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，但它们可以在不损害正常细胞的情况下清除病毒感染的细胞和癌细胞。在靶细胞识别中提供特异性的一个重要组成部分是识别被称为主要组织相容性复合体(MHC)I类表面分子的抑制性受体对NK细胞的抑制作用。MHC特异性识别NK细胞上的抑制性受体可防止正常健康细胞的杀伤。本项目的主要目的是阐明抑制性受体阻断NK细胞激活的机制。 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，SHP-1使鸟氨酸交换因子Vav去磷酸化。由于Vav对于正确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失活提供了一种有效的方法来阻断NK细胞的细胞毒作用。然而，我们最近已经表明，抑制信号通路更加复杂，涉及依赖于酪氨酸磷酸化步骤的第二组分。小接头Crk在被MHC I类特异性受体抑制的过程中被磷酸化。Crk的磷酸化导致其从肌动蛋白细胞骨架相关的信号复合体中解离。 自然杀伤(NK)细胞抑制受体招募酪氨酸磷酸酶来阻止激活，诱导小接头CRK的磷酸化和从细胞骨架支架复合体中解离，并维持NK细胞对后续激活事件的应答状态。Crk如何对抑制起作用尚不清楚。我们在携带IgG1Fc的脂双层上成像了原代NK细胞，以刺激CD16，并通过CD94-NKG2A受体抑制人类白细胞抗原(HLA)-E。HL A-Eone诱导NKG2A+NK细胞Crk磷酸化。在单独使用Fc激活突触时，Fc微簇的运动及其触发激活信号的能力需要Crk。在抑制性突触，HLA-E促进Fc和磷酸化Crk的中枢聚集，并阻断Fc诱导的F-肌动蛋白的积聚。我们提出了一个抑制性受体功能的统一模型：Crk的磷酸化阻止了必要的Crk依赖的激活信号，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参与的限制。

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.