Immune Tolerence to Transplanted Myoblasts

该项目的目标是使用同种异体成肌细胞治疗实验性Duchenne肌营养不良症 无持续免疫抑制的同种异体成肌细胞移植。这将是 要求诱导对同种异体成肌细胞以及由此产生的新抗原的免疫耐受 来自供体成肌细胞与宿主肌肉纤维(包括供体细胞表达的肌营养不良蛋白)的融合 携带野生型基因)。在小鼠中，MT已被证明对有效的耐受策略具有抵抗力，这些策略在 其他移植模型。出于这个原因，我们将利用新出现的策略，将mAbs结合起来对抗 高MR异构体的CD45蛋白酪氨酸磷酸酶(抗CD45RB)。短程抗CD45RB可以 诱导小鼠同种异体肾移植和胰岛移植耐受，并能促进移植肾的长期植入 猴子。此外，与抗CD40L抗体联合使用，抗CD45RB抗体可诱导小鼠长时间植入 高免疫原性菌株组合中的皮肤移植。在这里，我们将利用抗CD45RB来帮助诱导中枢 营养不良mdx小鼠对MT的耐受性。我们的初步数据显示，抗CD45Rb与 抗CD154和骨髓移植诱导稳定的高水平混合嵌合体(即共存 供者和受者的造血细胞)，并允许对移植的成肌细胞有强大的耐受性。在这 利用这种方法，我们获得了营养不良小鼠同种异体成肌细胞100天的存活率，且存活率高 肌营养不良蛋白的表达水平。在目标1中，我们将扩展和改进我们的方案，试图将毒性降至最低 实现混合嵌合体所需的条件化方案。具体的容忍度将通过以下方式展示 再次移植相同品系和不同品系(“第三方”)的成肌细胞。肌肉的改善 将对生理学进行评估。在目标2中，我们将确定抗CD45RB和BMT的机制 根据我们目前的理解，为这一模式中的宽容做出贡献。研究将包括：决心 针对同种异体抗原和包括抗肌营养不良蛋白在内的新抗原的特异性免疫反应性； 显示CD45异构体表达的转变；确定CTLA-4上调的作用；诱导 调节性T细胞和改变的炎性细胞因子；改变的体液免疫反应；以及 嵌合体和胸腺缺失。这些研究的结果将有助于制定一种 治疗这种一致致命的人类疾病。此外，对免疫反应的理解 将获得以更高水平和天然形式表达的抗肌营养不良蛋白。

The goal of this project is to treat experimental Duchenne Muscular Dystrophy using allogeneic myoblast transplantation without sustained immunosuppression through allogeneic myoblast transplantation (MT). This will require the induction of immunological tolerance towards allogeneic myoblasts, as well as to neoantigens resulting from the fusion of donor myoblasts with host muscle fibers (including dystrophin expressed by donor cells bearing the wild-type gene). MT in mice, has proved resistant to potent tolerogenic strategies that are successful in other transplantmodels. For this reason, we will utilize newly emerging strategies that incorporate mAbs against the higher Mr isoforms of the CD45 protein tyrosine phosphatase (anti-CD45RB). A short course of anti-CD45RB can induce tolerance to murine renal and islet allografts and can promote long-term engraftment of renal allografts in monkeys. Moreover, in combination with anti-CD40L, anti-CD45RB can induce prolonged engraftment of murine skin grafts in highly immunogenic strain combinations. Here, we will utilize anti-CD45RB to help induce central tolerance to MT in dystrophic mdx mice. Our preliminary data reveals that the combination of anti-CD45RB with anti-CD154 and bone marrow transplantation (BMT) induces stable high level mixed chimerism (i.e. co-existence of both donor and recipient hematopoietic cells) and allows robust tolerance toward transplanted myoblasts. In this regard, we obtain >100 day survival of allogeneic myoblasts in dystrophic mice using this approach, with high levels of dystrophin expression. In Aim 1, we will extend and refine our protocol in attempts to minimize toxicity of the conditioning regimen required to achieve mixed chimerism. Specific tolerance will be demonstrated by retransplantation of both same strain and different strain ("third-party") myoblasts. Improvement in muscle physiology will be assessed. In Aim 2, we will determine the mechanisms by which anti-CD45RB and BMT contribute towards tolerance in this model, based on our current understanding. Studies will include:determination of the specific immunological reactivity towards both allogeneic and neoantigens including dystrophin; demonstrating a shift in CD45 isoform expression; determining the role of CTLA-4 upregulation; induction of regulatory T cells and altered inflammatory cytokines; altered humoral immune responses; and the requirement for chimerism and thymic deletion. The results of these studies will contribute to development of an approach towards treating this uniformly fatal human disease. Moreover, an understanding of the immune response towards dystrophin, expressed at higher levels and in its native form, will be attained.