Early diagnosis and mechanistic studies of type 1 diabetes using single cell analysis

项目摘要 目前的项目提出了一个原始的框架，以翻译基本的小鼠免疫学的临床前 1型糖尿病（T1 D）阶段的人，使用先进的单细胞技术。过去四 多年来，我们已经解剖了抗原特异性CD 4 + T细胞反应在NOD小鼠模型中使用单细胞 基因谱分析、TCR测序和RNAseq，以了解自身反应性T细胞活化的过程， 靶器官。在确定了胰岛特异性CD 4 + T细胞的独特活化程序后，我们分离了相同的 来自糖尿病前期小鼠的外周血的细胞。我们的论点是，类似的研究现在可以在 并帮助在非常早期阶段诊断疾病、跟踪疾病演变和监测治疗 干预此外，同样的方法将在CD 4 + T细胞的作用中提供重要的机制见解。 细胞在T1 D发病和进展。我们的工作将集中在两个特定的目标：SA #1：扩展鼠标 优化其翻译价值。在NOD小鼠模型中，胰岛CD 4 + T细胞的单细胞分析 CD 4 + T细胞的研究已经允许鉴定外周血中再循环的自身反应性CD 4 + T细胞。探测 两种抗原特异性与一系列pMHC四聚体，致病细胞的概况被解剖的基因 表达谱分析、RNAseq和TCR αβ对测序。使用相同的方法，进一步表征 将使我们能够获得机制的见解，并确定潜在的新的治疗靶点。我们 假设来自疾病过程所有阶段的再循环T细胞可以在血液中发现， 通过单细胞技术进行分析，并用于诊断和跟踪疾病演变。SA #2：分析 来自T1 D患者外周血的活化的CD 4 + T细胞。使用相同的方法和单一 细胞技术，我们将表征循环CD 4 +HLA-DR+PD-1+ CXCR 3+细胞群，我们有 在T1 D患者中发现，而在对照组中未发现。将使用HLA-DQ四聚体检查抗原特异性， 以及在测序的αβ对的TCR再表达后使用T细胞活化测定以及新的 人源化小鼠模型。一个单一的抗原，胰岛素，和共同的小鼠-人表位将用于此 在检查额外的抗原反应性之前，翻译。该方法将被测试其能力， 测量“高危”、“刚诊断”和“确诊”T1 D患者的抗胰岛自身反应性，以及 与经典的抗胰岛抗体检测相比。这两个目标还将评估“P9”的确切作用， 分别在小鼠和人T1 D中T细胞识别的“开关”模式，并可能回答为什么这一点 疾病与单一HLA II类β57多态性有关。 我们希望通过询问外周血中的稀有细胞来证明单细胞技术， 在临床前阶段诊断T1 D的能力。这种革命性的方法将 允许监测旨在维持β细胞群的进化和新疗法。

Project Summary The current project proposes an original framework to translate basic mouse immunology of the pre-clinical phase of type 1 diabetes (T1D) to human, using sophisticated single cell technologies. Over the past four years, we have dissected the antigen-specific CD4+ T cell response in the NOD mouse model using single cell gene profiling, TCR sequencing, and RNAseq to understand the process of activation of autoreactive T cells in target organs. After defining a unique program of activation for islet-specific CD4+ T cells, we isolated the same cells from peripheral blood of pre-diabetic mice. Our contention is that similar studies can now be done in human and help diagnose disease at a very early stage and follow disease evolution and monitor therapeutic intervention. In addition, the same approach will deliver important mechanistic insights in the role of CD4+ T cells in T1D onset and progression. Our work will be focused on two specific aims: SA #1: Expand the mouse studies to optimize their translational value. In the NOD mouse model, single cell analysis of islet CD4+ T cells has allowed the identification of recirculating autoreactive CD4+ T cells in the peripheral blood. Probing two antigen specificities with a series of pMHC tetramers, the profile of pathogenic cells was dissected by gene expression profiling, RNAseq, and TCR αβ pair sequencing. Using the same approach, further characterization of these cells will allow us to gain mechanistic insights and identify potential new therapeutic targets. We hypothesize that recirculating T cells from all stages of the disease process can be found in blood and analyzed by single cell technologies, and used to diagnose and follow disease evolution. SA #2: Analysis of activated CD4+ T cells from the peripheral blood of T1D patients. Using the same approaches and single cell technology, we will characterize a circulating CD4+HLA-DR+PD-1+CXCR3+ cell population that we have identified in T1D patients and not in controls. Antigen specificity will be examined using HLA-DQ tetramers, and functionally using T cell activation assays after TCR re-expression of sequenced αβ pairs as well as new humanized mouse models. A single antigen, insulin, and common mouse-human epitopes will be used for this translation before additional antigen reactivities are examined. The approach will be tested for its ability to measure the anti-islet autoreactivity in “at-risk”, “just-diagnosed”, and “established” T1D patients, and compared to the classic anti-islet antibody detection. The two aims will also evaluate the precise role of the “P9 switch” mode of T cell recognition in mice and human T1D, respectively, and potentially answer why this disease is linked to the single HLA class II β57 polymorphism. Hopefully, we will demonstrate that single cell technologies by interrogating rare cells in peripheral blood, have the power to diagnose T1D in its pre-clinical phase in at risk patients. Such a revolutionary approach would permit to monitor evolution and new therapies aimed at maintaining β cell mass.