GENETIC ANALYSIS OF THYMOCYTE DEVELOPMENT

研究的目的是调查细胞和遗传事件 控制着正常T细胞的发育转基因和基因打靶 方法用于分析已知基因的功能和各种 分子技术（例如，RT-PCR，基因克隆）被用于 鉴定参与胸腺细胞发育的新基因。电流 研究集中在三个主要领域：（一）T细胞抗原的作用 受体（TCR）信号转导在胸腺细胞成熟。在成熟的T 细胞，TCR转导对T细胞活化重要的信号， 细胞介导的免疫和未成熟T细胞中的TCR信号是必需的 胸腺细胞发育和胸腺（阳性和阴性） 选择. TCR由多个潜在的信号转导通路组成， 亚基（CD 3链和一个或多个ζ家族成员， 蛋白质; ζ、η和Fc γ），其将TCR偶联至细胞内 通过保守功能序列的信号转导途径 （基于免疫受体酪氨酸的活化基序; ITAM）。以确定是否 TCR信号转导亚单位执行不同或类似的功能， 我们已经：a）检查了它们在T细胞中的作用， 通过产生zeta/eta/Fc γ缺陷小鼠进行个体发育， 通过基因靶向的CD 3-γ δ缺陷小鼠，B）遗传学上 用编码野生型或 单个zeta家族或CD 3蛋白的信号传导缺陷形式， c）检测胸腺细胞中3种ζ-链ITAM的功能 通过转基因重建的zeta缺陷型 小鼠这些研究表明，zeta家族和CD 3链的表达 是正常T细胞发育所必需的，但ζ链信号是 没有特别要求。因此，CD 3亚基ITAM可以与CD 3亚基ITAM结合， 胸腺细胞成熟所必需的信号。 然而，一个直接 TCR-（zeta）ITAM的总数与TCR-ITAM的表达之间存在相关性。 和胸腺细胞选择的效率表明， TCR内的ITAM起放大信号传导应答的作用。 这些 结果证明了以前未实现的多个TCR ITAM的作用 在胸腺细胞选择和鉴定信号放大功能中 在T细胞库的选择中。 (II)其他信号的作用 转导蛋白在T细胞发育中的作用正在由 转基因/敲除小鼠的产生。其中包括CD 5， 与TCR不同的受体，也含有ITAM样序列 但作为TCR信号传导的负调节剂。(III)新的基因， 在胸腺细胞发育或T细胞活化中具有潜在功能 正在被确认。一种新的淋巴特异性激酶Txk， 磷酸酶PTPK 1已被克隆，并被生化和转基因 正在采用各种方法来分析这些蛋白质的功能。 一个类似的方法被用来识别同源异型盒的成员 在T细胞个体发育过程中表达的基因家族。

Research is directed at investigating the cellular and genetic events that control normal T cell development. Transgenic and gene-targeting approaches are used to analyze the function of known genes and various molecular techniques (e.g.,RT-PCR, gene cloning) are being employed to identify novel genes that participate in thymocyte development. Current studies are focused in three major areas: (I) Role of T cell antigen receptor (TCR) signal transduction in thymocyte maturation. In mature T cells, the TCR transduces signals important for T cell activation and cell mediated immunity and in immature T cells TCR signals are required for thymocyte development and for thymic (positive and negative) selection. The TCR is composed of multiple potential signal transducing subunits (the CD3 chains and one or more members of the zeta-family of proteins; zeta, eta and Fcgamma) that couple the TCR to intracellular signal transduction pathways via conserved functional sequences (Immunoreceptor Tyrosine based Activation Motifs; ITAMs). To determine if the TCR signal transducing subunits perform distinct or analogous functions in development we have: a) examined their role in T cell ontogeny by generating zeta/eta/Fcgamma deficient mice and CD3-gammadeltaepsilon deficient mice by gene targeting, b) genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient forms of the individual zeta-family or CD3 proteins, and c) examined the function of the 3 zeta-chain ITAMs in thymocyte development and selection by transgene reconstitution of zeta-deficient mice. These studies reveal that expression of zeta-family and CD3 chains is required for normal T cell development but that zeta chain signals are not specifically required. Thus the CD3 subunit ITAMs can transduce all of the signals necessary for thymocyte maturation. However, a direct relationship was observed between the total number of TCR-(zeta) ITAMs and the efficiency of thymocyte selection demonstrating that the multiple ITAMs within the TCR function to amplify the signaling response. These results demonstrate a previously unrealized role for multiple TCR ITAMs in thymocyte selection and identify a function for signal amplification in selection of the T cell repertoire. (II) The role of other signal transducing proteins in T cell development is being examined by the generation of transgenic/knockout mice. These include CD5, a surface receptor distinct from the TCR that also contains an ITAM-like sequence but acts as a negative regulator of TCR signaling. (III) Novel genes that have potential functions in thymocyte development or T cell activation are being identified. A new lymphoid-specific kinase, Txk, and phosphatase PTPK1 have been cloned and biochemical and transgenic approaches are being employed to analyze the function of these proteins. A similar approach is being employed to identify members of the homeobox gene family that are expressed during T cell ontogeny.