Activation of Human Natural Killer Cell Function

人类自然杀伤细胞功能的激活

• 批准号: 8555771
• 负责人: Eric O Long
• 金额: $ 49.57万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555771
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adaptor Signaling Protein; Address; Adhesions; Antibodies; Antigens; Autoimmunity; B-Lymphocytes; Binding; Binding Sites; Biochemical; Cell Degranulation; Cell Line; Cell physiology; Cells; Communicable Diseases; Complement; Complex; Cytoplasmic Granules; Data; Drosophila genus; Elements; FCGR3B gene; Family; Fc Receptor; Flow Cytometry; Genomics; Goals; Guanine; Guanine Nucleotide Exchange Factors; Hematopoietic; Hematopoietic Neoplasms; Homeostasis; Host Defense; Human; Immune; Immune System Diseases; Immune Tolerance; Immunophenotyping; Individual; Infection; Inflammation; Inherited; Insecta; LCP2 gene; Leukocytes; Ligands; Lymphocyte Activation; Molecular; Mutation; NK Cell Activation; Natural Killer Cells; Normal Cell; PLCG2 gene; PLCgamma2; Pathway interactions; Phenotype; Phospholipase; Phosphorylation; Predisposition; Process; Production; Protein Tyrosine Kinase; Proteins; Receptor Activation; Receptor Signaling; Recruitment Activity; Regulation; Resistance; Rest; Role; Signal Transduction; Signaling Molecule; Site; Step Tests; Sum; Synapses; System; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Tyrosine; Urticaria; Vertebral column; Work; base; cancer immunotherapy; cell killing; cytokine; cytotoxicity; functional outcomes; immune function; insight; killings; mast cell; neoplastic cell; pathogen; receptor; reconstitution; release of sequestered calcium ion into cytoplasm; ubiquitin ligase

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.

NK细胞激活自然细胞毒性（杀死靶细胞）需要组合受体对，这些受体对传递协同信号，以激活细胞内Ca2+释放，细胞因子产生和细胞毒性。我们解决的主要问题是，如何整合与共激活受体的不同信号以实现协同作用，以及哪些分子检查点控制了此过程。在静息NK细胞中提供协同激活的受体组合中，NKG2D（CD314）和2B4（CD244）。在用自然配体（例如ULBP分子）刺激后，NKG2D募集磷脂酰肌醇-3-激酶（PI3K）和GRB2-VAV1通过与DAP10的关联。 2B4结合其在造血细胞上表达的配体CD48，并通过基于细胞质酪氨酸的基序募集小型适配器汁液。 SAP与酪氨酸激酶FYN结合。由于协同作用需要整合如此多样化的信号，因此了解协同作用的基础将揭示出不同的信号如何融合到同性恋发生的某个点。
通过两个共同激活受体的协同NK细胞激活伴随着增强的VAV1磷酸化，这等同于单独由每个受体诱导的磷酸化之和，并被要求克服C-CBBL泛素连接酶的抑制作用。直到最近，还没有关于如何汇合与调节VAV1及其下游信号的不同信号的信息。基于在协同过程中VAV1的添加剂而不是协同磷酸化的基础上，我们假设VAV1的不同库可能相互补充以实现协同作用。 作为第一步，我们测试了已知的衔接子的作用，该适配器有助于淋巴细胞激活的信号传导。我们研究了NK细胞协同作用是否需要适配器蛋白（例如SLP-76）和用于激活T细胞（LAT）的链接。 SLP-76和LAT是构成信号复合物T细胞的主链的重要适配器。 
通过互补的独立信号，可以最好地实现受体之间的协同作用。刺激天然杀手（NK） - 细胞细胞毒性和细胞因子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依赖性信号的抑制。是否涉及独特信号的互补，如果是的，则未知级别。我们在这里已经表明，协同作用需要适配器SLP-76，并且协同对的每个受体都控制SLP-76中特定酪氨酸的磷酸化。构成VAV1的单独结合位点的两个磷酸化酪氨酸中的每一种都是Ca2+的协同动员。 SLP-76中酪氨酸113或酪氨酸128的选择性磷酸化是由天然细胞毒性共激活受体的信号传导所独有的，因为FC受体CD16刺激NK细胞，导致两个部位在两个部位都在T-Cell受体刺激的T细胞上磷酸化。我们的数据揭示了NK细胞共激活受体在两种SLP-76酪氨酸的磷酸化中的选择性意外程度，这表明两个VAV1分子与SLP-76的结合可能对VAV1功能具有协同作用，并显示NK细胞效应函数的触发效应受互补的触发触发。
 孟德尔对免疫调节疾病的分析可以为用于宿主防御和免疫耐受性的途径提供分子见解。乔什·米尔纳（Josh Milner）组鉴定出了三个具有遗传性的冷荨麻疹，抗体缺乏症以及对感染和自身免疫性的易感性的家庭。表型是由于PLCG2中的突变引起的，编码在B，NK和肥大细胞中表达的信号分子。 使用流式细胞仪的免疫表型，我们表明NK细胞脱粒化，B细胞和NK细胞中的NK细胞脱粒和配体诱导的钙通量受损，但在T细胞中受损。位于自身抑制域内的PLCG2中的缺失产生具有组成型磷脂酶活性的蛋白质产物。 PLCG2中的基因组缺失导致磷脂酶C-gamma2功能的增加，导致多个白细胞子集的信号异常，以及涵盖过度和受损的免疫功能的表型。