Immune Tolerence to Transplanted Myoblasts

描述（由申请人提供）：本项目的目的是通过同种异体成肌细胞移植（MT），在不持续免疫抑制的情况下，使用同种异体成肌细胞移植治疗实验性杜氏肌营养不良症。这将需要诱导对同种异体成肌细胞以及对由供体成肌细胞与宿主肌纤维融合产生的新抗原（包括由携带野生型基因的供体细胞表达的肌营养不良蛋白）的免疫耐受。MT在小鼠中的应用已被证明对在其他移植模型中成功的强效致耐受性策略具有抗性。出于这个原因，我们将利用新出现的策略，纳入针对CD45蛋白酪氨酸磷酸酶（抗CD45RB）的高Mr亚型的mAb。短期抗CD45 RB治疗可诱导对小鼠肾和胰岛同种异体移植物的耐受，并可促进猴肾同种异体移植物的长期植入。此外，在高免疫原性菌株组合中，抗CD45 RB与抗CD40 L组合可诱导小鼠皮肤移植物的长期植入。在这里，我们将利用抗CD45RB来帮助诱导营养不良mdx小鼠对MT的中枢耐受。我们的初步数据表明，抗CD45 RB与抗CD154和骨髓移植（BMT）的组合诱导稳定的高水平混合嵌合体（即供体和受体造血细胞共存），并允许对移植的成肌细胞的强大耐受性。在这方面，我们使用这种方法获得了营养不良小鼠中同种异体成肌细胞的> 100天存活，具有高水平的肌营养不良蛋白表达。在目标1中，我们将扩展和完善我们的方案，以尽量减少达到混合嵌合体所需的预处理方案的毒性。将通过再次移植相同品系和不同品系（"第三方"）成肌细胞来证明特异性耐受性。将评估肌肉生理学的改善。在目标2中，我们将根据我们目前的理解，确定抗CD45RB和BMT在该模型中有助于耐受的机制。研究将包括：确定对同种异体抗原和新抗原（包括肌营养不良蛋白）的特异性免疫反应性;证明CD45同种型表达的转变;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;改变的体液免疫应答;以及嵌合和胸腺缺失的要求。这些研究的结果将有助于开发一种治疗这种致命的人类疾病的方法。此外，对抗肌萎缩蛋白的免疫反应的理解，在更高的水平和其天然形式表达，将达到。

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.