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

项目摘要 由于T1 D引起的血糖失调导致两种短期后果，例如低血糖症 诱发损伤和死亡，以及长期并发症，包括截肢、失明、肾衰竭，以及 神经病变胰岛移植具有为所有T1 D患者提供生物血糖调节的潜力， 有效地治愈疾病。然而，这种有前途的疗法受到对无效的治疗的要求的困扰。 免疫抑制疗法。目前，只有50%至70%的移植物在移植后5年仍然存活， 这主要是由于免疫抑制疗法引起毒性并且不能完全保护移植物。现时 一种昂贵的免疫抑制药物鸡尾酒，在很少成功的尝试提供广泛的免疫， 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 这些免疫抑制方案可以维持胰岛移植存活而没有脱靶副作用。 本提案的目的是从机制上理解如何重新利用这些组件中的单个组件， 免疫抑制鸡尾酒，雷帕霉素，使用工程纳米药物载体可以提供持续的胰岛 保护发现囊泡聚合物纳米载体（即聚合物囊泡，PS）包封雷帕霉素（rPS）， 独特地改变雷帕霉素的细胞生物分布，以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，并通过直接抑制 T细胞增殖，但rPS完全避开T细胞，而是切换免疫抑制机制 涉及抗原呈递细胞（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在于CD 8+调节性T细胞的上调 和双阳性CD 4 + CD 8 + T细胞，在严格的完全主要 组织相容性复合体（MHC）不匹配同种异体门静脉内（肝）胰岛移植小鼠模型。 在这里，本建议将调查和基准的免疫机制rPS对临床 相关的治疗性贝拉西普，一种CTLA 4-IgG融合蛋白，通过免疫抑制剂诱导共刺激阻断， 另一种方法：阻断APC上的CD 80/86辅助受体。将实现以下目标： 目的1：确定rPS是否诱导全身免疫抑制或抗原特异性耐受。目前 尚不清楚rPS是否诱导抗原特异性或全身性免疫抑制。一种新的离体混合物， 淋巴细胞反应测定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2：比较rPS和CTLA 4-IgG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。使用T分布随机近邻的高参数流式细胞术 完全MHC错配胰岛移植后移植物长期存活的包埋（tSNE）分析和评估 将进行移植。

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.