Immune Tolerence to Transplanted Myoblasts

描述(申请人提供)：该项目的目标是通过同种异体成肌细胞移植(MT)治疗实验性Duchenne肌营养不良症，而不需要持续免疫抑制。这将需要诱导对同种异体成肌细胞的免疫耐受，以及对供体成肌细胞与宿主肌纤维融合产生的新抗原的免疫耐受(包括携带野生型基因的供体细胞表达的抗肌营养不良蛋白)。在小鼠中，MT已被证明对在其他移植模型中成功的有效的耐受策略具有抵抗力。为此，我们将利用新出现的策略，结合抗CD45蛋白酪氨酸磷酸酶(抗CD45RB)的较高MR异构体的单抗。短程抗CD45RB可诱导小鼠同种异体肾和胰岛移植物的耐受，并可促进猴移植肾的长期植入。此外，与抗CD40L抗体联合使用，抗CD45RB抗体可在高免疫原性品系组合中诱导小鼠皮肤移植物的长时间植入。在这里，我们将利用抗CD45RB来帮助诱导营养不良的MDX小鼠对MT的中枢耐受。我们的初步数据显示，联合应用抗CD45RB、抗CD154和骨髓移植(BMT)可诱导稳定的高水平混合嵌合体(即供受者造血细胞共存)，并允许对移植的成肌细胞有很强的耐受性。在这方面，我们使用这种方法获得了营养不良小鼠同种异体成肌细胞100天的存活，并获得了高水平的肌营养不良蛋白表达。在目标1中，我们将扩展和改进我们的方案，试图将实现混合嵌合体所需的条件化方案的毒性降至最低。通过再次移植相同菌株和不同菌株(“第三方”)的成肌细胞，将显示出特定的耐受性。将对肌肉生理学的改善进行评估。在目标2中，我们将根据我们目前的理解，在这个模型中确定抗CD45RB和BMT促进耐受的机制。研究将包括：测定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亚型表达的变化；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；改变体液免疫反应；以及嵌合体和胸腺缺失的要求。这些研究的结果将有助于开发一种治疗这种一致致命的人类疾病的方法。此外，还将了解对dystrophin的免疫反应，该免疫反应在较高水平和以其天然形式表达。

DESCRIPTION (provided by applicant): 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 transplant models. 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.