Local regulation and deletion of T cells to induce tolerance and establish long-term survival of high-risk corneal transplants

免疫排斥是同种异体角膜移植失败的最常见原因，尤其是当 受者有高危的血管化(HRV)角膜床。事实上，在每年接受治疗的40,000名患者中 对于视力康复，4-6000被认为是HRV，成功率很低(&lt；30%)。诱导 根据NEI的说法，这些移植的免疫耐受是一个关键的、未得到满足的医疗需求。 (https://nei.nih.gov/news/scienceadvances/advances/corneal_transplantation).在这篇文章中提出的研究 应用程序将开发一种独特的组合策略，从未在固体器官移植中测试过，以诱导长期... 高危带血管角膜移植(HRVCT)受者的长期耐受性 环磷酰胺(Cy)缺失抗原(Ag)特异性效应T细胞(目标1)，b)通过 标记的体内Treg扩增(AIM 2)和c)通过溴结构域的炎症基因表观遗传调控 抑制剂Beti(Aim 3)。我们的初步数据显示，使用简单、低剂量的环磷酰胺 CT后给予(Cy)可显著延长同种异体移植物的存活时间，其机制是消除效应T细胞和 角膜新生血管逐渐减少。我们新的针对肿瘤坏死因子受体的双途径策略 使用融合蛋白(TL1A-Ig)的超级家族25(TNFRSF25)诱导CD4+FoxP3+显著扩张 Tregs系统地和在眼房内。重要的是，这些树显示出非常强大的效应器/ 抑制性活动。初步数据表明，使用溴域抑制剂的表观遗传调节 (Beti)可在体内减少促炎细胞因子的产生，同时不干扰Treg 扩展和功能。令人兴奋的是，在这项提议的背景下，Beti显著减少了临床变化 局部给药时会对角膜造成伤害。我们假设高危角膜是检验这一假设的理想组织。 因为有机会局部调节免疫和炎症，我们将在体内应用优雅 用OUR研究、监测和分离抗原特异性T细胞和抗原特异性树突状细胞的模型 新培育的B6-Nur77GFPFoxP3RFP小鼠。本申请中提出的研究将提供一种独特的 开发和测试三步组合机制(CT-Cy、Treg和Beti) 建立同种异体移植耐受以维持永久HRVCT。值得注意的是，因为HRVCT行为 类似于其他带血管的器官移植，这里的工作可以应用于其他组织以诱导移植 接受。因此，我们将首先通过Treg操作在皮肤移植中测试我们的方法， 使用小鼠原位移植，然后继续测试我们的策略在人类身上的翻译应用 在免疫缺陷小鼠模型中使用人类同种异体皮肤移植，并利用新型人类试剂(MAb) PTX-35)来操纵人类Treg细胞。总而言之，这种独特而强大的方法将把不同的 诱导对角膜移植抗原耐受的免疫调节机制 失明和其他需要实体器官移植的情况。

Immunological rejection is the most common cause of corneal allo-transplant failure particularly when the recipient has a high risk vascularized (HRV) corneal bed. In fact, of the 40,000 patients per year receiving a CT for vision rehabilitation, 4-6,000 are considered HRV and the success rate is dismal (<30%). Induction of immune tolerance for these transplants is a critical and unmet medical need according to the NEI. (https://nei.nih.gov/news/scienceadvances/advances/corneal_transplantation). The studies proposed in this application will develop a unique combinatorial strategy never tested in a solid organ transplant to induce long- term tolerance in recipients of high-risk vascularized corneal transplants (HRVCT) through sequential: a) cyclophosphamide (Cy) deletion of antigen (Ag) specific effector T cells (Aim 1), b) immune suppression via marked in vivo Treg expansion (Aim 2) and c) inflammatory gene epigenetic regulation via bromodomain inhibitors BETi (Aim 3). Our preliminary data demonstrate that the use of brief, low dose cyclophosphamide (Cy) administered post-CT can dramatically prolong allograft survival by deleting effector T cells and diminishing corneal neovascularization. Our novel two-pathway strategy additionally targeting the TNF receptor super family 25 (TNFRSF25) using a fusion protein (TL1A-Ig) induces marked expansion of CD4+FoxP3+ Tregs systemically and within the ocular compartment. Importantly, these Tregs exhibit highly potent effector / suppressive activity. Preliminary data demonstrates that epigenetic regulation using a bromodomain inhibitor (BETi) can diminish pro-inflammatory cytokine production in vivo while simultaneously not interfering with Treg expansion and function. Excitingly in the context of this proposal, BETi significantly diminished clinical changes to the cornea when locally administered. We posit high-risk cornea is the ideal tissue to test this hypothesis because there is opportunity to locally modulate immunity and inflammation and we will apply elegant in vivo models to study, monitor and isolate antigen specific effector T cells and antigen specific Tregs using our newly developed B6-Nur77GFPFoxP3RFP mice. The studies proposed in this application will provide a unique opportunity to develop and test a 3-step combinatorial mechanism (CT-Cy, Treg, and BETi) for the establishment of allograft tolerance to maintain permanent HRVCT. Significantly, because HRVCT behave similarly to other vascularized organ transplants, work here can be applied to other tissues to induce transplant acceptance. Therefore, we will begin to test our approach through Treg manipulation in skin transplants first, using orthotopic mouse grafts and then continue by testing our strategy for translational application in humans by using human skin allografts in an immunodeficient mouse model and utilizing novel human reagents (mAb PTX-35) to manipulate human Treg cells. In total, this unique and powerful approach will bring together distinct immune regulatory mechanisms to induce tolerance to transplant antigens for the treatment of corneal blindness and other conditions requiring solid organ transplantation.