Regulation of NK Cell Activation by DOCK8-Mediated Pathways

描述(由申请人提供)：NK细胞是淋巴细胞的一个亚群，其独特的受体有助于检测感染、转化或‘应激’细胞。这种免疫识别随后导致NK细胞介导的细胞毒作用的发展，或产生激活免疫系统其他组成部分的细胞因子和趋化因子。高IgE综合征(HIEs)患者存在免疫失调，可受到顽固性皮肤疱疹病毒和乳头瘤病毒感染的影响。在这些患者中发现的一个主要的遗传异常是编码胞质分裂决定因子8(DOCK8)的基因的缺失或功能丧失突变，DOCK8是CDC42的一种鸟嘌呤核苷酸交换因子(Gef)。DOCK8缺陷患者反复皮肤病毒感染的高发生率提示自然杀伤(NK)细胞功能缺陷。事实上，其他人和我们最近的研究表明，DOCK8缺陷的NK细胞的溶解功能降低，粘附性降低，F-肌动蛋白在细胞毒性突触聚集，并且不能极化溶解颗粒朝向靶细胞。然而，DOCK8如何调控NK细胞杀伤发展中的许多关键步骤尚不清楚。我们的中心假设是DOCK8相互作用体协调肌动蛋白和微管细胞骨架的调节，以促进NK细胞的极化和作用 NK细胞杀伤活性。根据这一建议中包括的初步数据，我们假设：(A)CDC42的DOCK8激活对于NK细胞杀伤的发展至关重要；(B)DOCK8与WASP的相互作用对于其在NKIS的定位和F-肌动蛋白的产生至关重要；(C)DOCK8的TALI募集介导了NK细胞与靶细胞的黏附；(D)CCDC88B是一种与DOCK8相互作用的蛋白质，它与DOCK8相互作用，参与MTOC极化和溶解颗粒聚集；(E)CCDC88B通过其钩状结构域与微管直接结合；(F)Septins是DOCK8相互作用蛋白，通过其对微管和细胞骨架的影响来调节NK细胞的杀伤。为了验证这些假说，我们将：(1)确定DOCK8调节F-肌动蛋白动力学和细胞黏附的机制；(2)确定CCDC88B在NK细胞介导的杀伤中的作用；(3)确定Septins在调节NK细胞中的作用 细胞毒性。这些实验结果将为理解参与调节NK细胞效应器功能的分子事件提供实验基础，并将在更广泛的背景下促进我们对细胞激活中导致F-肌动蛋白调节、MTOC极化和颗粒吐出的基本过程的理解。此外，通过这些研究获得的信息可能会指导DOCK8如何在其他免疫细胞中发挥作用，并为其在HIE中的作用提供新的见解。

DESCRIPTION (provided by applicant): NK cells are a subpopulation of lymphocytes whose unique receptors facilitate the detection of infected, transformed, or 'stressed' cells. This immune recognition subsequently leads to the development of NK cell- mediated cytotoxicity or the generation of cytokines and chemokines that activate other components of the immune system. Patients with hyper-IgE syndrome (HIES) have immune dysregulation and can be affected by recalcitrant cutaneous herpes virus and papillomavirus infections. A major genetic abnormality found in these patients is deletion or loss-of-function mutations in the gene encoding Dedicator of Cytokinesis 8 (DOCK8), a guanine nucleotide exchange factor (GEF) for Cdc42. The high incidence of recurrent cutaneous viral infections in DOCK8-deficient patients is suggestive of defects in natural killer (NK) cell function. In fact, others and we have recently shown that NK cells deficient in DOCK8 have reduced lytic function, decreased adhesion, F-actin accumulation at the cytotoxic synapse and an inability to polarize lytic granules toward the target cell. However, how DOCK8 can regulate so many critical steps in the development of NK cell killing is not known. It is our central hypothesis that the DOCK8-interactome coordinates the regulation of the actin and microtubule cytoskeletons to facilitate NK cell polarization and effect NK cellular cytotoxicity. Based on the preliminary data included in this proposal, we hypothesize that: (a) DOCK8 activation of CDC42 is critical to the development of NK cell killing; (b) DOCK8 interaction with WASP is critical for its localization and F-actin generation at the NKIS; (c) tali recruitment by DOCK8 mediates NK cell - target adhesion; (d) CCDC88B, a hematopoietically expressed protein that interacts with DOCK8 is involved in MTOC polarization and lytic granule clustering; (e) CCDC88B directly binds microtubules through its hook-like domain; (f) septins are DOCK8 interacting proteins that regulate NK cell killing through their effects on the microtubule and actin cytoskeletons. In order to test these hypotheses we will: (1) Determine the mechanism by which DOCK8 regulates F-actin dynamics and cell adhesion; (2) Define the role of CCDC88B during NK cell-mediated killing; (3) Determine the role of septins in the regulation of the NK cell cytotoxicity. The outcome of the proposed experiments will provide an experimental basis for understanding the molecular events that are involved in the regulation of NK cell effector functions, and will, in a broader context, advance our understanding of fundamental processes in cellular activation leading to F-actin regulation, MTOC polarization and granule exocytosis. Moreover, the information obtained through these studies will likely instruct how DOCK8 is functioning in other immune cells and provide new insight into its role in HIES.