Immune Tolerence to Transplanted Myoblasts

描述（由申请人提供）：该项目的目标是通过同种异体成肌细胞移植（MT）治疗实验性杜氏肌营养不良症，而不需要持续的免疫抑制。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。小鼠MT已被证明对其他移植模型中成功的强效耐受性策略具有抗性。因此，我们将利用新出现的策略，结合针对CD45蛋白酪氨酸磷酸酶（抗cd45rb）的高Mr亚型的单克隆抗体。短疗程的抗cd45rb可诱导对小鼠肾和胰岛移植的耐受，并可促进猴子肾移植的长期植入。此外，与抗cd40l联合，抗cd45rb可以诱导小鼠皮肤移植长时间植入高免疫原性菌株组合。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。我们的初步数据显示，抗cd45rb和抗cd154与骨髓移植（BMT）的结合可诱导稳定的高水平混合嵌合（即供体和受体造血细胞共存），并对移植的成肌细胞具有强大的耐受性。在这方面，我们使用这种方法在营养不良小鼠中获得了100天的同种异体成肌细胞存活，并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，以尽量减少实现混合嵌合所需的调节方案的毒性。特异性耐受性将通过同一株和不同株（“第三方”）成肌细胞的再移植来证明。将评估肌肉生理的改善情况。在Aim 2中，我们将根据我们目前的理解，确定抗cd45rb和BMT在该模型中促进耐受性的机制。研究将包括：对异体抗原和新抗原（包括肌营养不良蛋白）的特异性免疫反应性的测定；证明CD45异构体表达发生了变化；确定CTLA-4上调的作用；调节性T细胞的诱导和炎性细胞因子的改变；体液免疫反应改变；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统一致命的人类疾病的方法。此外，对抗肌营养不良蛋白的免疫反应的理解，表达在更高的水平和在其天然形式，将获得。

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.