Project 3

项目3

• 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
• 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

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手臂提供了对病原体的精致保护，但高度免疫系统的免疫系统也是如此。 当免疫调节检查点由于遗传风险而被破坏时， 和炎症事件。耐受性的破坏影响T细胞被认为是一个关键的免疫 在1型糖尿病（T1 D）的自然史检查点。在大约150个独立的基因座中， 通过全基因组关联研究确定为促成多基因T1 D风险，主要组织相容性 由于其对T细胞的胸腺选择的影响，MHC复合体仍然是最大的风险因素 受体（TCR）库和对外周T细胞活化的影响。尽管有这一关键环节， HLA-DR 4等高危等位基因如何影响TCR选择、激活阈值和组织分布。 我们令人信服的初步数据表明，风险相关的DR 4/DQ 8单倍型，这已被链接到 具有胰岛素自身反应性，在抗原呈递细胞（APC）表面高表达， 具有独特的TCR信号影响T细胞共刺激和分化的另外的基因变体也被发现。 在T1 D受试者中富集，但它们对T1 D自身免疫的机制贡献仍然很差 表征了项目3提议填补影响技术合作报告的这些关键方面的知识空白 在健康和疾病中的所有功能和激活需求。我们假设MHC风险，以及额外的 非MHC蛋白编码的风险变体，导致异常的T细胞活化和分化阈值， T细胞耐受性的丧失。具体地说，T1 D相关的风险变异改变了免疫球蛋白的功能亲合力。 MHC：肽：TCR复合物，以及SIRPG和CD 226分子中的风险变体改变T细胞信号传导，导致 自身反应性效应T细胞的活化以及调节性T细胞（Treg）的免疫调节缺陷。 这些研究旨在探讨MHC II类（Aim 1）和非MHC风险变异体在人类免疫系统中的作用机制。 (Aim 2）通过分析胰腺引流淋巴结（pLN）、脾和淋巴结转移来控制自身反应性T细胞。 T1 D受试者和有疾病发生风险的受试者的外周血（通过核心A采集）。 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 选择和分析与国家的最先进的单细胞分析和集中的功能测定利用这两个 基因型选择和基因编辑的样本。为了研究个体克隆和风险等位基因，我们将修改 通过慢病毒基因表达系统和CRISPR/Cas9的原代人T细胞特异性和功能 用于离体胰腺切片培养和体外同基因细胞建模系统的基因组工程， 分别与项目1和项目2合作。该项目的数据有望为T细胞 参与T1 D发病机制的激活检查点，将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，并确定靶向的途径， 治疗干预以阻止T1 D中β细胞的自身免疫破坏。