助者，从谁可以产生 供体特异性T淋巴细胞;和3）研究效应和调节性T细胞在移植前的命运。 IKs受体（目的3），以确定耐受机制并识别潜在的生物标志物 预测耐受性和排斥性。这些研究将涉及评估效应子的缺失或扩增。 和调节性T细胞，使用Core B中开发的高通量TCR测序方法。小岛将是 由核心A提供，与项目1合作，两个核心将通过核心C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.