Achieving Xenograft Tolerance through Thymic Programming in Primates

项目1旨在通过带血管胸腺的联合移植(TX)来诱导狒狒对猪肾的耐受性。 我们已经诱导了供者特异性T细胞无反应，并防止了抗供者诱导的抗体(Abs)，但 尚未实现异种移植肾的长期存活。在前一个项目期间，我们确定了两个 这是实现这一目标的主要障碍，并制定了克服这些障碍的战略。它们是：(I)异种的早期损失。 由于潜伏的猪巨细胞病毒(PCMV)的激活导致胸腺肾(TKS)；移植物的长期存活通过以下方法恢复 通过剖腹产捐赠者消除pCMV；和(Ii)出现严重的蛋白尿；这 通过防止依赖SMPDL-3b的猪足细胞破坏，问题可以延迟(但不能防止) 围TX期应用利妥昔单抗治疗。当前解决的剩余主要障碍 建议是：1)由进行性蛋白尿导致的肾病综合征的最终发展；以及2)发展 由于蛋白尿中免疫球蛋白的丢失以及推测的恢复不足而引起的感染 猪胸腺移植物的保护性T细胞免疫。基于该团队开发的数据(项目2、3、 和4)，我们设计了创新的战略来克服这些障碍。在目标1中，我们将首先确定 利妥昔单抗治疗后持续蛋白尿的机制及有效治疗 战略。我们发现足细胞上SMPDL-3b的缺失和猪CD80的上调都是 与异种移植肾病有关。我们的初步数据表明，与猪结合的利妥昔单抗 足细胞SMPDL-3B和抑制CD80激活的belatacept都可以部分稳定蛋白丢失。这个 使用这种综合疗法的最新移植目前存活70天，肌酐水平正常，仅 最少的蛋白尿。我们的数据表明，CD47和SIRP-α在物种之间的不相容可能促进 先天免疫激活，最终导致足细胞激活。在目标1中，我们将通过以下方式克服蛋白尿 优化利妥昔单抗/CTLA-4免疫球蛋白治疗及hCD47转基因(TG)GalT-KO猪TK供体预防 狒狒巨噬细胞激活。在目标2中，我们将通过更快地重建 宿主和供体受限的保护性T细胞免疫。项目3中的研究表明人类T细胞的局限性 猪胸腺发育后的功能和动态平衡。我们将解决这样的假设：这些 通过将受体TEC添加到猪胸腺移植物中，结合混合 嵌合体。我们现在将优化混合胸腺移植物的结构，评估它们对免疫功能的影响 并将该方案与项目2中开发的策略相结合，以实现持久的混合异种嵌合体 (目标3)。混合胸腺移植和持久的混合嵌合体的结合将确保两者的耐受性。 先天和获得性免疫反应。这种结合的方法还将优化T细胞的功能，提供 对供体、移植物和受体的保护性免疫，以及对残留物的保护性免疫 供者反应性T细胞和自身免疫。

Project 1 aims to induce tolerance of porcine kidneys in baboons by co-transplantation (Tx) of vascularized thymus. We have induced donor-specific T cell unresponsiveness and prevented anti-donor elicited antibodies (Abs), but have not achieved long-term 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. The remaining major obstacles addressed in the current proposal are: 1) eventual development of nephrotic syndrome due to progressive proteinuria; and 2) development of infections due both to loss of immunoglobulins from proteinuria and presumably insufficient recovery of protective T cell immunity from porcine thymic grafts. Based upon the data developed by this team (Projects 2, 3, and 4), we have designed innovative strategies to overcome these obstacles. In Aim 1, we will first identify the mechanism responsible for continuing proteinuria despite Rituximab treatment and develop effective treatment strategies. We have found both loss of SMPDL-3b and upregulation of porcine CD80 on podocytes to be associated with xenograft nephropathy. Our preliminary data indicate that Rituximab, which binds to porcine podocyte SMPDL-3B and Belatacept, which inhibits CD80 activation, can both partially stabilize protein loss. The latest transplant with this combined therapy currently survives >70 days, with normal creatinine levels and only minimal proteinuria. Our data suggest that CD47 and SIRP-alpha incompatibility between species may promote innate immune activation that culminates in podocyte activation. In Aim 1, we will overcome proteinuria by optimizing Rituximab/CTLA-4 Ig therapy and using hCD47 transgenic (Tg) GalT-KO pig TK donors to prevent baboon macrophage activation. In Aim 2, we will achieve xenograft tolerance with more rapid 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 TEC to the porcine thymus graft combined with mixed chimerism. We will now optimize the construction of hybrid thymic grafts, assess their impact on immune function and combine this protocol with strategies developed in Project 2 to achieve durable mixed xenogeneic chimerism (Aim 3). The combination of hybrid thymic grafting and durable mixed chimerism will assure tolerance of both innate and adaptive immune responses. The combined approach will also optimize functions of T cells providing protective immunity against infections for the donor graft and recipient and of Tregs that protect against residual donor-reactive T cells and autoimmunity, respectively.