Achieving Xenograft Tolerance through Thymic Programming in Primates

项目1的总体目标是通过共同移植来诱导狒狒对猪肾的耐受性 (Tx)。到目前为止，我们已经诱导了供者特异性T细胞无应答，并防止了抗- 供者诱导抗体，但尚未实现异种移植的长期无免疫抑制存活 肾脏。在前一个项目期间，我们确定了实现这一目标的两个主要障碍，并制定了策略 去克服它们。它们是：(I)由于猪CMV潜伏病毒的激活而导致异种胸腺肾(TKS)的早期丧失 (PCMV)；通过对捐赠者剖腹产消除pCMV，恢复了移植物的长期存活；以及 (Ii)出现严重蛋白尿；这一问题可以通过预防SMPDL来延缓(但不能预防)- 3B依赖的猪足细胞的破坏，通过处理利妥昔单抗在周围期。我们也 猪CD80在足细胞上的上调与异种移植肾病有关。我们的 初步数据显示，与猪足细胞SMPDL结合的利妥昔单抗的联合治疗- 3B和贝拉坦抑制CD80激活，极大地减少了蛋白尿的发展，并显著 延长存活期(90天、93天和155天)，在体外供者特异性无反应。最新的动物(现在 &gt；155天)从猪胸腺移植物中培养出新的移植物T细胞。根据由该机构开发的数据 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和 允许完全停止免疫抑制。在目标1中，我们将首先研究负责 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途径外，CD47和SIRP-α在物种之间是不相容的 可能促进先天免疫激活，最终导致足细胞激活，从而导致CD80上调 在TKS的足细胞上。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 活体，采用hCD47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 耐受性，试图通过重建受宿主和供体限制的受体来改善受者的免疫状态 保护性T细胞免疫。项目3中的研究表明人类T细胞功能和动态平衡的局限性 跟随着猪胸腺的发育。我们将解决这样的假设，即这些异常将通过以下方式得到纠正 将受体胸腺上皮细胞(TECs)添加到猪胸腺移植物中。我们最近成功地准备了 移植NHP TECs的杂交猪胸腺。我们将评估使用这些杂交胸腺的影响 对狒狒的免疫功能的影响。在目标3中，我们将把这一策略与开发的持久混合嵌合体结合起来 在项目2中，杂交胸腺移植和持久的混合嵌合体的组合应该会诱导两者的耐受 先天和获得性免疫反应，包括NK和B细胞。这种结合的方法还将优化 T细胞的功能，提供对受体和供体移植物感染的保护性免疫力 树突状细胞既能保护残留的供者反应性T细胞，又能保护自身免疫。

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.