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（由适用提供）：尽管在揭示了预防自身免疫性的细胞和分子调节机制方面取得了数十年的进展，但我们仍然只有对淋巴细胞如何“破坏规则”来逃避这些自我耐受机制以参与自身免疫性疾病的边际理解。几乎所有对疾病病因的研究都集中在家庭遗传学和环境触发因素上，从感染和异种生物损伤到维生素缺乏症。然而，这些都没有为自身免疫的随机性提供足够的机械解释，而自身免疫性仍以相对粗略的方式处理而且通常无效的疗法。在这个项目中，我们建议开发一个模型来测试在很大程度上忽略的第三个选择。我们将测试以下假设：淋巴细胞中的体细胞诱变是自身免疫性疾病的主要因素。尽管许多证据表明了这一想法，但相关研究已经死亡。原因很简单：迄今为止，假设在很大程度上与研究很棘手。但是，DNA测序和干细胞生物学/再生药物的最新技术进步已融合到迄今锁定的门。现在，我们可以在I型糖尿病的非肥胖糖尿病小鼠（NOD）模型的背景下解决突变/自身免疫性假设。我们的实验室在独特的位置，可以直接访问源自糖尿病NOD小鼠脾细胞的大型糖尿病性CD4 T细胞克隆。在两个目的中，我们将利用这些克隆来检验以下假设，即它们在功能上因体细胞突变而在功能上改变了，从而使它们能够摆脱自我耐受性并促进其胰岛特异性病理学。我们将使用NextGen测序来发现糖尿病性CD4 T细胞克隆外部的体细胞突变，并进行生物信息学途径分析，以识别受突变影响的信号通路。为了完整的目的，我们将通过产生诱导的多能干细胞（IPSC）来评估一个克隆BDC-2.5的整个基因组中的体细胞突变，以消除BDC-2.5克隆的表观遗传程序。随后衍生自BDC-2.5 IPSC的CD4 T细胞将在功能上与共有BDC-2.5克隆»T细胞受体的转基因小鼠的CD4 T细胞进行比较，而不是BDC-2.5克隆特异性的体细胞突变。在自适应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克隆病理特性的影响。揭示糖尿病中体细胞诱变的作用将改变这种和其他自身免疫性疾病的投资，开辟新的途径，以识别理性治疗干预的目标。无论结果如何，我们的研究将是第一个直接评估体细胞遗传学与表观遗传学对自身免疫性的贡献的研究。