The role of Cdc42 and Rac in NK cell activation

描述(申请人提供)：自然杀伤(NK)细胞是免疫系统的中心效应器。它们在形成紧密的NK细胞/靶细胞对后杀死病毒感染和肿瘤靶细胞。有效的杀伤需要NK细胞极化到细胞界面。NK细胞极化的调节机制目前仍知之甚少。密切相关的Rho GTP酶CDc42和Rac是多种细胞类型中细胞极化的中央调节因子。CDc42和Rac的激活剂Vav和CDc42的即时效应蛋白Wiskott-Aldrich综合征蛋白(WASP)是有效的NK细胞杀伤所必需的。在IL-2培养的NK细胞系中，RAC是杀伤细胞所必需的。然而，一般情况下，CDC42和RAC在原代NK细胞中的作用尚不清楚。作为本申请的重点，我们将阐明这些角色。我们已经做好了这样做的准备。多年来，我们一直在研究原代细胞中NK细胞的极化和激活。在研究原代T细胞中的CDC42时，我们已经产生了多种用于操纵CDC42/RAC活性的试剂和活细胞生物传感器来确定它们的活性的亚细胞定位。在初步数据中，对CDC42和RAC活性的操纵影响了NK细胞的杀伤，支持了它们在初级NK细胞激活中的重要性。此外，CDC42和RAC在原代NK细胞/肿瘤细胞对中以重叠但不同的模式被激活。由于定位对活细胞的功能至关重要，这些模式可以指导对CDC42和Rac作用的有效研究。在第一个目标中，将评估NK细胞表面受体对CDC42和RAC的激活，重点是NKG2D。多种受体可激活NK细胞。NKG2D尤其令人感兴趣，因为它似乎偶联到在其他细胞类型中激活CDC42/RAC的信号通路，并且在抗癌NK活性中作为激活受体发挥着重要的生理作用。在第二个目标中，将通过短期和定量地操纵CDC42/RAC的活性来识别受CDC42/RAC调节的T细胞极化和激活的元件。总之，这些实验将表征CDC42/RAC的激活，并识别受CDC42/RAC调控的NK细胞功能的各个方面，重点是NKG2D。除了对CDC42/RAC功能的直接了解外，这些数据将为未来更全面地分析控制原代NK细胞中Rho GTP酶的分子途径奠定基础。此外，由于NK细胞极化是NK杀伤肿瘤细胞的关键和限制因素，我们的研究有望建立合理的方法来增强NK细胞极化，以促进抗肿瘤NK活性的治疗，我们将在未来进行测试。自然杀伤细胞是免疫系统的中心效应器，可以杀死病毒感染的细胞和肿瘤细胞。在这里，我们将研究它们的激活调节。对NK细胞功能调节的了解的提高有望导致治疗方法的改进，特别是在癌症的治疗方面。

DESCRIPTION (provided by applicant): 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.