IDENTIFICATION OF GENES THAT REGULATE T CELL DEVELOPMENT

作为复杂细胞相互作用和受体介导的部位 导致成熟T细胞产生的信号事件，胸腺 是导致原发免疫缺陷的突变的可能靶点。 尽管有许多细胞表面标记物可以用来描绘阶段 关于胸腺中T细胞的发育，我们知道的很少 调节这些过程的潜在分子事件。这是 对于正向选择来说尤其如此，区分 T细胞抗原受体(TCR)导致胸腺细胞发育 中介激活。我们组装了一套独特的工具，并提供 缩小关键基因搜索范围的独特方法 在积极选择中。而不是试图分离所有其基因 表达与T细胞的特定发育阶段有关 成熟，我们的方法是为了识别受调控的基因 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，我们之前分离和 特色化的。这些细胞保持分化的能力。 文化，我们已经表明，这种区别既是RAS，也是 钙调神经磷酸酶依赖，与正常胸腺细胞的阳性选择一样。在……里面 此外，表达活性RAS的DPK细胞表现出部分 分化的表型。利用抑制性消减杂交 和表达活性RAS或显性负性突变体的DPK细胞 RAS，我们将分离由TCR激活特异性诱导的基因 双阳性胸腺细胞中RAS/MAP激酶通路的表达。一种类似的 描述了一种分离特定诱导基因的方法 TCR介导钙调神经磷酸酶激活。一个筛选这些人的策略 候选基因的差异表达序列 概述了它在T细胞发育中的重要作用。体外基因 转基因转移、转基因和基因靶向策略将 最终被用来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.