ORIGIN AND DEVELOPMENT OF HUMAN GD T-CELLS

这项研究建议的长期目标是确定 人γ-βT细胞的生物学功能。伽马三角洲 人类的功能谱偏向于带有T细胞受体的细胞 (TCR)由一组可变(V)基因(V-Gamma9和V-Gamma9)编码 增量2)。对小鼠的伽马德尔塔T细胞的研究表明 靶向V基因重排、随机事件和胸腺选择可能 解释了伽马-德尔塔T亚型的高度有序生产 细胞。然而，伽马三角洲细胞生物学上的几个显著差异 在啮齿动物和人类身上的研究表明，伽马-德尔塔T的研究结果 在小鼠中进行的细胞个体发育不能外推到Gamma-Delta T 人类的细胞发育和功能。根据我们自己的初步调查 我们正在进行的研究表明，成年人的伽马-三角洲T细胞库 作为至少部分地发生的选择事件的结果， 在胎儿发育期间胸腺以外的组织中。一旦形成，这些 细胞的功能主要是作为胎儿免疫系统的一部分。这 因此，假设意味着V-Gamma9/V-的个体发育- Delta2+Gamma-Delta曲目与他们的专业 功能。为了验证这一假设，我们建议进行研究，以检验 人V-Gamma9/V-delta2+γ-Delta T的发育及其特异性 具有以下特定目标的细胞：1.确定处于什么阶段 在妊娠和胎儿组织中，V-Gamma9/V-Delta2+Gamma-Delta T 首先生成单元格。特别是，我们将确定这一点是否 细胞群来自胸腺或胸腺外，如果 胎儿来源的γ-Delta T细胞群是成人的一部分 曲目。2.确定V-Gamma 9/V-识别的配体(S) Delta2-TCR由一群出现的伽马-Delta T细胞表达， 在正常的胎儿发育过程中占主导地位。 在胎儿组织中原位检测和可视化表达该基因的细胞 编码V-Gamma9/V-delta2-TCR敏感聚合酶链的基因 反应(PCR)/Southern印迹和原位杂交分析将 分别使用。DNA测序将用于验证PCR- 扩增的TCR，以确定结构多样性，并识别独特的 区分表达这一点的细胞谱系的序列 在胎儿发育过程中不同组织中的受体。一个 免疫磁分离程序将有助于分离这种病毒 细胞群体，允许TCRGamma-Delta基因表达 与受体蛋白表达及其表型的相关性 人物刻画。S抗原与猪传染性支气管炎病毒的V-Gamma9/V-Delta2 TCR反应 胎儿T细胞将被鉴定，并随后在 根据它们与完整的、组装的伽马-增量TCR的反应性 以可溶形式，或由胎儿伽马表达的细胞相关形式- 德尔塔T细胞克隆。确定人类伽马-三角洲T细胞的起源 (特定目标1)和它们的分子相互作用 生成(特定目标2)不仅对确定 γ-DeltaTCR识别抗原的性质和人类的功能 伽马-德尔塔T细胞，也是为了了解 以维持伽马-德尔塔T细胞亚群内的自我耐受性。这是 尤其重要的是最近的临床研究表明 γ-Delta T细胞在某些疾病发病机制中的作用 自身免疫性疾病。

The long-term objective of this research proposal is to determine the biological function of gamma-delta T cells in man. The gamma-delta repertoire in humans is biased towards cells bearing a T cell receptor (TCR) encoded by a single set of variable (V) genes (V-gamma9 and V- delta2). Studies of gamma-delta T cells in mice have suggested that targeted V-gene rearrangement, stochastic events and thymic selection may account for the highly ordered production of subtypes of gamma-delta T cells. However, several striking differences in gamma-delta cell biology in rodents and man suggest that the results from studies of gamma-delta T cell ontogeny carried out in mice cannot be extrapolated to gamma-delta T cell development and function in man. Based upon our own preliminary studies we are proposing that the gamma-delta T cell repertoire of adults is generated as a result of selection events that occur, at least in part, in tissues outside the thymus during fetal development. Once formed, these cells function primarily as part of the fetal immune system. This hypotheses implies, therefore, that the ontogeny of the V-gamma9/V- delta2+gamma-delta repertoire is intimately related to their specialized function. To test this hypothesis, we propose to perform studies examining the development and specificity of human V-gamma9/V-delta2+ gamma-delta T cells which have the following specific aims: 1. To identify at what stage in gestation and in which fetal tissues V-gamma9/V-delta2+ gamma-delta T cells are first generated. In particular, we will determine whether this population of cells is thymically- or extrathymically-derived and, if fetal-derived gamma-delta T cell populations are part of the adult repertoire. 2. To identify the ligand(s) recognized by a V-gamma9/V- delta2-TCR expressed by a population of gamma-delta T cells that arise, and predominate, during normal fetal development. To detect and visualize cells in situ, in fetal tissues, that express the genes encoding the V-gamma9/V-delta2-TCR, sensitive polymerase chain reaction (PCR)/Southern blotting and in situ hybridization assays will be used, respectively. DNA-sequencing will be used to authenticate PCR- amplified TCRs, to determine the structural diversity and, identify unique sequences that distinguish the repertoire of cells that express this receptor in different tissues throughout fetal development. An immunomagnetic separation procedure will facilitate the isolation of this population of cells, allowing TCRgamma-delta gene expression to be correlated with receptor protein expression and for their phenotypic characterization. The antigen(s) reactive with a V-gamma9/V-delta2 TCR of fetal T cells will be identified and subsequently characterized on the basis of their reactivity with either an intact, assembled gamma-deltaTCR in soluble form, or the cell-associated form expressed by fetal gamma- delta T cell clones. Defining the origin of human gamma-delta T cells (specific aim 1) and the molecular interactions involved in their generation (specific aim 2) are not only essential for determining the nature of antigens recognized by gamma-deltaTCR and the function of human gamma-delta T cells but also for understanding the mechanisms that operate to maintain self-tolerance within the gamma-delta T cell subset. This is of particular importance in light of recent clinical studies demonstrating the involvement of gamma-delta T cells in the pathogenesis of certain autoimmune disorders.