Thymic selection abnormalities in Type 1 Diabetes

项目摘要 1型糖尿病(T1D)是一种自身免疫性疾病，T细胞以产生胰岛素的β细胞为靶细胞。至少60 每一种非人类白细胞抗原基因变异都会导致T1D风险的小幅增加。而反常的负选择有 在NOD小鼠中被牵连，胸腺选择异常在驱动人类T1D中的可能作用尚不清楚。 人类免疫系统(HIS)小鼠，病人的免疫系统是从病人的 造血干细胞(HSCs)被称为个性化免疫小鼠(PIM)模型，为人类提供了一种独特的 评估导致T1D的胸腺选择事件的机会，允许评估免疫 曲目开发和实验操作，如T细胞受体(TCR)转基因。我们的 初步数据显示，与人类T1D易感性相关的非人类白细胞抗原基因变异 自身反应性胸腺细胞的负选择受损和Treg分流受损。通过比较T1D和 健康对照(HC)来源的PI小鼠，我们已经确定了两个胸腺选择的异常 T1D免疫系统中TCR的结构变化。一种(类型I)的特点是时间长于平均水平 CDR3βS具有更多的疏水氨基酸和更高的自我亲和力，通常是Treg中最丰富的 子集。将高亲和力I型胰岛素反应性TCR(Clone 5)插入HC HSCs导致克隆性缺失 或用这种TCR分流胸腺细胞。然而，这两个过程在T1D免疫中都存在缺陷 系统，导致胰岛自身反应性T细胞仅在T1D来源的情况下进入外周谱系 免疫系统。第二类(类型II)具有较短的CDR3β区和较少的疏水性 与HC免疫系统相比，T1D组的胰岛自身反应性克隆存活率更高。 单细胞RNAseq鉴定了经历负选择的主要胸腺细胞群体， 表达TCR信号和促凋亡基因。这一簇在T1D胸腺细胞中几乎不存在， 这表明调节胸腺缺失的远端信号通路存在严重缺陷。这一损害是 SH2B3和Erk/MAP激酶通路单核苷酸多态性与T1D易感性的关系 基因。这项建议的总体目标是更好地了解胸腺的基因控制和异常。 选择参与了调节T1D的自身免疫谱系的发展。我们建议：1)： 进一步表征有缺陷的胸腺细胞选择，并从基因上鉴定HSC固有的、非人类白细胞抗原- 确定T1D中导致负选择和Treg受损的TCR信号缺陷 差异化。我们还将定义I型和II型TCRs在T1D免疫系统中的致病作用 自体IPSC来源的β细胞移植；2)利用转基因自身反应性TCR和特异性抗原MHC 四聚体直接评估T1D来源的异常负选择和Treg重定向的程度 免疫系统。总而言之，这些研究将导致对人类白细胞抗原(HLA)和人类白细胞抗原(HL A) 非人类白细胞抗原风险等位基因倾向于胸腺选择导致胰岛自身免疫的T细胞谱系。

Project Summary Type 1 Diabetes (T1D) is an autoimmune disease in which T cells target insulin-producing β cells. At least 60 non-HLA genetic variants each confer small increases in T1D risk. While abnormal negative selection has been implicated in NOD mice, the possible role of aberrant thymic selection in driving human T1D is unknown. Human immune system (HIS) mice in which a patient’s immune system is generated de novo from their hematopoietic stem cells (HSCs), known as the personalized immune mouse (PIM) model, provide a unique opportunity to evaluate thymic selection events that contribute to T1D, permitting assessment of immune repertoire development and experimental manipulations such as T cell receptor (TCR) transgenesis. Our preliminary data suggest that non-HLA genetic variants associated with human T1D susceptibility confer both impaired negative selection and impaired Treg diversion of autoreactive thymocytes. By comparing T1D- and healthy control (HC)-derived PI mice, we have identified abnormalities in the thymic selection of two structural varieties of TCR in T1D immune systems. One (Type I) is characterized by longer than average CDR3βs with more hydrophobic amino acids and higher self-affinity and is normally most abundant in the Treg subset. Inserting a high affinity Type I insulin-reactive TCR (Clone 5) into HC HSCs resulted in clonal deletion or Treg diversion of thymocytes with this TCR. However, both processes are defective in T1D immune systems, resulting in entry of the islet autoreactive T cells into the peripheral repertoire only in T1D-derived immune systems. The second (Type II) is characterized by shorter CDR3β regions with few hydrophobic amino acids and shows greater islet autoreactive clonal survival in T1D compared to HC immune systems. Single cell RNAseq identified a major thymocyte population undergoing negative selection that highly expresses TCR signaling and pro-apoptotic genes. This cluster was virtually absent among T1D thymocytes, indicating a profound defect in the distal signaling pathways regulating thymic deletion. This impairment was associated with T1D susceptibility single nucleotide polymorphisms in SH2B3 and Erk/MAP kinase pathway genes. The overall goal of this proposal is to better understand the genetic control and abnormalities in thymic selection involved in the development of the autoimmune repertoires that mediate T1D. We propose to: 1): Further characterize defective thymocyte selection and identify the HSC-intrinsic, non-HLA genetically- determined TCR signaling defects in T1D that result in impaired negative selection and impaired Treg differentiation. We will also define pathogenic roles of Type I and Type II TCRs in T1D immune systems with autologous iPSC-derived β-cell grafts; 2) Utilize transgenic autoreactive TCRs and specific antigen-MHC tetramers to directly assess the extent of aberrant negative selection and Treg redirection in T1D-derived immune systems. Collectively, these studies will lead to novel insights into the mechanisms by which HLA and non-HLA risk alleles predispose to thymic selection of a T cell repertoire that causes islet autoimmunity.