ROLE OF RAS IN T-CELL FATE DETERMINATION

我已经提出了一系列实验，旨在了解在T细胞发育的特定时间点控制细胞命运的细胞内信号，当时对未成熟的双阳性细胞进行TCR活性筛选。基于我们之前的结果，潜在的假设是，不同的细胞命运决定是由本质上不同的信号事件驱动的。在第一个目标中，我们更详细地研究了RAS及其效应器在正选择中的作用，使用我们以前产生的显性阴性RAS小鼠与表达MAPK超敏变体的转基因小鼠相结合，以解决MAPK是否是唯一对正选择重要的RAS效应器。如果不是，我们建议进行一系列实验，使用逆转录病毒感染系统，测试不同的RAS效应突变体拯救dnRas的能力。利用在这些实验中获得的结果，我们将更详细地定义参与正选择的RAS信号通路的组成部分，并试图识别由RAS诱导并参与正选择的基因。第二个目的是为了了解不同的信号通路对CD4或CD8谱系的承诺的贡献。实验的重点是Lck在这一过程中的作用，并利用生化和遗传学方法测试RAS/MAPK级联是否是Lck在这一过程中的下游靶点。这项资助的第三个目的是通过产生表达这种酶的显性、负性和结构性活性突变的转基因小鼠来分析PI3-K在T细胞发育中的作用。我们利用转基因模型研究了pI3-K在正选择和负选择中的可能作用。鉴于PI3-K在细胞存活中的作用，我们将使用体内和体外方法分析它是否影响胸腺细胞的存活。这些研究应该能让我们更好地理解在T细胞发育过程中控制正负选择的机制，更广泛地说，可以为如何通过TCR实现差异信号提供线索。拥有不同的小鼠品系将允许直接比较它们的表型，并打开进行遗传实验的可能性，以揭示T细胞发育过程中不同信号转导途径之间的关系。

I have proposed a set of experiments aimed to understand the intracellular signals that control cell fate determination at a defined point in T cell development, when immature double positive cells are screened for the reactivity of their TcR. The underlying hypothesis, based on our previous results, is that alternative cell fate decisions are driven by qualitatively different signaling events. In the first aim, we investigate in more detail the role of Ras and its effectors in positive selection using our previously generated dominant negative Ras mice in combination with transgenic mice that express a hypersensitive variant of MAPK, to address whether MAPK is the only Ras effector important for positive selection. In case it is not, we propose a series of experiments that, using a retroviral infection system, test different Ras effector mutants for their ability to rescue dnRas. Using the results obtained in these experiments, we will define in more detail the components of the Ras signaling pathway involved in positive selection and try to identify genes induced by Ras and involved in positive selection. The second aim is directed at understanding the contribution of different signaling pathways to the commitment to either the CD4 or CD8 lineages. The experiments focus on the role of Lck in this process, and test whether the Ras/MAPK cascade is a downstream target of Lck in this process using biochemical and genetic approaches. The third aim of this grant proposes to analyze the role of PI 3-K in T cell development by generating transgenic mice expressing dominant negative and constitutively active mutants of this enzyme. We address the possible role of PI 3-K in positive and negative selection using transgenic models. Given the role of PI 3-K in cell survival we will analyze whether it affects thymocyte survival, using both in vivo and in vitro approaches. These studies should provide us with a better understanding of the mechanisms that control positive and negative selection during T cell development, and, more widely, could provide clues as to how differential signaling through the TCR is achieved. Having the different mice strains will allow a direct comparison of their phenotype, as well as open the possibility of performing genetic experiments to unravel the relationships among the different signal transduction pathways during T cell development.