Project 3

项目3

基本信息

批准号：
10549504
负责人：
Todd Michael Brusko
金额：
$ 30.01万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-09-30 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10549504
关键词：
Address Adhesions Age Alleles Antigen-Presenting Cells Autoimmunity Automobile Driving Avidity Beta Cell Biological Biological Assay Biological Markers Biological Models Biometry CD4 Positive T Lymphocytes CD8B1 gene CRISPR/Cas technology Cell model Cells Cellular Indexing of Transcriptomes and Epitopes by Sequencing Cellular biology Circulation Clinical Clone Cells Code Collaborations Complex Coupled Data Defect Development Disease Disease Progression Donor Selection Elements Event Flow Cytometry Gene Delivery Gene Expression Genes Genetic Genetic Polymorphism Genetic Risk Genome engineering Genotype HLA Antigens Haplotypes Health Histocompatibility Antigens Class II Human Immune Immune system Immunogenetics Immunophenotyping In Situ In Vitro Individual Inflammatory Infrastructure Insulin Insulin-Dependent Diabetes Mellitus Intake Knock-out Knowledge Laboratories Lead Link Lymphatic Major Histocompatibility Complex Memory Metadata Minor Molecular Monitor Natural History Pancreas Pathogenesis Pathogenicity Pathway interactions Peptides Peripheral Phenotype Population Receptor Signaling Regulation Regulatory T-Lymphocyte Resistance Risk Risk Factors Sampling Signal Transduction Single Nucleotide Polymorphism Slice Specificity Spleen Standardization Surface Surface Antigens System Systems Biology T cell differentiation T cell receptor repertoire sequencing T-Cell Activation T-Cell Immunologic Specificity T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets T-cell receptor repertoire TNFRSF10A gene Testing Therapeutic Therapeutic Intervention Thymus Gland Tissues Vascular Endothelium arm autoimmune pathogenesis autoreactive T cell autoreactivity cell killing checkpoint receptors cytotoxicity data acquisition data integration diabetes pathogenesis diabetes risk disorder risk draining lymph node effector T cell genetic variant genome wide association study genome-wide analysis high risk human imaging immune checkpoint immune function immunoregulation insight islet lymph nodes multiple omics pathogen peripheral blood polygenic risk score precision medicine programs protein complex receptor risk variant single-cell RNA sequencing synergism targeted treatment

项目摘要

The adaptive arm of the human immune system provides exquisite protection from pathogens, but the highly variable receptors can turn on self-tissues when immunoregulatory checkpoints are broken due to genetic risk and inflammatory events. A breakdown in tolerance impacting T cells is thought to be a critical immune checkpoint during the natural history of type 1 diabetes (T1D). Of the approximately 150 independent loci identified by genome-wide association studies as contributing to polygenic T1D risk, the major histocompatibility complex (MHC) remains the largest factor conferring risk due to its influence over thymic selection of the T cell receptor (TCR) repertoire and impact on peripheral T cell activation. Despite this critical link, little is known about how high-risk alleles like HLA-DR4 impact TCR selection, activation thresholds, and distribution across tissues. Our compelling preliminary data suggest that the risk-associated DR4/DQ8 haplotype, which has been linked with insulin autoreactivity, is hyper-expressed on the surface of antigen presenting cells (APCs) and associated with a unique TCR signature. Additional gene variants impacting T cell co-stimulation and differentiation are also enriched in subjects with T1D, yet their mechanistic contributions toward T1D autoimmunity remain poorly characterized. Project 3 proposes to address knowledge gaps governing these key aspects influencing the TCR repertoire and activation requirements in health and disease. We hypothesize that MHC risk, and additional non-MHC protein-coding risk variants, lead to aberrant T cell activation and differentiation thresholds and result in the loss of T cell tolerance in T1D. Specifically, T1D-associated risk variants alter the functional avidity of the MHC:peptide:TCR complex, and risk variants in the molecules SIRPG and CD226 alter T cell signaling, resulting in activation of autoreactive effector T cells as well as defective immunoregulation by regulatory T cells (Treg). These studies aim to investigate the mechanisms by which MHC class II (Aim 1) and non-MHC risk variants (Aim 2) control autoreactive T cells through the analysis of pancreatic draining lymph nodes (pLN), spleen, and peripheral blood from subjects with T1D and those at-risk for disease development (collected through Core A). We will leverage polygenic risk scoring and broad immunophenotyping data generated by Core B to direct case selection and analysis with state-of-the-art single-cell profiling and focused functional assays utilizing both genotype-selected and gene-edited samples. To investigate individual clones and risk alleles, we will modify primary human T cell specificity and function through lentiviral gene expression systems and CRISPR/Cas9 genome engineering for use in ex vivo pancreas slice culture and in vitro isogenic cellular modeling systems, in collaboration with Projects 1 & 2, respectively. Data from this Project are expected to inform on the T cell activation checkpoints involved in T1D pathogenesis, linking pathogenic clones and phenotypes in lymphatics to peripheral blood, ultimately, to develop biomarkers of disease progression and identify pathway-targeted therapeutic interventions to halt the autoimmune destruction of β-cells in T1D.

人类免疫系统的适应性臂提供了针对病原体的精致保护，但高度当免疫调节检查点因遗传风险而被破坏时，可变受体可以开启自身组织影响 T 细胞的耐受性崩溃被认为是一种关键的免疫。 1 型糖尿病 (T1D) 自然史期间的检查点大约 150 个独立位点。全基因组关联研究确定其会导致多基因 T1D 风险，这是主要的组织相容性由于其对 T 细胞胸腺选择的影响，MHC 复合体 (MHC) 仍然是最大的风险因素尽管存在这一关键联系，但人们对 TCR 受体（TCR）的组成部分及其对外周 T 细胞激活的影响知之甚少。 HLA-DR4 等高风险等位基因如何影响 TCR 选择、激活阈值和跨组织分布。我们令人信服的初步数据表明，与风险相关的 DR4/DQ8 单倍型，已与具有胰岛素自身反应性，在抗原呈递细胞 (APC) 表面过度表达并相关还具有影响 T 细胞共刺激和分化的其他基因。在 T1D 受试者中丰富，但其对 T1D 自身免疫的机制贡献仍然很差项目 3 建议解决影响 TCR 的这些关键方面的知识差距。我们勇于承担健康和疾病方面的全部功能和激活要求。非 MHC 蛋白编码风险变异，导致 T 细胞激活和分化阈值及结果异常具体而言，T1D 相关的风险变异会改变 T 细胞耐受性的丧失。 MHC：肽：TCR 复合物以及 SIRPG 和 CD226 分子中的风险变异会改变 T 细胞信号传导，从而导致自身反应性效应 T 细胞的激活以及调节性 T 细胞 (Treg) 的免疫调节缺陷。这些研究旨在调查 MHC II 类（目标 1）和非 MHC 风险变异的机制（目标 2）通过分析胰腺引流淋巴结 (pLN)、脾脏和来自 T1D 受试者和有疾病发展风险的受试者的外周血（通过核心 A 收集）。我们将利用 Core B 生成的多基因风险评分和广泛的免疫表型数据来指导病例通过最先进的单细胞分析和重点功能分析进行选择和分析为了研究个体克隆和风险等位基因，我们将修改基因型选择和基因编辑的样本。通过慢病毒基因表达系统和 CRISPR/Cas9 实现原代人类 T 细胞特异性和功能用于离体胰腺切片培养和体外等基因细胞建模系统的基因组工程，分别与项目 1 和 2 合作，预计该项目的数据将提供有关 T 细胞的信息。参与 T1D 发病机制的激活检查点，将淋巴管中的致病性克隆和表型与外周血最终开发疾病进展的生物标志物并确定靶向途径阻止 T1D β 细胞自身免疫破坏的治疗干预措施。