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创的框架来翻译临床前的基本小鼠免疫学 使用复杂的单细胞技术，将1型糖尿病(T1D)阶段转移到人类。在过去四年中 多年来，我们已经使用单细胞解剖了NOD小鼠模型中抗原特异性的CD4T细胞反应 基因图谱、TCR测序和RNAseq以了解自体反应性T细胞的激活过程 靶器官。在为胰岛特异的CD4T细胞定义了一个独特的激活程序后，我们分离出了同样的 糖尿病前期小鼠外周血中的细胞。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，跟踪疾病演变并监测治疗情况 干预。此外，同样的方法将对CD4T的作用提供重要的机械论见解 细胞在T1D的发生和发展。我们的工作将集中在两个具体目标上：SA#1：扩展鼠标 研究如何优化它们的翻译价值。在NOD小鼠模型中，胰岛CD4T的单细胞分析 细胞可以识别外周血中循环的自体反应的CD4T细胞。探查 与一系列pMHC四聚体的两种抗原特异性，用基因解剖了致病细胞的图谱 表达谱分析、RNAseq和TCRαβ对测序。使用相同的方法，进一步描述 对这些细胞的研究将使我们能够获得机械性的见解，并确定潜在的新治疗靶点。我们 假设疾病过程中所有阶段的循环T细胞都可以在血液和 通过单细胞技术进行分析，用于诊断和跟踪疾病演变。SA#2：分析 T1D患者外周血中活化的CD4T细胞。使用相同的方法和单一 细胞技术，我们将描述我们拥有的循环中的CD4HLA-DR PD-1CXCR3细胞群 在T1D患者中发现，而在对照组中未发现。抗原特异性将使用人类白细胞抗原-DQ四聚体进行检测， 并在功能上使用TCR重新表达测序的αβ对以及新的 人性化的老鼠模型。一种单一的抗原、胰岛素和常见的鼠-人表位将用于这一点 在检查额外的抗原反应性之前进行翻译。该方法将接受测试，以确定其是否有能力 测量“风险”、“刚诊断”和“确诊”的T1D患者的抗胰岛自身反应性，以及 与经典的抗胰岛抗体检测方法相比。这两个目标还将评估“P9”的确切作用 分别在小鼠和人类T1D中转换T细胞识别模式，并潜在地回答了为什么 这种疾病与单一的人类白细胞抗原II类β57多态有关。 希望我们将通过询问外周血中的稀有细胞来展示单细胞技术 高危患者在临床前阶段诊断T1D的能力。这种革命性的方法将 允许监测旨在维持β细胞团的进化和新疗法。

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.