Inhibition of Natural Killer Cell Function

与B和T细胞不同，NK细胞不表达抗原特异性受体，但它们可以消除病毒感染的细胞和癌细胞，而不伤害正常细胞。在靶细胞识别中提供特异性的一个重要组成部分是通过识别称为I类主要组织相容性复合体（MHC）的表面分子的抑制性受体抑制NK细胞。NK细胞上抑制性受体的MHC特异性识别阻止了正常健康细胞的杀伤。本项目的主要目标是阐明抑制性受体阻断NK细胞活化的机制。 MHC I类特异性抑制性受体对天然细胞毒性的控制涉及酪氨酸磷酸酶SHP-1的募集，其使鸟嘌呤交换因子Vav去磷酸化。由于Vav对于适当的肌动蛋白重塑和突触形成是必不可少的，通过去磷酸化使Vav失活提供了阻断NK细胞细胞毒性的有效方式。然而，我们最近已经表明，抑制性信号通路更复杂，并涉及第二个组件，这依赖于酪氨酸磷酸化步骤。小衔接子Crk在由MHC I类特异性受体抑制期间被磷酸化。Crk磷酸化导致其从肌动蛋白细胞因子相关信号复合物中解离。 自然杀伤（NK）细胞抑制性受体募集酪氨酸磷酸酶，以防止激活，诱导磷酸化和解离的小接头Crk从细胞骨架支架复合物，并保持NK细胞在响应于随后的激活事件的状态。Crk如何促进抑制尚不清楚。我们在脂质双层上对原代NK细胞进行成像，脂质双层携带IgG 1 Fc以刺激CD 16，并通过受体CD 94-NKG 2A抑制人类白细胞抗原（HLA）-E。在NKG 2A + NK细胞中，HLA-E单独诱导Crk磷酸化。在单独用Fc激活突触时，Crk是Fc微簇的运动及其触发激活信号的能力所必需的。在抑制性突触中，HLA-E促进了Fc和磷酸化Crk在中枢的积累，并阻断了Fc诱导的F-肌动蛋白的积累。我们提出了抑制性受体功能的统一模型：Crk磷酸化阻止了必需的Crk依赖性激活信号并阻止了F-肌动蛋白网络的形成，从而减少了对激活受体后续接合的限制。

Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component that provides specificity in target cell recognition is inhibition of NK cells by inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents killing of normal healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Control of natural cytotoxicity by MHC class I-specific inhibitory receptors involves recruitment of the tyrosine phosphatase SHP-1, which dephosphorylates the guanine exchange factor Vav. As Vav is essential for proper actin remodeling and synapse formation, Vav inactivation through dephosphorylation provides an efficient way to block NK cell cytotoxicity. However, we have shown recently that the inhibitory signaling pathway is more complex and involves a second component, which relies on a tyrosine phosphorylation step. The small adapter Crk is phosphorylated during inhibition by MHC class I-specific receptors. Crk phosphorylation results in its dissociation from actin cytoskeleton-associated signaling complexes. Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16, and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-Ealone induced Crk phosphorylation in NKG2A+ NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk, and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors.