Micro-nanotechnologies for the analysis of islet-derived extracellular vesicles implicated in Type 1 Diabetes

摘要 1型糖尿病（T1 D）中β细胞的破坏由胰岛反应性细胞毒性CD 8 + T细胞介导， 渗透到胰岛细胞外囊泡（EV）是直径为30 - 40 μ m的双层膜结构。 1000 nm，由全身细胞释放到血液中，浓度约为1010/ml。 它们的蛋白质、双链DNA寡聚体、微小RNA（miRNA）、mRNA和其他分析物的分子含量， 可以通过EV贩运发挥多种功能作用，也可以提供诊断报告， 疾病部位或组织的起源。因此，EV可以作为T1 D生物标志物的潜在来源，甚至可能是T1 D生物标志物的潜在来源。 提供对胰岛微环境的遗传和功能特征的深入了解。 我们提出了一个从胰岛衍生囊泡中寻找T1 D生物标志物的计划， 微流控纳米颗粒富集技术的一套多参数分析方法。在AIM 1中，我们 利用一套独特的批量和单EV（sEV）转录组学和蛋白质组学方法， 研究来自健康供体和患有T1 D的个体的胰岛细胞的分子含量， 从这些培养的细胞中。这将产生一组蛋白质和RNA，可以独特地识别来自β细胞的EV， 以及对胰岛EV异质性的认识。在这里，我们也开始解决分子 T1 D发病机制所特有的特征。胰岛浸润性CD 8 + T细胞的研究经常受到以下因素的阻碍： 获取捐赠胰岛组织的途径有限胰岛浸润性CD 8 + T细胞将EV排入血液的事实 流式细胞术提供了一个独特的机会，用于监测胰岛浸润T细胞的活动， 电动车在AIM 2中，我们提出了一种纳米颗粒技术来捕获携带T细胞受体（TCR）的EV， 流通我们计划对来自健康和T1 D血清的EV进行表征，以确定 来源于胰岛浸润性CD 8 + T细胞。监测这些EV人群可以揭示关键的当地 胰腺中的事件，如T细胞活化和细胞毒性。因为我们预计 在AIM 3中，我们提供了一种微流体使能的解决方案来富集来自胰腺的EV。 来自人血清的β细胞和浸润性CD 8 + T细胞。这种集成的样品处理工作流程允许 从有限体积的血清中自动分离胰岛衍生EV，只需最少的手动时间。分析 库存血清标本将产生一组与T1 D发展相关的候选生物标志物， 用一个独立的队列进行验证和完善。重要的是，拟议的研究不仅提供EV- 基于T1 D生物标志物，而且还生成对设计和开发具有高价值的数据集， T1 D的抗原特异性免疫疗法。 我们的多学科团队由具有EV生物学、T1 D、 发病机理、免疫学、多组学疾病诊断、计算生物学和测定开发。

ABSTRACT The destruction of β cells in Type 1 Diabetes (T1D) is mediated by islet-reactive cytotoxic CD8+ T cells that infiltrate the pancreatic islet. Extracellular Vesicles (EVs) are bilayer membrane structures of diameters 30 – 1000 nm released into the blood by cells throughout the body, at concentrations on the order of 1010 per ml. Their molecular content of proteins, dsDNA oligomers, microRNAs (miRNAs), mRNAs, and other analytes, may play multiple functional roles via EV trafficking, and may also provide a diagnostic report back on the disease site or tissue of origin. As such, EVs can serve as potential sources of T1D biomarkers, perhaps even providing insights into the genetic and functional characteristics of the pancreatic islet microenvironment. We propose a program pursuing T1D biomarkers from islet-derived vesicles that couples our cutting-edge, microfluidic nanoparticle enrichment technology to a suite of multiparameter analytical methods. In AIM 1, we utilize a distinct set of bulk and single-EV (sEV) transcriptomic and proteomic methods to comprehensively investigate the molecular contents of islet cells from healthy donors and individuals with T1D, and EVs derived from those cultured cells. This will yield a set of proteins and RNAs that can uniquely identify EVs from β cells, as well as insights on the heterogeneity of the islet EVs. Here we also begin to resolve the molecular signatures that are unique to T1D pathogenesis. The study of islet-infiltrating CD8+ T cells is often impeded by the limited access to donor islet tissues. The fact that islet-infiltrating CD8+ T cells shed EVs into the blood stream provides a unique opportunity for monitoring the activities of the islet-infiltrating T cells with circulating EVs. In AIM 2, we propose a nanoparticle technology to capture T-cell receptor (TCR) bearing EVs in circulation. We plan to characterize these EVs from healthy and T1D sera to determine the EV populations that arise from pancreatic islet infiltrating CD8+ T cells. Monitoring these EV populations can reveal key local events in the pancreas such as T cell activation and cytotoxicity. As we anticipate a low blood abundance of islet-derived EVs, in AIM 3, we provide a microfluidics-enabled solution to enrich EVs derived from pancreatic β cells and infiltrating CD8+ T cells from human sera. This integrated sample processing workflow allows automatic isolation of islet-derived EVs from limited volumes of serum, with minimal hands on time. Analysis of banked serum specimens will yield a set of candidate biomarkers associated with T1D development, which will be validated and refined with an independent cohort. Importantly, the proposed study will not only provide EV- based T1D biomarkers, but also generate datasets that are of high value to the design and development of antigen-specific immunotherapies for T1D. Our multidisciplinary team is comprised of scientists and clinicians with expertise in EV biology, T1D pathogenesis, immunology, multi-omic disease diagnostics, computational biology, and assay development.