Impact of MHC Genotype on Ex Vivo T cell Function in Type 1 Diabetes

MHC 基因型对 1 型糖尿病离体 T 细胞功能的影响

• 批准号: 8435673
• 负责人: John Christopher Love
• 金额: $ 387.68万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2017-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435673
• 关键词: Accounting; Address; Affinity; Alleles; Antigens; Autologous; B-Lymphocytes; Binding; Biological Assay; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Death; Cell physiology; Cells; Clonal Expansion; Cloning; Coculture Techniques; Cytolysis; Data; Dendritic Cells; Detection; Devices; Disease; Elements; Epitopes; Ethnic group; Frequencies; Future; Genes; Genetic; Genetic Risk; Genotype; Glass; Haplotypes; IL2RA gene; Image; Incidence; Individual; Insulin-Dependent Diabetes Mellitus; Interferons; Interleukin-10; Label; Ligands; Link; Linkage Disequilibrium; Love; Lymphocyte; MHC Class I Genes; MHC Class II Genes; Measures; Methods; Microbe; Molds; Molecular; Pathogenesis; Patients; Pattern; Peptide/MHC Complex; Peptides; Phenotype; Physiologic pulse; Population; Predisposition; Prevention approach; Proliferation Marker; Property; Resistance; Risk; Slide; Specificity; Staining method; Stains; Surface; System; T cell response; T-Cell Activation; T-Lymphocyte; TNFRSF10A gene; Technology; Testing; Thymus Gland; Virus; Work; cytokine; cytotoxicity; design; dimer; elastomeric; high risk; improved; interest; monocyte; new technology; novel; novel strategies; prevent; response

DESCRIPTION (provided by applicant): The MHC locus contributes significantly to the genetic risk for type 1diabetes (T1D), but the molecular mechanisms are not well understood. The central problem is that current experimental methods for characterization of self-reactive T cell populations are highly inadequate. These approaches, including tetramer labeling, intracellular cytokine staining (ICS) and ELISpot assays, enable sensitive detection of high- affinity microbe-specific T cell populations but are suboptimal for self-reactive T cells which tend to have low affinities for their peptide-MHC ligands. We will use a novel single-cell technology that enables sensitive detection of self-reactive T cells and generates a comprehensive body of data on surface phenotype, cytokine release and other functions, such as proliferation and cytotoxicity. A dense, elastomeric array of wells with subnanoliter volumes (nanowells) is generated by replica molding, and individual T cells are co-cultured with autologous mature dendritic cells pulsed with antigen. Many different cytokines are captured on a glass slide and quantified on a microarray scanner, while CD8 T cell cytotoxicity is quantified by imaging lysis of co- cultured target cells. T cells of interest can also be isolated for subsequent clonal expansion. This system provides a rapid and high-throughput method for ex vivo characterization of lymphocytes. Preliminary data show that this approach greatly increases the sensitivity of detection for self-reactive T cells and enables comprehensive assessment of their ex vivo functions. We will use this novel technology to address three fundamental questions on the mechanisms by which MHC genes confer susceptibility and resistance to T1D. First, it remains unknown whether there are important differences in CD4 and/or CD8 T cell functions in patients with distinct MHC haplotypes that confer different degrees of risk. We will compare cytokine patterns and CD8 T cell cytotoxicity in response to B cell antigens in patients who carry either DR3-DQ2 (DQ2) or DR4- DQ8 (DQ8) haplotypes or the highest risk DQ2/DQ8 haplotype. Distinct, yet complementary functions could account for the high risk conferred by heterozygosity for DQ2 and DQ8 haplotypes. Second, genetic data suggest that DQ trans-dimers (encoded in trans by different haplotypes) contribute to the high risk of patients with heterozygosity for DQ2/DQ8 haplotypes. We will directly test this hypothesis by cloning CD4 T cells from nanowells and testing their MHC-peptide specificity. Third, several haplotypes are known to confer dominant protection from T1D. Particularly important is the DR15-DQ6 haplotype because it reduces risk more than 30- fold and is common in populations with a high incidence of T1D. We will assess three possible mechanisms for dominant protection: epitope capture, deletion of particular effector T cell populations, or induction of b cell- specific T cels with regulatory functions that can control effector T cell responses. This highly novel approach will thus allow us to address central questions on the function of MHC genes in T1D. PUBLIC HEALTH RELEVANCE: This project focuses on the mechanisms for MHC-linked susceptibility to T1D. A novel single cell approach will be used to interrogate the function of ¿ cell specific CD4 and CD8 T cell populations in patients and normal subjects with different predisposing or protective MHC genotypes. These studies have the potential to impact the design of future prevention approaches and to improve prediction of T1D in susceptible populations.

描述（由申请人提供）：MHC 基因座对 1 型糖尿病 (T1D) 的遗传风险有显着影响，但其分子机制尚不清楚。核心问题是目前用于表征自身反应性 T 细胞群的实验方法非常不足。这些方法，包括四聚体标记、细胞内细胞因子染色 (ICS) 和 ELISpot 测定，能够灵敏地检测高亲和力的微生物特异性 T 细胞群，但对于自身反应性 T 细胞来说并不是最理想的，因为自身反应性 T 细胞往往对其肽-MHC 配体具有较低的亲和力。我们将使用一种新型单细胞技术，能够灵敏地检测自身反应性 T 细胞，并生成有关表面表型、细胞因子释放和其他功能（如增殖和细胞毒性）的全面数据。通过复制模制生成具有亚纳升体积（纳米孔）的致密弹性体阵列，并将单个 T 细胞与用抗原脉冲的自体成熟树突细胞共培养。许多不同的细胞因子被捕获在载玻片上并在微阵列扫描仪上进行定量，而 CD8 T 细胞的细胞毒性则通过共培养的靶细胞的成像裂解来定量。还可以分离感兴趣的 T 细胞用于随后的克隆扩增。该系统为淋巴细胞的离体表征提供了一种快速、高通量的方法。初步数据表明，这种方法大大提高了自身反应性T细胞检测的灵敏度，并能够对其离体功能进行全面评估。我们将利用这项新技术来解决有关 MHC 基因赋予 T1D 易感性和抗性机制的三个基本问题。首先，尚不清楚具有不同风险程度的不同 MHC 单倍型患者的 CD4 和/或 CD8 T 细胞功能是否存在重要差异。我们将比较携带 DR3-DQ2 (DQ2) 或 DR4-DQ8 (DQ8) 单倍型或最高风险 DQ2/DQ8 单倍型的患者对 B 细胞抗原的细胞因子模式和 CD8 T 细胞细胞毒性。不同但互补的功能可以解释 DQ2 和 DQ8 单倍型杂合性带来的高风险。其次，遗传数据表明，DQ 反式二聚体（由不同单倍型反式编码）导致 DQ2/DQ8 单倍型杂合性患者的高风险。我们将通过从纳米孔克隆 CD4 T 细胞并测试其 MHC 肽特异性来直接检验这一假设。第三，已知有几种单倍型可对 T1D 提供显性保护。特别重要的是 DR15-DQ6 单倍型，因为它可以将风险降低 30 倍以上，并且在 T1D 发病率高的人群中很常见。我们将评估显性保护的三种可能机制：表位捕获、特定效应 T 细胞群的删除或诱导具有可控制效应 T 细胞反应的调节功能的 b 细胞特异性 T 细胞。因此，这种高度新颖的方法将使我们能够解决 T1D 中 MHC 基因功能的核心问题。 公共卫生相关性：该项目重点关注与 MHC 相关的 T1D 易感性机制。将使用一种新颖的单细胞方法来探究 ¿ 具有不同易感性或保护性 MHC 基因型的患者和正常受试者中的细胞特异性 CD4 和 CD8 T 细胞群。 这些研究有可能影响未来预防方法的设计，并改善易感人群对 T1D 的预测。