ROLE OF RAS IN T-CELL FATE DETERMINATION

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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.