IN VIVO ANALYSIS OF INTRACELLULAR SIGNALS CONTROLLING LYMPHOCYTE

我们目前的努力集中在细胞内 控制淋巴细胞相关现象的信号 发育分化，如胸腺阳性和阴性 选择、淋巴细胞活化和免疫记忆的产生 in vivo.先前的分析表明，所有这些现象 通常需要细胞表面抗原受体之间的相互作用 和它们的配体。正是这些相互作用启动了一个激活过程 包括酪氨酸激酶和G 协调作用以决定细胞反应的蛋白质。 在过去一年，我们主要集中在功能分析， c-cbl和Grb 2缺陷小鼠的研究 模型cb 1是原癌基因c-cbl的产物， 已显示参与T细胞受体（TCR）介导的 发信号。c-cbl缺陷小鼠的胸腺细胞显示 多种蛋白质上的高酪氨酸磷酸化，包括 Zap 70酪氨酸激酶，MAP激酶活性的上调和 磷脂酶C-g1（PLC-g1）和PI-3激酶活性抑制 通过TCR刺激。而负选择的 胸腺细胞保持完整，TCR阳性选择 转基因T细胞在这些小鼠中显著增强。从 这些结果表明，Cbl在 协调TCR介导的多种信号传导功能 途径和微调的信号阈值胸腺 选择.我们还建立了另一种缺乏 衔接分子Grb 2。分析表明，这种突变 在胚胎发育早期导致胚胎死亡。我们 初步分析显示没有纯合Grb 2突变胚胎 怀孕第10天后这一结果表明， grb 2分子与早期胚胎发生。我们现在还在 产生具有条件Grb 2的突变小鼠的过程 缺陷最后，为了研究体内T细胞活化，我们 通过引入一种cDNA， 通过基因打靶将GFP编码入IL-2基因位点。我们 研究表明，GFP荧光在T 细胞活化，并且主要与IL-2共表达于单个- 细胞水平。在通过T细胞抗原受体刺激后， 抗原特异性CD 4+细胞分离成不同的GFP+和GFP- 群体，表明活化的幼稚T细胞可以被分裂为 IL-2生产者和非生产者。因此，原代T细胞 通常双等位基因表达IL-2基因，并且在 IL-2的生产。另外，这些老鼠 通过单细胞非侵入性检测IL-2的产生， 分析这些细胞随后的分化命运， 免疫反应发展。在未来，我们计划 从这些突变动物中建立各种细胞系。详细 将进行单个信号通路的分析 利用这些体外系统进行生化反应。此外，我们将 我还将这些突变动物与其他动物模型杂交， 了解这些分子对免疫系统的贡献 生理学和病理学。

Our current effort is focusing on the intracellular signals controlling phenomena associated with lymphocyte development differentiation, such as thymic positive and negative selection, lymphocyte activation and generation of immune memory in vivo. Previous analysis indicated that all these phenomena generally require interactions between cell surface antigen receptors and their ligands. It is these interactions that initiate an activation cascade of signaling molecules including tyrosine kinases and G proteins that act concordantly to determine the cellular responses. In the past year, we focused mainly on functional analysis of the c-cbl and Grb2 in using established c-cbl and Grb2 deficient mouse models. Cb1 is the product of the proto-oncogene c-cbl and has been shown to be involved in T cell receptor (TCR) mediated signaling. Thymocytes from c-cbl deficient mice showed hyper-tyrosine phosphorylation on multiple proteins including Zap70 tyrosine kinase, up- regulation of MAP kinase activity and inhibition of phospholipase C-g1(PLC-g1) and PI-3 kinase activities upon stimulation through TCR. While negative selection of the thymocytes remained intact, positive selection of the TCR transgenic T cells was significantly enhanced in these mice. From these results we concluded that Cbl plays a novel role in orchestrating functions of the TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection. We also generated another mouse model deficient in the adaptor molecules Grb2. Analysis indicated that this mutation causes an embryonic lethality at early embryo development. Our preliminary analysis revealed no homozygous Grb2 mutant embryo after day 10 of the pregnancy. This result indicates a critical role of Grb2 molecules for the early embryo-genesis. We are now still in the process of generating a mutant mice with conditional Grb2 deficiency. Finally, in order to study T cells activation in vivo we have generated a mutant mouse strain by introducing a cDNA encoding GFP into the IL-2 gene locus through gene targeting. Our studies indicated that GFP fluorescence is readily detectable upon T cell activation, and is mostly co-expressed with IL-2 at the single- cell level. Upon stimulation through the T cell antigen receptor, antigen-specific CD4+ cells separate into distinct GFP+ and GFP- populations, indicating that activated naive T cells can be divided into IL-2 producers and non-producers. Thus, primary T cells usually express the IL-2 gene biallelically, and are heterogeneous in terms of IL-2 production. Besides, these mice uniquely allow non-invasive detection of IL-2 production by single cells and analysis of the subsequent differentiative fate of these cells as an immune response develops. In the future, we are planning to establish various cell lines from these mutant animals. Detailed analysis of individual signaling pathway will be carried out biochemically using these in vitro systems. Additionally, we will also cross these mutant animals to other animal models in order to understand the contribution of these molecules to immune system physiology and pathology.