ORIGIN AND DEVELOPMENT OF HUMAN GD T-CELLS

本研究建议的长期目标是确定 人类γ-δ T细胞的生物学功能。 在人类中的所有成分偏向于携带T细胞受体的细胞 (TCR)由单组可变（V）基因（V-γ 9和V-γ 9）编码， δ 2）。对小鼠γ-δ 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γ-δ T亚型的高度有序产生 细胞然而，在γ-δ细胞生物学中， 在啮齿动物和人类中的研究结果表明， 在小鼠中进行的细胞个体发育不能外推到γ-δ T细胞 细胞的发育和功能。基于我们自己的初步研究， 研究表明，成年人的γ-δ T细胞库 作为选择事件的结果而产生，至少部分地， 在胎儿发育期间胸腺外的组织中。一旦形成，这些 细胞主要作为胎儿免疫系统的一部分发挥作用。这 因此，假设意味着，V-gamma 9/V- delta 2 +gamma-delta库与它们的特化密切相关， 功能为了验证这一假设，我们建议进行研究， 人V-gamma 9/V-delta 2 + gamma-deltaT基因构建及其特异性研究 具有以下具体目标的细胞：1.确定处于哪个阶段 妊娠期且胎儿组织V-gamma 9/V-delta 2 + gamma-deltaT 首先生成细胞。特别是，我们将确定这是否 细胞群是胸腺或胸腺外来源的，如果 胎儿来源的γ-δ T细胞群是成人的一部分， 保留曲目。2.为了鉴定由V-γ 9/V- 由产生的γ-δ T细胞群表达的δ 2-TCR， 在正常的胎儿发育过程中占主导地位。 为了检测和可视化胎儿组织中原位细胞， 编码V-γ 9/V-δ 2-TCR敏感聚合酶链的基因 将进行PCR/Southern印迹和原位杂交试验， 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， 区分表达这种基因的细胞库的序列， 在整个胎儿发育过程中不同组织中的受体。一个 免疫磁性分离程序将有助于分离这种 细胞群，允许TCR γ-δ基因表达， 与受体蛋白表达和表型相关 特征化所述抗原与V-gamma 9/V-delta 2 TCR反应， 胎儿T细胞将被识别并随后在 基于它们与完整的、组装的γ-δ TCR的反应性 以可溶形式，或由胎儿γ- δ T细胞克隆。定义人类γ-δ T细胞的起源 （具体目标1）和分子相互作用参与其 （具体目标2）不仅是确定 γ-δ TCR识别的抗原的性质和人免疫球蛋白的功能 γ-δ T细胞，同时也有助于理解 以维持γ-δ T细胞亚群内的自身耐受性。这是 鉴于最近的临床研究表明， γ-δ T细胞参与某些 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.