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杜氏肌营养不良症 通过同种异体成肌细胞移植（MT）进行无持续免疫抑制的移植。这将 需要诱导对同种异体成肌细胞的免疫耐受，以及对新抗原的免疫耐受， 来自供体成肌细胞与宿主肌纤维（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）。MT在小鼠中，已被证明对有效的致耐受性策略具有抗性，这些策略在小鼠中成功地 其他移植模型。出于这个原因，我们将利用新出现的策略，包括针对 CD45蛋白酪氨酸磷酸酶（抗CD45RB）的高Mr亚型。短期抗CD45RB治疗可以 诱导对小鼠肾和胰岛同种异体移植物耐受，并可促进肾同种异体移植物的长期植入， 猴子此外，与抗CD40 L组合，抗CD45 RB可诱导小鼠移植物的植入延长。 高免疫原性菌株组合中的皮肤移植物。在这里，我们将利用抗CD45RB来帮助诱导中枢神经系统的 在营养不良mdx小鼠中的MT耐受性。我们的初步数据显示，抗CD45RB与 抗CD154和骨髓移植（BMT）诱导稳定的高水平混合嵌合（即共存 供体和受体造血细胞），并允许对移植的成肌细胞的强耐受性。在这 关于这一点，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率， 抗肌萎缩蛋白表达水平。在目标1中，我们将扩展和改进我们的方案，以尽量减少毒性 达到混合嵌合体所需的预处理方案。将通过以下方式证明特定公差： 相同品系和不同品系（"第三方"）成肌细胞的再移植。改善肌肉 将评估生理学。在目标2中，我们将确定抗CD45RB和BMT的机制， 基于我们目前的理解，在这个模型中为宽容做出贡献。研究将包括： 对同种异体抗原和新抗原（包括抗肌萎缩蛋白）的特异性免疫反应性; 证明了CD45亚型表达的变化;确定了CTLA-4上调的作用; 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 嵌合体和胸腺缺失。这些研究的结果将有助于制定一种方法， 治疗这种致命的人类疾病此外，了解免疫反应， 将获得以更高水平和天然形式表达的肌营养不良蛋白。

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.