PTPN22 R619W in NOD mice

项目总结： 1型糖尿病(T1D)是由于免疫耐受性崩溃而导致的 胰岛分泌胰岛素的β细胞。大量的遗传和环境因素 增加了疾病的发病率。最强的非MHC遗传风险因素之一是单核苷酸 编码酪氨酸磷酸酶非受体22型的基因PTPN22的多态性导致 在单一氨基酸变化的蛋白质中，R620W。PTPN22在多种来源的骨髓中均有表达 细胞，包括淋巴细胞和髓系细胞。尽管R620W增加了许多 与自身抗体产生有关的自身免疫性疾病，包括T1D、类风湿性关节炎、 系统性红斑狼疮、Graves病、白癜风和其他疾病，对它是如何增强的知之甚少 疾病。在一定程度上，这是因为它在许多不同类型的细胞中发挥作用，这些细胞影响免疫系统。在……里面 为了制作一种可以用来识别其作用机制的小鼠模型，我们使用了Crispr/Cas9 基因组编辑将PTPN22(619W)促自身免疫等位基因的小鼠同源基因引入非 肥胖糖尿病(NOD)小鼠，自发发展为T1D。携带该突变的NOD小鼠 表现出抗胰岛素抗体的加速产生和更高外显率的加速T1D， 与表达R620W的人表现出的表型相同。该提案将使用619W NOD型号 通过追求以下具体目标来揭示疾病加重的机制： AIM1将鉴定内源性表达619W促进IAA产生和T1D的免疫细胞。 这需要过继转移实验来产生表达KEY中PTPN22 619W突变的小鼠 免疫细胞。 目的2探讨619W小鼠体内IAA和T1D增强的机制。免疫细胞的类型 根据目标1进行鉴定，以确定受等位基因差异影响其表达水平的基因 在619。基因和途径将通过RNAseq或通过分析单个 细胞水平。将通过改变shRNA的表达水平来测试候选基因的功能 逆转录病毒载体。 目的研究R619W小鼠对胰岛素特异性B淋巴细胞的耐受性。 Aim 4将使用TCR转基因小鼠和单细胞表达分析来检测胰岛抗原的命运 在表达619W或619R的小鼠中，特异性T细胞被激活时。 这些研究的成功完成将与开发可能预防艾滋病的治疗方法相关 与PTPN22相关的许多不同的自身免疫性疾病。

Project Summary: Type 1 diabetes (T1D) results from a breakdown in immune tolerance that progresses to the destruction of the insulin producing beta cells of the pancreatic islets. A large number of genetic and environmental factors contribute to the incidence of disease. One of the strongest non-MHC genetic risk factors is a single nucleotide polymorphism in the gene that encodes the tyrosine phosphatase non-receptor type 22, PTPN22, that results in a single amino acid change in the protein, R620W. PTPN22 is expressed in many bone marrow derived cells, including lymphocytes and myeloid derived cells. Although R620W increases the risk of many autoimmune diseases associated with production of autoantibodies, including T1D, rheumatoid arthritis, systemic lupus erythematosus, Graves disease, vitiligo, and others, little is know about how it enhances disease. In part, this is because it functions in many different types of cells that affect the immune system. In order to produce a mouse model that can be used to identify its mechanism of action, we used Crispr/Cas9 genome editing to introduce the mouse ortholog of the pro-autoimmune allele of PTPN22 (619W) into the non- obese diabetes (NOD) mouse that spontaneously develops T1D. NOD mice harboring this mutation demonstrate accelerated production of anti-insulin antibodies and accelerated T1D of higher penetrance, which is the same phenotype exhibited by humans expressing R620W. This proposal will use the 619W NOD model to reveal the mechanism responsible for enhanced disease by pursuing the following specific aims: Aim1 will identify immune cells in which endogenous expression of 619W enhances IAA production and T1D. This entails adoptive transfer experiments to produce mice expressing the 619W mutation of PTPN22 in key immune cells. Aim 2 will explore the mechanistic basis for enhanced IAA and T1D in 619W mice. The types of immune cells identified under Aim 1 will be probed to identify genes affected in their level of expression by allelic difference at 619. Genes and pathways will be identified by either RNAseq or by analyzing gene expression at the single cell level. Candidate genes will be tested for function by altering level of expression with shRNAs delivered by retroviral vectors. Aim 3 will examine tolerance of insulin specific B lymphocytes in R619W mice. Aim 4 will use TCR transgenic mice and single cell expression analysis to examine the fate of islet antigen specific T cells as they become activated in either 619W or 619R expressing mice. Successful completion of these studies will have relevance to development of treatments that may prevent the many different autoimmune disease associated with PTPN22.