IDENTIFICATION OF GENES THAT REGULATE T CELL DEVELOPMENT

作为复杂的细胞相互作用和受体介导的位点， 导致成熟T细胞产生的信号事件，胸腺 是导致原发性免疫缺陷的突变的可能靶点。 虽然许多细胞表面标记物可用于描绘阶段 关于胸腺中T细胞的发育，我们知之甚少。 调节这些过程的潜在分子事件。这是 特别是对于正选择， T细胞抗原受体（TCR）的结果是胸腺细胞的发育- 介导的激活。我们已经组装了一套独特的工具，并提供一个 一种独特的方法来缩小对基因的搜索， 在积极的选择。而不是试图分离所有的基因， 表达与T细胞的特定发育阶段相关 成熟，我们的方法旨在确定基因的调控， 通过TCR激活T细胞所需的特定信号通路， 细胞发育为了实现这一目标，我们将利用 未成熟的胸腺细胞系DPK，我们以前已经分离， 表征了这些细胞保持分化的能力， 文化，我们已经表明，这种分化是Ras和 钙调磷酸酶依赖性，如正常胸腺细胞的阳性选择。在 此外，表达活性Ras的DPK细胞显示出部分的 分化表型利用抑制消减杂交 和表达活性Ras或显性失活突变体的DPK细胞， Ras，我们将分离出由TCR激活特异性诱导的基因， Ras/MAP激酶通路的双阳性胸腺细胞。类似的 描述了一种用于分离特异性诱导的基因的方法， TCR介导的钙调磷酸酶激活。筛查这些的策略 基因的差异表达序列， 概述了在T细胞发育中的重要作用。体外基因 转基因转移以及转基因和基因靶向策略将 最终用于确定这些基因的功能。

As the site of complex cellular interactions and receptor mediated signaling events that lead to the production of mature T cells, the thymus is a likely target for mutations that result in primary immunodeficiency. Although many cell surface markers are available to delineate the stages of T cell development in the thymus, we know very little about the underlying molecular events that regulate these processes. This is particularly true for positive selection, the differentiation of developing thymocytes as a consequence of T cell antigen receptor (TCR)- mediated activation. We have assembled a unique set of tools and offer a unique approach to narrow the search for genes that play a critical role in positive selection. Rather than attempt to isolate all genes whose expression is associated with a particular development stage of T cell maturation, our approach is designed to identify genes that are regulated by TCR activation of specific signaling pathways that are required for T cell development. In order to accomplish this, we will take advantage of the immature thymocyte cell line DPK, that we have previously isolated and characterized. These cells maintain the ability to differentiate in culture, and we have shown that this differentiation is both Ras and calcineurin dependent, as is positive selection of normal thymocytes. In addition, DPK cells that express active Ras show a partially differentiated phenotype. Utilizing suppression subtractive hybridization and DPK cells that expresses active Ras or a dominant negative mutant of Ras, we will isolate genes that are specifically induced by TCR activation of the Ras/MAP kinase pathway in double positive thymocytes. A similar approach is described for isolating genes that are specifically induced by TCR mediated Calcineurin activation. A strategy for screening these differentially expressed sequences for genes that are candidates to play an important role in T cell development is outlined. In vitro gene transgene transfer and transgenic and gene targeting strategies will ultimately be used to determine the function of these genes.