Thymic selection abnormalities in Type 1 Diabetes

项目概要 1 型糖尿病 (T1D) 是一种自身免疫性疾病，其中 T 细胞靶向产生胰岛素的 β 细胞。至少 60 每个非 HLA 基因变异都会导致 T1D 风险小幅增加。虽然异常阴性选择 尽管在 NOD 小鼠中存在相关性，但异常胸腺选择在驱动人类 T1D 中的可能作用尚不清楚。 人类免疫系统（HIS）小鼠，其中患者的免疫系统是从患者的免疫系统中重新产生的。 造血干细胞（HSC），称为个性化免疫小鼠（PIM）模型，提供了独特的 有机会评估导致 T1D 的胸腺选择事件，从而可以评估免疫 曲目开发和实验操作，例如 T 细胞受体 (TCR) 转基因。我们的 初步数据表明，与人类 T1D 易感性相关的非 HLA 遗传变异赋予 自身反应性胸腺细胞的阴性选择受损和 Treg 转向受损。通过比较 T1D- 和 健康对照 (HC) 衍生的 PI 小鼠中，我们发现两种小鼠的胸腺选择异常 T1D 免疫系统中 TCR 的结构变异。一种（I 型）的特点是比平均长度更长 CDR3β 具有更多疏水性氨基酸和更高的自亲和力，通常在 Treg 中最丰富 子集。将高亲和力 I 型胰岛素反应性 TCR（克隆 5）插入 HC HSC 导致克隆缺失 或用该 TCR 调节胸腺细胞的 Treg 转移。然而，这两个过程在 T1D 免疫中都有缺陷 系统，导致胰岛自身反应性 T 细胞仅在 T1D 衍生的细胞中进入外周细胞库 免疫系统。第二种（II 型）的特点是 CDR3β 区域较短，疏水性很少。 与 HC 免疫系统相比，T1D 中的胰岛自身反应性克隆存活率更高。 单细胞 RNAseq 鉴定了一个主要的胸腺细胞群，该群体正在经历负选择，并且高度 表达 TCR 信号传导和促凋亡基因。 T1D 胸腺细胞中实际上不存在该簇， 表明调节胸腺缺失的远端信号通路存在严重缺陷。这种损害是 SH2B3 和 Erk/MAP 激酶通路中与 T1D 易感性单核苷酸多态性相关 基因。该提案的总体目标是更好地了解胸腺的遗传控制和异常 选择涉及介导 T1D 的自身免疫系统的发展。我们建议：1）： 进一步表征有缺陷的胸腺细胞选择并鉴定 HSC 内在的、非 HLA 遗传性的 确定 T1D 中的 TCR 信号传导缺陷会导致负选择受损和 Treg 受损 差异化。我们还将定义 I 型和 II 型 TCR 在 T1D 免疫系统中的致病作用： 自体 iPSC 衍生的 β 细胞移植物； 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 衍生的异常阴性选择和 Treg 重定向的程度 免疫系统。总的来说，这些研究将对 HLA 和 HLA 的机制产生新的见解。 非 HLA 风险等位基因易导致 T 细胞库的胸腺选择，从而导致胰岛自身免疫。

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.