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

免疫排斥反应是同种异体角膜移植失败的最常见原因，特别是当 受体具有高风险血管化（HRV）角膜床。事实上，在每年接受治疗的40，000名患者中， 视力康复的CT，4- 6000被认为是HRV，成功率很低（<30%）。诱导 根据NEI，对这些移植的免疫耐受是一个关键的和未满足的医疗需求。 （https：//nei.nih.gov/news/scienceparces/advances/corneal_transplantation）。本报告中提出的研究 应用将开发一种独特的组合策略，从未在实体器官移植中进行过测试，以诱导长期的 高危血管化角膜移植（HRVCT）接受者的长期耐受性：a） 环磷酰胺（Cy）缺失抗原（Ag）特异性效应T细胞（Aim 1），B）免疫抑制， 显著的体内Treg扩增（Aim 2）和c）通过布罗莫结构域的炎性基因表观遗传调节 抑制剂BETi（Aim 3）。我们的初步数据表明，使用短期，低剂量环磷酰胺， (Cy)CT后给药可通过删除效应T细胞显著延长同种异体移植物存活， 减少角膜新生血管形成。我们的新的双途径策略，另外靶向TNF受体 使用融合蛋白（TL 1A-Ig）的超家族25（TNFRSF 25）诱导CD 4 + FoxP 3+的显著扩增 全身性和眼室内的过敏。重要的是，这些TdR表现出高度有效的效应子/ 抑制活性。初步数据表明，使用布罗莫结构域抑制剂的表观遗传调控 （BETi）可以减少体内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，在这个提议的背景下，BETi显著减少了临床变化 局部给药时，角膜。我们认为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， 模型研究，监测和分离抗原特异性效应T细胞和抗原特异性T细胞，使用我们的 新开发的B6-Nur 77 GFPFoxP 3RFP小鼠。本申请中提出的研究将提供一个独特的 有机会开发和测试3步组合机制（CT-Cy，Treg和BETi）， 建立同种异体移植物耐受以维持永久性HRVCT。值得注意的是，由于HRVCT表现为 类似于其他血管化器官移植，这里的工作可以应用于其他组织以诱导移植 验收因此，我们将开始首先通过皮肤移植中的Treg操纵来测试我们的方法， 使用原位小鼠移植物，然后继续测试我们在人类中的翻译应用策略 通过在免疫缺陷小鼠模型中使用人皮肤同种异体移植物并利用新型人试剂（mAb PTX-35）来操纵人Treg细胞。总的来说，这种独特而强大的方法将汇集不同的 诱导对移植抗原耐受以治疗角膜炎的免疫调节机制 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.