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

描述（由申请人提供）：MHC位点对1型糖尿病（T1D）的遗传风险有显著影响，但其分子机制尚不清楚。核心问题是，目前的实验方法表征自身反应性T细胞群是非常不足的。这些方法，包括四聚体标记，细胞内细胞因子染色（ICS）和ELISpot检测，能够灵敏地检测高亲和力的微生物特异性T细胞群，但对于对其肽- mhc配体具有低亲和力的自反应性T细胞来说，这是次优的。我们将使用一种新的单细胞技术，能够灵敏地检测自反应性T细胞，并生成关于表面表型、细胞因子释放和其他功能（如增殖和细胞毒性）的综合数据。通过复制模塑产生亚纳升体积的密集弹性孔阵列（纳米孔），单个T细胞与抗原脉冲的自体成熟树突状细胞共培养。许多不同的细胞因子在载玻片上被捕获，并在微阵列扫描仪上被量化，而CD8 T细胞的细胞毒性是通过成像裂解共培养的靶细胞来量化的。感兴趣的T细胞也可以被分离出来进行后续克隆扩增。该系统为淋巴细胞的体外鉴定提供了一种快速、高通量的方法。初步数据表明，这种方法大大提高了检测自身反应性T细胞的灵敏度，并能够全面评估其体外功能。我们将使用这项新技术来解决MHC基因赋予T1D易感性和抗性机制的三个基本问题。首先，在具有不同MHC单倍型的患者中，CD4和/或CD8 T细胞功能是否存在重要差异尚不清楚，从而导致不同程度的风险。我们将比较携带DR3-DQ2 （DQ2）或DR4- DQ8 （DQ8）单倍型或高风险DQ2/DQ8单倍型的患者对B细胞抗原的细胞因子模式和CD8 T细胞毒性。不同的，但互补的功能可以解释DQ2和DQ8单倍型的杂合性所赋予的高风险。其次，遗传数据表明，DQ反式二聚体（由不同的单倍型编码）是DQ2/DQ8单倍型杂合性患者的高风险因素。我们将通过从纳米孔中克隆CD4 T细胞并测试其mhc肽特异性来直接验证这一假设。第三，已知几种单倍型具有对T1D的显性保护。尤其重要的是DR15-DQ6单倍型，因为它降低了30倍以上的风险，并且在T1D高发人群中很常见。我们将评估三种可能的显性保护机制：表位捕获，特定效应T细胞群的删除，或诱导具有控制效应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关于MHC基因在T1D中的功能的核心问题。

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.