Achieving Xenograft Tolerance through Thymic Programming in Primates

项目1的总体目标是通过共移植在狒狒中诱导猪肾耐受 (Tx)血管化的胸腺到目前为止，我们已经诱导了供体特异性T细胞无反应性，并阻止了抗- 供体引发抗体，但尚未实现异种移植物的长期无免疫抑制存活 肾脏在上一个项目期间，我们确定了实现这一目标的两个主要障碍，并制定了战略 来克服它们。这些是：（i）由于潜伏的猪CMV激活而导致的异种胸腺肾（TK）早期丢失 （pCMV）;通过供体的剖腹产术消除pCMV来恢复长期移植物存活;以及 (ii)严重蛋白尿的发展;这个问题可以通过预防SMPDL来延迟（但不能预防）- 通过在围Tx期用利妥昔单抗处理的猪足细胞的3b依赖性破坏。我们也 证实足细胞上的猪CD 80上调与异种移植肾病相关。我们 初步数据表明，结合猪足细胞SMPDL- 3b和抑制CD 80活化的贝拉西普，大大减少了蛋白尿的发生， 存活期延长（90、93和>155天），体外供体特异性无反应性。最新的动物（现在） >155天）从猪胸腺移植物中产生新的迁移T细胞。根据这一数据， 团队（项目2，3和4），我们设计了创新的策略，旨在完全消除蛋白尿， 允许完全停止免疫抑制。在目标1中，我们将首先研究 持续性蛋白尿，以便制定适当的策略来消除它。根据我们的初步数据， 我们假设除了SMPDL-3b通路，物种间CD 47和SIRP-α的不相容性 可能促进先天免疫激活，最终导致足细胞激活，导致CD 80上调 对足细胞的影响。我们将在体外研究足细胞破坏的机制，并优化目前的治疗方法， 体内，使用hCD 47转基因GalT-KO猪TK供体。在目标2中，我们将继续努力实现异种移植 免疫耐受，试图通过重建宿主和供体限制性的 保护性T细胞免疫项目3中的研究证明了人类T细胞功能和稳态的局限性 在猪胸腺中发育。我们将讨论的假设，这些异常将得到纠正， 将受体胸腺上皮细胞（TEC）加入猪胸腺移植物中。我们最近成功地 移植了NHP TEC的杂交猪胸腺移植物。我们将评估使用这些混合胸腺的影响， 对狒狒免疫功能的影响在目标3中，我们将联合收割机与开发的持久混合嵌合体相结合 在项目2中。杂交胸腺移植和持久的混合嵌合体的结合应诱导两者的耐受 先天性和适应性免疫应答，包括NK和B细胞。这种组合方法还将优化 T细胞的功能，提供针对受体和供体移植物感染的保护性免疫， 保护抵抗残余供体反应性T细胞和自身免疫的T细胞。

SUMMARY The overall goal of Project 1 is to induce tolerance of porcine kidneys in baboons by co-transplantation (Tx) of vascularized thymus. To date, we have induced donor-specific T cell unresponsiveness and prevented anti- donor elicited antibodies, but have not yet achieved long-term, immunosuppression-free survival of xenograft kidneys. During the previous project period, we identified two major obstacles to this goal and developed strategies to overcome them. These are: (i) early loss of xeno-thymokidneys (TKs) due to activation of latent porcine CMV (pCMV); long-term graft survival was restored by elimination of pCMV through cesarean section of donors; and (ii) development of severe proteinuria; this problem could be delayed (but not prevented) by preventing SMPDL- 3b-dependent disruption of pig podocytes through treatment with Rituximab in the peri-Tx period. We also demonstrated upregulation of porcine CD80 on podocytes to be associated with xenograft nephropathy. Our preliminary data demonstrated that a combination therapy of Rituximab, which binds to porcine podocyte SMPDL- 3b and Belatacept, which inhibits CD80 activation, greatly minimized development of proteinuria and markedly prolonged survival (90, 93 and >155 days), with in vitro donor-specific unresponsiveness. The latest animal (now >155 days) developed new emigrant T cells from the porcine thymic graft. Based upon the data developed by this team (Projects 2, 3, and 4), we have designed innovative strategies that aim to completely eliminate proteinuria and permit total discontinuation of immunosuppression. In Aim 1, we will first study the mechanism responsible for persistent proteinuria in order to develop an appropriate strategy to eliminate it. On the basis of our preliminary data, we hypothesize that in addition to a SMPDL-3b pathway, CD47 and SIRP-alpha incompatibility between species may promote innate immune activation that culminates in podocyte activation, which causes upregulation of CD80 on podocytes in TKs. We will study mechanisms of podocyte destruction in vitro and optimize the current therapy in vivo, using hCD47 transgenic GalT-KO pig TK donors. In Aim 2, we will continue our efforts to achieve xenograft tolerance, attempting to improve the immune status of recipients by reconstitution of host- and donor-restricted protective T cell immunity. Studies in Project 3 demonstrated limitations in human T cell function and homeostasis following development in a pig thymus. We will address the hypothesis that these abnormalities will be corrected by adding recipient thymic epithelial cells (TECs) to the porcine thymus graft. We have recently succeeded in preparing hybrid thymic porcine grafts in which NHP TECs engrafted. We will assess the impact of using these hybrid thymi on immune function in baboons. In Aim 3, we will combine this strategy with durable mixed chimerism developed in Project 2. The combination of hybrid thymic grafting and durable mixed chimerism should induce tolerance of both innate and adaptive immune responses, including NK and B cells. The combined approach will also optimize functions of T cells, providing protective immunity against infections of both the recipient and the donor graft and of Tregs that protect both against residual donor-reactive T cells and against autoimmunity.