Somatic Mutagenesis in Autoimmunity

自身免疫中的体细胞突变

基本信息

批准号：
8851517
负责人：
Lawrence J Wysocki
金额：
$ 20.28万
依托单位：
NATIONAL JEWISH HEALTH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8851517
关键词：
Address Adoptive Transfer Affect Antigens Autoimmune Diseases Autoimmunity B-Lymphocytes Behavior Bioinformatics Bone Marrow CD4 Positive T Lymphocytes CD8B1 gene Characteristics Chimera organism DNA DNA Sequence Diabetes Mellitus Diabetic mouse Dideoxy Chain Termination DNA Sequencing Disease Effector Cell Epigenetic Process Etiology Evolution Exocrine pancreas Experimental Models Face Genetic Genome Genomics Head Health Immunocompetent Inbred NOD Mice Infection Infiltration Insulin-Dependent Diabetes Mellitus Investigation Islets of Langerhans Laboratories Lymphocyte Malignant Neoplasms Mediating Modeling Molecular Mus Mutagenesis Mutation Nature Outcome Pancreas Pathology Pathway interactions Peripheral Play Positioning Attribute Property Recruitment Activity Regenerative Medicine Research Personnel Role Self Tolerance Sequence Analysis Signal Pathway Somatic Mutation Splenocyte T-Cell Receptor T-Lymphocyte Testing Therapeutic Intervention Tissues Transgenes Transgenic Mice Vitamin Deficiency Xenobiotics autoreactive T cell diabetic exome exome sequencing genome-wide immune self tolerance induced pluripotent stem cell islet macrophage mouse model next generation sequencing perforin programs research study stem cell biology

项目摘要

DESCRIPTION (provided by applicant): Despite decades of progress in unraveling cellular and molecular regulatory mechanisms that protect against autoimmunity, we still have only a marginal understanding of how lymphocytes "break the rules" in escaping these self-tolerance mechanisms to participate in autoimmune disease. Nearly all investigations of disease etiology have focused on familial genetics and environmental triggers that range from infections and xenobiotic insults to vitamin deficiencies. Yet none of these have provided sufficient mechanistic explanation for the stochastic nature of autoimmunity, which is still treated with relatively crude and often ineffective therapies. In this project, we propose to develop a model to test a largely ignored third alternative. We will test the hypothesis that somatic mutagenesis in lymphocytes is a major contributor to autoimmune disease. Though many lines of evidence point to this idea, there has been a dearth of associated study. The reason is simple: to date the hypothesis has been largely intractable to investigation. However, recent technical advances in DNA sequencing and stem cell biology/regenerative medicine have converged to open a heretofore locked door. We can now address the mutation/autoimmunity hypothesis in the context of the nonobese diabetic mouse (NOD) model of type I diabetes. Our laboratory is in the unique position of having direct access to a large panel of diabetogenic CD4 T cell clones derived from splenocytes of diabetic NOD mice. In two aims, we will make use of these clones to test the hypothesis that they have been functionally altered by somatic mutations, enabling their escape from self-tolerance and promoting their pancreatic islet-specific pathology. We will use NextGen sequencing to uncover somatic mutations in the exomes of diabetogenic CD4 T cell clones and perform bioinformatic pathways analyses to identify signaling pathways affected by mutations. In a complementary aim, we will evaluate somatic mutations in the entire genome of one clone, BDC-2.5, by generating induced pluripotent stem cells (iPSC) in order to erase the epigenetic program of the BDC-2.5 clone. CD4 T cells subsequently derived from BDC-2.5 iPSC will be functionally compared head-to-head with CD4 T cells of transgenic mice that share the BDC-2.5 clone ¿¿T cell receptor but not the BDC-2.5 clone- specific somatic mutations. In the context of adoptive transfer and bone marrow chimera studies, this comparison will enable us to determine the effects of genome-wide somatic mutations on pathological properties of the BDC-2.5 clone. Uncovering a role for somatic mutagenesis in diabetes would transform investigation of this and other autoimmune diseases, opening new avenues to identify targets for rational therapeutic intervention. Regardless of outcome, our study will be the first to directly assess the contributions of somatic genetics versus epigenetics to autoimmunity.

描述（由申请人提供）：尽管在阐明防止自身免疫的细胞和分子调节机制方面取得了数十年的进展，但我们对细胞淋巴如何“打破规则”逃避这些自我耐受机制参与自身免疫性疾病的了解仍然有限几乎所有疾病病因学的研究都集中在家族遗传学和环境触发因素上，从感染和外源性损伤到维生素缺乏，但这些都没有提供足够的机制解释。自身免疫的随机性，目前仍然用相对粗糙的方法来处理在这个项目中，我们建议开发一个模型来测试很大程度上被忽视的第三种选择，尽管许多证据都表明这一观点，但淋巴细胞的体细胞突变是导致自身免疫性疾病的主要原因。原因很简单：迄今为止，这一假设在很大程度上难以进行研究，然而，DNA 测序和干细胞生物学/再生医学的最新技术进步已经打开了此前紧闭的大门。我们可以现在，我们的实验室处于独特的地位，可以直接获得大量源自糖尿病脾细胞的致糖尿病 CD4 T 细胞克隆。 NOD 小鼠有两个目标，我们将利用这些克隆来测试它们的功能已被体细胞突变改变的假设，使它们能够逃避自我耐受并促进其胰岛特异性。我们将使用 NextGen 测序来揭示致糖尿病 CD4 T 细胞克隆的外显子组中的体细胞突变，并进行生物信息学通路分析以识别受突变影响的信号通路。作为补充目标，我们将评估一个克隆的整个基因组中的体细胞突变。，BDC-2.5，通过产生诱导多能干细胞（iPSC）以消除随后从 BDC-2.5 iPSC 衍生的 BDC-2.5 克隆的表观遗传程序。与共享 BDC-2.5 克隆的转基因小鼠的 CD4 T 细胞进行功能上的头对头比较 ¿ ¿ T 细胞受体而非 BDC-2.5 克隆特异性体细胞突变在过继转移和骨髓嵌合体研究的背景下，这种比较将使我们能够确定全基因组体细胞突变对 BDC-2.5 病理特性的影响。揭示体细胞突变在糖尿病中的作用将改变对这种疾病和其他自身免疫性疾病的研究，为确定合理治疗干预的目标开辟新途径，无论结果如何，我们的研究都将是第一个直接评估的研究。体细胞遗传学与表观遗传学对自身免疫的贡献。