Exosome-induced innate and antigen-specific immune responses in Type 1 Diabetes

描述(申请人提供)：1型糖尿病(T1D)是一种T细胞介导的组织特异性自身免疫性疾病，其中II型MHC基因在控制疾病易感性方面发挥主要作用。这表明，胰岛自身抗原及其在MHC分子上的短肽是导致胰岛分泌胰岛素的β细胞被破坏的关键因素。由于胸腺中的自我耐受机制通常能有效地清除强大的自身反应性T细胞，因此外周循环中的T细胞会被清除以避免自身反应性；然而，尽管自身免疫性疾病的发生率很低，但也会发生。我们推测，某些炎症因子可能通过增强胰岛自身抗原的免疫原性来启动胰岛自身免疫反应。我们对候选胰岛抗原谷氨酸脱羧酶65 KDa(GAD65)进行了多年的研究，了解到诱导致病或致糖尿病效应T细胞需要独特的抗原递送和处理途径，并证明与糖尿病相关的候选基因SLc11a1可以改变树突状细胞(DC)处理胰岛抗原的途径。我们最近从小鼠胰岛素瘤和原代胰岛神经胶质细胞的培养上清液中分离出一种微粒子/囊泡，即外切体(Exosome，EXO)，发现EXO含有强烈的促炎物质，可以刺激包括DC在内的各种抗原提呈细胞产生炎症细胞因子，上调MHC和共刺激分子；更重要的是，从原代胰岛神经胶质细胞收集的EXO还表达包括GAD65在内的几种已知的胰岛自身抗原。因此，我们提出了一个新的假设，即EXO可能作为一种独特的内源性佐剂和抗原载体，调节APC改变自身抗原的处理/提呈，从而激活自身反应性T细胞。在这个方案中，我们将首先对初级胰岛细胞释放的EXO进行基本的生化分析，并研究EXO诱导的先天性反应中不同TLRs的需求，以表征EXO中包含的促炎物质的性质。我们将比较糖尿病易感株和耐药株之间的胰岛神经胶质细胞及其EXO，以确定异常或过量的EXO产生是否可能是导致糖尿病的一个事件。然后，我们将使用EXO作为抗原载体，研究在DC中处理EXO后，如何诱导针对胰岛抗原，特别是GAD65的自身反应性效应T细胞。我们预测，在应激或炎症情况下，通过EXO分泌增加的胰岛抗原泄漏是激活高度促糖尿病T细胞的关键。最后，我们将继续研究SLc11a1基因在DC激活和抗原处理中的作用。我们将使用EXO作为激活DC和运送抗原的载体。我们预计EXO可以上调DC中SLc11a1的功能，从而促进致病Th1细胞的诱导。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is a T cell-mediated, tissue-specific autoimmune disease, in which the class II MHC genes play a major role controlling the disease susceptibility. This suggests that islet autoantigens and their short peptides presented on the MHC molecules are the key elements leading to the destruction of the insulin-producing beta cells in the pancreatic islets. Since self-tolerance mechanism in the thymus normally is effective in removing strong autoreactive T cells, peripheral circulating T cells are purged to avoid self- reactivity; however, autoimmune disease does occur despite of rare incidence. We hypothesized that certain inflammatory triggers may initiate the autoimmune responses in the islets by enhancing the immunogenic activity of the islet autoantigens. We have studied one candidate islet antigen, glutamic acid decarboxylase 65 KDa (GAD65), for several years and learned that unique antigen delivery and processing pathways are required to induce disease-causing or diabetogenic effector T cells, and demonstrated that a diabetes- associated candidate gene, Slc11a1, could alter the pathways of processing islet antigens in dendritic cells (DC). We recently isolated one type of microparticles/vesicles, namely exosomes (EXO), from the culture supernatants of mouse insulinomas and primary islet glial cells, and found that the EXO contains strong proinflammatory materials that could stimulate various antigen presenting cells including DC to produce inflammatory cytokines and upregulate MHC and costimulatory molecules; more importantly, the EXO collected from the primary islet glial cells also expresses several known islet autoantigens including GAD65. We thus propose a novel hypothesis that EXO may act as a unique type of endogenous adjuvant and antigen carrier that can condition APC to alter the processing/presenting of the autoantigens and thus, to activate the autoreactive T cells. In this proposal, we will first perform basic biochemistry analysis for the EXO released by the primary islet cells, and study the requirements of different TLRs in the EXO-induced innate responses to characterize the nature of the proinflammatory materials enclosed in the EXO. We will compare the islet glial cells and their EXO between diabetes-susceptible NOD strain and resistant strains to address whether abnormal or excess EXO production could be one diabetes-causative event. Then, we will use EXO as an antigen carrier to study how autoreactive effector T cells specific for islet antigens, particularly GAD65, are induced following processing the EXO in DC. We predict that increased leakage of islet antigens via EXO secretion under stress or inflammatory situation is the key for activating highly diabetogenic T cells. Finally, we will continue studying the Slc11a1 gene for its roles in DC activation and antigen processing. We will use EXO as a vehicle for activating DC and delivering antigens. We anticipate that EXO can upregulate Slc11a1 function in DC and thus promote induction of the disease-causing Th1 cells.