Natural killer (NK) cells are central effectors of the immune system. They kill virally infected and tumor target cells following formation of tight NK cell/target cell couples. Effective killing requires NK cells to polarize towards the cellular interface. The regulation of NK cell polarization is still poorly understood. The closely related Rho GTPases Cdc42 and Rac are central regulators of cellular polarization in numerous cell types. An activator of Cdc42 and Rac, Vav, and an immediate effector of Cdc42, Wiskott-Aldrich Syndrome protein (WASP), are required for effective NK cell killing. Rac is required for killing in IL-2-cultured NK cell lines. However, roles of Cdc42 in general and of Rac in primary NK cells are less clear. As the focus of this application, we will elucidate such roles. We are well positioned to do so. We have studied NK cell polarization and activation in primary cells for years. Studying Cdc42 in primary T cells, we have generated multiple reagents for the manipulation of Cdc42/Rac activity and live cell biosensors to determine the sub-cellular localization of their activity. In preliminary data, manipulation of Cdc42 and Rac activity impacted NK cell killing, supporting their importance in primary NK cell activation. In addition, Cdc42 and Rac were activated in primary NK cell/tumor cell couples in overlapping yet distinct patterns. As localization is critical for function in live cells, these patterns can guide an efficient investigation of roles of Cdc42 and Rac. In the first aim, activation of Cdc42 and Rac by NK cell surface receptors will be assessed with an emphasis on NKG2D. Numerous receptors can active NK cells. NKG2D is of particular interest, as if couples to signaling pathways known to active Cdc42/Rac in other cell types, and as it plays a major physiological role as an activating receptor in anti-cancer NK activity. In the second aim, elements of T cell polarization and activation that are regulated by Cdc42/Rac will be identified by manipulating Cdc42/Rac activity short-term and quantitatively. Together these experiments will characterize Cdc42/Rac activation and identify aspects of NK cell function that are regulated by Cdc42/Rac with an emphasis on NKG2D. Beyond the immediate insight into Cdc42/Rac function, these data will generate the foundation for a future, more comprehensive analysis of the molecular pathways governing Rho GTPases in primary NK cells. Moreover, as NK cell polarization is critical for and limiting in NK killing of tumor cells, our studies can be expected to establish rational means to enhance NK cell polarization to promote anti-tumor NK activity therapeutically, as we will test in the future. Natural killer cells are central effectors of the immune system that kill virally infected and tumor cells. Here the regulation of their activation will be studied. An improved understanding of the regulation of NK cell function can be expected to lead to improved therapies, in particular in the treatment of cancers. ? ? ? ?