Activation of Human Natural Killer Cell Function

NK细胞激活天然的细胞毒作用(杀伤靶细胞)需要受体对的组合，这些受体对为激活细胞内钙离子释放、细胞因子产生和细胞毒作用提供协同信号。我们已经解决的主要问题是来自共激活受体的不同信号如何整合以实现协同作用，以及哪个分子检查点(S)控制这一过程。在提供静息NK细胞协同激活的受体组合中，有NKG2D(CD314)和2B4(CD244)。在其天然配体如ULBP分子的刺激下，NKG2D通过与DAP10的结合来招募磷脂酰肌醇-3-激酶(PI3K)和Grb2-Vav1。2B4与其在造血细胞上表达的配体CD48结合，并通过胞浆酪氨酸基序招募小接头SAP。SAP与酪氨酸激酶Fyn结合。由于协同作用需要整合这些不同的信号，了解协同作用的基础将揭示不同的信号如何汇聚到协同发生的特定时间点。两个共激活受体协同激活NK细胞伴随着Vav1磷酸化的增强，这相当于每个受体单独诱导的磷酸化的总和，需要克服c-Cbl泛素连接酶的抑制。直到最近，还没有关于协同受体的不同信号如何汇聚来调节Vav1及其下游信号的信息。基于Vav1在协同过程中的相加而不是协同磷酸化，我们假设不同的Vav1池可能相互补充以实现协同作用。 作为第一步，我们测试了已知对淋巴细胞激活信号有贡献的适配器的作用。我们研究了诸如SLP-76和激活T细胞的连接物(LAT)等适配蛋白是否是NK细胞协同所必需的。SLP-76和LAT是构成信号复合体T细胞骨架的重要适配子。受体之间的协同作用最好是通过独立信号的互补来实现。刺激自然杀伤(NK)细胞的细胞毒作用和细胞因子的分泌需要来自共激活受体的协同信号。我们以前的工作表明，需要协同作用来克服泛素连接酶c-Cbl对鸟嘌呤交换因子Vav1依赖的信号的抑制。是否涉及独特信号的互补，如果涉及，在什么水平上，尚不清楚。我们在这里已经证明了适配器SLP-76是协同作用所必需的，并且协同对的每个受体控制着SLP-76中特定酪氨酸的磷酸化。构成Vav1单独结合位点的两个磷酸化酪氨酸中的每一个都是协同动员钙离子所必需的。SLP-76中酪氨酸113或128的选择性磷酸化是天然细胞毒性共激活受体所特有的信号，因为Fc受体CD16刺激NK细胞会导致这两个位点的磷酸化，就像T细胞受体刺激T细胞一样。我们的数据显示，NK细胞共激活受体对两个SLP-76酪氨酸的磷酸化具有意想不到的选择性，提示两个Vav1分子与SLP-76结合可能对Vav1功能具有协同作用，并表明NK细胞效应功能的触发受到互补信号的严格调控。 孟德尔对免疫调节紊乱的分析可以提供对用于宿主防御和免疫耐受的途径的分子洞察。乔什·米尔纳的研究小组鉴定了三个家系，这些家系具有以遗传为主的寒冷性荨麻疹、抗体缺乏、感染易感性和自身免疫的复合体。这种表型是由于PLCG2的突变所致，PLCG2编码一种在B细胞、NK细胞和肥大细胞中表达的信号分子。用流式细胞仪进行免疫表型分析，发现NK细胞脱颗粒，配体诱导的钙离子通量在B细胞和NK细胞中受损，但在T细胞中受损。PLCG2中的缺失位于自身抑制区域内，产生具有结构性磷脂酶活性的蛋白质产物。PLCG2的基因组缺失导致磷脂酶C-Gamma2功能增强，导致多个白细胞亚群的信号异常，以及包括免疫功能过度和受损的表型。

Activation of natural cytotoxicity (killing of target cells) by NK cells requires combinations of receptor pairs that deliver synergistic signals for activation of intracellular Ca2+ release, cytokine production, and cytotoxicity. The main question we have addressed is how distinct signals from co-activation receptors are integrated to achieve synergy, and which molecular checkpoint(s) control this process. Among the receptor combinations that provide synergistic activation in resting NK cells are NKG2D (CD314) and 2B4 (CD244). Upon stimulation with its natural ligands, such as ULBP molecules, NKG2D recruits phosphatidylinositol-3-kinase (PI3K) and Grb2-Vav1 through its association with DAP10. 2B4 binds to its ligand CD48, which is expressed on hematopoietic cells, and recruits the small adaptor SAP through cytoplasmic tyrosine-based motifs. SAP binds to the tyrosine kinase Fyn. Because synergy requires the integration of such diverse signals, understanding the basis for synergy would disclose how disparate signals converge to a certain point at which synergy occurs.
Synergistic NK cell activation by two coactivation receptors was accompanied by enhanced Vav1 phosphorylation, which was equivalent to the sum of phosphorylation induced by each receptor alone and was required to overcome inhibition by c-Cbl ubiquitin ligase. Until recently, there was no information about how distinct signals from synergizing receptors converge to regulate Vav1 and its downstream signaling. Based on the additive, rather than synergistic phosphorylation of Vav1 during synergy, we hypothesized that different pools of Vav1 may complement each other to achieve synergy. As a first step, we tested the role of adaptors known to contribute to signaling for lymphocyte activation. We investigated whether adaptor proteins such as SLP-76 and linker for activation of T cells (LAT) are required for NK cell synergy. SLP-76 and LAT are important adaptors that form the backbone of signaling complexes T cells. 
Synergy among receptors is best achieved by complementation of independent signals. Stimulation of natural killer (NK)-cell cytotoxicity and cytokine secretion requires synergistic signals from coactivation receptors. Our previous work has shown that synergy is required to overcome inhibition of guanine exchange factor Vav1-dependent signals by the ubiquitin ligase c-Cbl. Whether complementation of unique signals is involved and, if so, at what level, is unknown. We have shown here that the adapter SLP-76 is required for the synergy and that each receptor of a synergistic pair controls the phosphorylation of a specific tyrosine in SLP-76. Each one of the two phosphorylated tyrosines that constitute separate binding sites for Vav1 is required for synergistic mobilization of Ca2+. The selective phosphorylation of either tyrosine 113 or tyrosine 128 in SLP-76 is unique to signaling by natural cytotoxicity co-activation receptors, as stimulation of NK cells by the Fc receptor CD16 resulted in phosphorylation at both sites, much like T cells stimulated by T-cell receptor. Our data reveal an unexpected degree of selectivity in the phosphorylation of two SLP-76 tyrosines by NK cell co-activation receptors, suggest that binding of two Vav1 molecules to SLP-76 may have a synergistic effect on Vav1 function, and show that triggering of NK cell effector function is tightly regulated by complementary signals.
 Mendelian analysis of disorders of immune regulation can provide molecular insight into pathways utilized for host defense and immune tolerance. Three families with a dominantly inherited complex of cold urticaria, antibody deficiency, and susceptibility to infection and autoimmunity were identified by the group of Josh Milner. The phenotype is due to mutations in PLCG2, encoding a signaling molecule expressed in B, NK, and mast cells. Using immunophenotyping by flow cytometry, we showed that NK cell degranulation, and ligand-induced calcium flux was impaired in B cells and NK cells, but in T cells. The deletions in PLCG2, located within an autoinhibitory domain, produce protein products with constitutive phospholipase activity. Genomic deletions in PLCG2 cause gain of phospholipase C-gamma2 function leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and impaired immune function.