Tuning peptide specifities for T cell tolerance in Type 1 diabetes

项目摘要/摘要 1型糖尿病(T1D)是一种经典的T细胞介导的自身免疫性疾病，大量数据表明 胰岛素是T1D病的主要自身抗原。在T1D的NOD小鼠模型中，值得注意的研究表明 缺乏天然胰岛素表达，但改变了胰岛素序列以维持血糖水平的小鼠， 完全不会患上胰腺炎和糖尿病。越来越多的证据也表明，胰岛素肽结合 和在MHC第二类(多肽寄存器)内的取向对于确定相互作用的强度和 自身反应性T细胞的识别。在与Kappler实验室的合作中，我们发现了一种不寻常的多肽 主要胰岛素表位InSb的结合特性：9-23。大多数InSb：9-23特异性的CD4+T细胞 在外周识别胰岛素结合在这个不寻常的寄存器3中，通过敲打单一的氨基酸 NOD小鼠(Ins2EE/+)胰岛素基因只有一个拷贝的变异(R到E)，小鼠完全受到保护 治疗糖尿病。 胰岛素优势表位的“超级激动剂”版本的开发使我们能够解决几个问题 围绕MHC分子产生、呈递多肽的生化途径的关键问题， 和自身反应性致病T细胞的识别。胰岛素基因主要表位的突变是否允许 这些可供选择的表位所特有的CD4+T效应细胞还是Treg细胞？这些都是模仿者吗？ 致病表位能够显著改变疾病结果吗？我们能微调这些表位吗？ 改变耐受机制，从缺失转变为Treg诱导？我们实验室最近的工作主要集中在 识别关键鼠标背景上的胰岛素专用库，我们计划利用这些工具 和特征良好的小鼠模型，以检查改变胰岛素表达的影响，从胸腺和 胸外之交。这些工具以及我们在产生大量TCR转基因小鼠方面的经验 LINE将允许我们在T1D的背景下解决这些问题。 因此，我们假设，表位呈现和TCR亲和力的改变推动了对 TCR的曲目是走向宽容，远离自我反应。使用胰岛素作为模型抗原，我们 建议通过以下具体目标来检验我们的假设： 目的1：确定中枢耐受在胰岛素反应性克隆缺失中的作用 目的2：研究外周耐受对胰岛素反应性T细胞的影响 目标3：探索支配容忍的机制，以了解翻译为 T1D的治疗方法 通过这些实验，我们希望对胰岛素表位的变化和 抗原性驱动糖尿病的发病机制，并确定未来免疫调节和 T1D治疗和预防的治疗性干预。

Project Summary/Abstract Type 1 Diabetes (T1D) is a classical T-cell mediated autoimmune disease and substantial data implicates insulin as a dominant autoantigen in T1D disease. In the NOD mouse model of T1D, notable studies have shown that mice lacking native insulin expression, but with an altered insulin sequence to maintain blood glucose levels, are completely protected from insulitis and diabetes. Growing evidence also indicates that insulin peptide binding and orientation within MHC Class II (peptide register) is important in determining the strength of interaction and recognition by autoreactive T cells. In collaboration with the Kappler lab, we have uncovered an unusual peptide binding characteristic of the dominant insulin epitope InsB:9-23. The majority of InsB:9-23-specific CD4+ T cells in the periphery recognize insulin bound in this unusual register 3, and by knocking in a single amino acid variation (R to E) into just one copy of the insulin gene in NOD mice (Ins2EE/+), the mice are completely protected against diabetes. The development of a “super agonist” version of the insulin dominant epitopes allows us to address several key questions surrounding the biochemical pathways of peptide generation, presentation by MHC molecules, and recognitions by auto-reactive pathogenic T cells. Do mutations of the major epitope in the insulin gene allow CD4+ T effectors or Treg cells specific for these alternative epitopes to develop? Are mimotopes of these pathogenic epitopes capable of dramatically altering disease outcomes? Are we able to fine tune these epitopes to alter tolerance mechanisms to shift from deletion to Treg induction? Recent work in our lab has focused on the identification of the insulin-specific repertoire on key mouse backgrounds, and we plan to utilize these tools and well-characterized mouse models to examine the effects of altering insulin expression, thymically and extrathymically. These tools as well as our experience with the generation of numerous TCR-transgenic mouse lines will allow us to address these questions in the context of T1D. Thus, we hypothesize that alterations to epitope presentation and TCR affinity drive the tunning of the TCR repertoire towards tolerance and away from self-reactivity. Using Insulin as a model antigen, we propose to test our hypothesis through the following specific aims: Aim 1: Define the role of central tolerance upon the deletion of insulin-reactive clones Aim 2: Characterize the effects of peripheral tolerance on insulin-reactive T cells Aim 3: Explore mechanisms of dominate tolerance to understand the potential for translation into therapeutic treatments for T1D Through these experiments, we hope to gain a nuanced understanding of how changes in insulin epitopes and antigenicity drive the pathogenesis of diabetes and identify targets for future immune modulation and therapeutic intervention for T1D treatment and prevention.