Identification of the immunomodulatory mechanisms of nanocarrier-enhanced costimulation blockade in an allogeneic portal vein islet transplantation model

项目总结 由于T1D引起的血糖调节失调会导致两种短期后果，如低血糖 致伤和死亡，以及包括截肢、失明、肾功能衰竭和 神经病。胰岛移植有可能为所有T1D患者提供生物血糖调节， 有效地治愈了这种疾病。然而，这种有希望的疗法受到无效要求的困扰。 免疫抑制疗法。目前，只有50%到70%的移植物在移植后5年仍能存活， 很大程度上是由于免疫抑制治疗引起的毒性和未能完全保护移植物。目前，一个 昂贵的免疫抑制药物鸡尾酒被用来提供广泛的免疫力，这是一次罕见的成功尝试 副作用缓解的覆盖范围。因此，需要更简单、更低成本和更有效的替代方案 这些免疫抑制疗法可以维持胰岛移植的存活率，而不会产生非靶向副作用。 该提案的目标是机械地理解如何重新调整这些组件的单个用途 使用基因工程纳米药物载体的免疫抑制鸡尾酒雷帕霉素可以提供持续的胰岛 保护。发现了包裹雷帕霉素(RPS)的囊状聚合物纳米载体(即聚合体，PS) 独特地改变雷帕霉素的细胞生物分布，避免副作用并显著改善 功效。重要的是，雷帕霉素通常具有广泛的细胞生物分布和功能，直接抑制 T细胞增殖，但RPS完全避免T细胞，而是切换免疫抑制机制 涉及对抗原提呈细胞(APC)的选择性和有效的共刺激阻断。这种新型的细胞选择性 纳米载体增强的共刺激阻断的特征是CD8+调节性T细胞上调 和双阳性的CD4+CD8+T细胞，实现持续正常血糖在严格的完全主要 组织相容性复合体(MHC)不相合同种异体门静脉(肝)胰岛移植小鼠模型。 在这里，这项建议将调查和基准RPS的免疫学机制与临床 相关治疗性Belatacept，一种CTLA4-Ig G融合蛋白，通过 替代方法：阻断APC上的CD80/86辅助受体。将实现以下目标： 目的1：确定RPS是否诱导全身性免疫抑制或抗原特异性耐受。它目前是 目前尚不清楚RPS诱导的是抗原特异性还是全身性免疫抑制。一种新型的体外混合制剂 淋巴细胞反应分析和体内双重抗原特异性/非特异性胰岛移植将解决这一问题 问题。 目的：比较RPS与CTLA4-Ig G诱导的共刺激阻断的免疫调节机制 以提高同种异体胰岛移植物的存活率。基于T分布随机邻域的高参数流式细胞术 完全MHC错配胰岛移植后长期存活的包埋(TSNE)分析和评估 将进行移植手术。

PROJECT SUMMARY Dysregulation of blood glucose due to T1D leads to both short term consequences such as hypoglycemia induced injury and death, and long-term complications including amputation, blindness, kidney failure, and neuropathy. Islet transplantation has the potential to provide biologic glycemic regulation for all T1D patients, effectively curing the disease. However, this promising therapy is plagued by the requirement for ineffective immunosuppressive therapies. Currently, only 50 to 70% of grafts remain viable at 5 years post-transplantation, largely due to the immunosuppressive therapy causing toxicity and failing to fully protect the graft. Currently, a costly cocktail of immunosuppressive drugs is given in a rarely successful attempt to provide broad immune coverage with side effect mitigation. Thus, a need exists for simpler, lower cost, and more effective alternatives to these immunosuppressive regimens that can maintain islet transplant survival without off-target side-effects. The objective of this proposal is to mechanistically understand how repurposing a single component of these immunosuppressive cocktails, rapamycin, using engineered nanoscale drug carriers can provide sustained islet protection. Vesicular polymeric nanocarriers (i.e. polymersomes, PS) encapsulating rapamycin (rPS) were found to uniquely change the cellular biodistribution of rapamycin to avoid side-effects and significantly improve efficacy. Importantly, rapamycin normally has a wide cellular biodistribution and functions by directly inhibiting T cell proliferation, but rPS completely avoids T cells and instead switches the immunosuppressive mechanism to a selective and potent costimulation blockade of antigen presenting cells (APCs). This novel cell-selective nanocarrier-enhanced costimulation blockade was characterized by an upregulation of CD8+ regulatory T cells and double positive CD4+CD8+ T cells, achieving sustained normoglycemia in a rigorous fully major histocompatibility complex (MHC) mismatched allogenic intraportal (liver) islet transplantation mouse model. Here, this proposal will investigate and benchmark the immunological mechanism of rPS against the clinically relevant therapeutic belatacept, a CTLA4-IgG fusion protein that induces costimulation blockade via an alternative method: the blocking of CD80/86 coreceptors on APCs. The following aims will be achieved: Aim 1: Determine whether rPS induces general immunosuppression or antigen-specific tolerance. It is currently not known whether rPS induce antigen-specific or general systemic immunosuppression. A novel ex vivo mixed lymphocyte reaction assay and in vivo dual antigen-specific / non-specific islet transplantation will address this question. Aim 2: Compare the immunomodulatory mechanisms of costimulation blockade induced by rPS vs. CTLA4-IgG for enhanced allogenic islet graft survival. High parameter flow cytometry using T-distributed Stochastic Neighbor Embedding (tSNE) analysis and assessment of long-term graft survival following fully-MHC mismatch islet transplantation will be performed.