Activation of Human Natural Killer Cell Function

人类自然杀伤细胞功能的激活

• 批准号: 8336065
• 负责人: Eric O Long
• 金额: $ 74.78万
• 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/8336065
• 关键词: 1-Phosphatidylinositol 3-Kinase; Activated Natural Killer Cell; Adaptor Signaling Protein; Address; Adhesions; Affect; Antigen-Presenting Cells; Antigens; Attention; B-Lymphocytes; Binding; Binding Sites; Biochemical; C-terminal; Cell Degranulation; Cell Line; Cell membrane; Cell physiology; Cells; Chimera organism; Cholesterol; Communicable Diseases; Complement; Complex; Cytoplasmic Granules; Data; Detergents; Diffusion; Drosophila genus; DsRed; Elements; Endocrine; Event; Exocytosis; FCGR3B gene; Fc Receptor; Fluorescence; Glycosphingolipids; Goals; Guanine; Guanine Nucleotide Exchange Factors; Hematopoietic; Hematopoietic Neoplasms; Homeostasis; Human; Image; Imaging technology; Immune; Immune response; Individual; Inflammation; Insecta; Ionomycin; LCP2 gene; Label; Lateral; Life; Ligands; Link; Lymphocyte; Lymphocyte Activation; Lytic; Mediating; Membrane; Membrane Microdomains; Molecular; Monitor; Mouse Cell Line; N-terminal; NK Cell Activation; Natural Killer Cells; Neurons; Normal Cell; Oncogenic Viruses; PHluorin; PTPRC gene; Phorbol Esters; Phosphorylation; Physiological; Play; Process; Production; Property; Proteins; Proto-Oncogene Proteins c-fyn; Receptor Activation; Receptor Signaling; Recruitment Activity; Recycling; Resistance; Resolution; Rest; Role; Signal Transduction; Site; Step Tests; Sum; Synapses; System; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Tail; Techniques; Testing; Total Internal Reflection Fluorescent; Transmembrane Domain; Tumor Necrosis Factor Ligand Superfamily Member 6; Tyrosine; Variant; Vertebral column; Vesicle; Work; base; cancer immunotherapy; cell killing; cytokine; cytotoxic; cytotoxicity; extracellular; functional outcomes; immunological synapse; interest; link protein; neoplastic cell; pathogen; peripheral blood; receptor; reconstitution; relating to nervous system; response; tool; ubiquitin ligase

An interesting aspect of natural cytotoxicity by NK cells is that several pairwise combinations of receptors, which have very distinct signaling properties, result in potent synergy for activation of intracellular Ca2+ release, cytokine production, and cytotoxicity. The main question we have addressed was how distinct signals from co-activation receptors are integrated to regulate such 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 bound to the tyrosine kinase Fyn through cytoplasmic tyrosine-based motifs. 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. At present, there is 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 adapters 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 co-activation 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. In studies of activation of lymphocytes most of the attention has been given to the signaling receptors on lymphocytes, and how these receptors are distributed at the plasma membrane once they bind to their ligands on antigen-presenting cells (APC) or target cells. In contrast, whether the distribution of ligands on the stimulating cells has an impact on lymphocyte responses has received little attention. Furthermore, while the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution within membrane domains on APC and target cells. Detergent-resistant membrane (DRM) domains, into which glycophosphatidylinositol (GPI)-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. NKG2D is one the main co-activation receptors on NK cells. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation independently of HLA class I ligands for inhibitory receptors, we expressed ULBP1 in a mouse cell line. A chimera consisting of the extracellular portion of ULBP1 and the transmembrane region of CD45 was generated. Its expression resulted in the localization of the normally GPI-linked ULBP1 from detergent-resistant membrane fractions to detergent-soluble fractions. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. This redistribution of ULBP1 caused a significant reduction in cytotoxicity and degranulation by NK cells, implying a role for receptor ligand distribution in the activation of NK cell responses. Our data suggests that ligand distribution into distinct membrane domains in general may play an underappreciated role in the activation of NK cells. Given the potential of tumor cells or virus-infected cells to alter ligand distribution at the plasma membrane and to escape immune responses, it will be important to investigate how the distribution of other ligands impacts the activation of lymphocytes. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Imaging and electrophysiological techniques have suggested that both complete and incomplete fusion occur in endocrine cells and several different neural synapses. To date, the precise steps of lytic granules fusion in cytotoxic lymphocytes such as CTL and NK cells have not been imaged in live cells. To test whether complete fusion or incomplete fusion of lytic granules with the plasma membrane occurs in cytotoxic lymphocytes, we used the human NK cell line NKL, as well as primary NK cells from human peripheral blood. We used TIRF microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptorligand interactions. FasL is present in the lytic granule membrane of CTL and NK cells. pHluorin is a pH sensitive variant of the green fluorescence protein (GFP) and has been used to monitor vesicle fusion in endocrine and neuronal cells. We attached pHluorin to the C-terminal, lumenal portion of Fas ligand (FasL), and DsRed to the N-terminal, cytosolic tail of FasL. By specifically labeling lytic granules with different fluorescent proteins, and by using TIRF microscopy, we have shown that NK cells use two distinct modes for exocytosis of lytic granules: complete fusion, characterized by loss of granule content and rapid diffusion of Fas ligand at the plasma membrane; and incomplete fusion, characterized by transient fusion pore opening and retention of Fas ligand at the fusion site. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the plasma membrane, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

NK细胞自然细胞毒性的一个有趣的方面是，具有非常不同的信号传导特性的几种成对组合会导致激活细胞内Ca2+释放，细胞因子产生和细胞毒性的有效协同作用。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来调节这种协同作用，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与其在造血细胞上表达的配体CD48结合，并通过基于细胞质酪氨酸的基序募集与酪氨酸激酶FYN结合的小型适配器SAP。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。目前，尚无有关如何共同信号来调节VAV1及其下游信号传导的信息。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。刺激天然杀伤（NK） - 细胞细胞毒性和细胞因子分泌需要共同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。 在淋巴细胞激活的研究中，大部分注意力都已对淋巴细胞上的信号受体给予，以及这些受体在质膜上与抗原呈递细胞（APC）或靶细胞上的配体结合后如何分布在质膜上。相反，配体在刺激细胞上的分布是否对淋巴细胞反应产生影响很少。此外，尽管已经建立了膜微域在受体介导的淋巴细胞激活中的重要性，但对APC和靶细胞上膜结构域中受体配体分布的作用知之甚少。 糖磷脂酰肌醇（GPI）连接的蛋白分析中耐洗涤剂的膜（DRM）结构域富含胆固醇和糖磷脂。 ULBP1是一种用于天然细胞毒性受体NKG2D的GPI连接配体。 NKG2D是NK细胞上的主要共激活受体之一。为了研究目标细胞上的ULBP1分布如何影响NKG2D依赖性的NK细胞激活，独立于HLA I类配体用于抑制受体，我们在小鼠细胞系中表达了ULBP1。产生了由ULBP1的细胞外部分和CD45的跨膜区域组成的嵌合体。它的表达导致正常与GPI相关的ULBP1定位，从耐洗涤剂的膜级分至洗涤剂可溶解的分数。 ULBP1的聚类和横向扩散不受膜锚的变化影响。 ULBP1的这种重新分布导致NK细胞的细胞毒性和脱粒显着降低，这意味着受体配体分布在NK细胞反应激活中的作用。 我们的数据表明，在NK细胞的激活中，通常在不同的膜结构域中的配体分布在不同的膜结构域中起作用。鉴于肿瘤细胞或病毒感染细胞改变质膜处的配体分布并避免免疫反应，研究其他配体的分布如何影响淋巴细胞的激活将很重要。 尽管在内分泌和神经元细胞中已经广泛研究了囊泡胞吐作用，但对细胞毒性淋巴细胞中裂解颗粒的融合知之甚少。成像和电生理技术已经表明，内分泌细胞和几种不同的神经突触都会发生完整和不完整的融合。迄今为止，在活细胞中尚未对细胞毒性淋巴细胞（例如CTL和NK细胞）的裂解颗粒融合的精确步骤。为了测试裂解颗粒与质膜的完全融合或不完整的融合发生在细胞毒性淋巴细胞中，我们使用了人类NK细胞系NKL以及人类外周血的主要NK细胞。我们使用TIRF显微镜检查了用Phorbol酯和Ionymycin刺激的NK细胞系NKL中用荧光标记的FAS配体标记的裂解颗粒，以及在生理受体相互作用激活的原代NK细胞中。 FASL存在于CTL和NK细胞的裂解颗粒膜中。 Phluorin是绿色荧光蛋白（GFP）的pH敏感变体，已用于监测内分泌和神经元细胞中的囊泡融合。我们将Phluorin连接到FAS配体（FASL）的C末端，Lumenal部分，并将DSRED与FASL的N末端，胞质尾部。 通过将裂解颗粒具有不同的荧光蛋白，并使用TIRF显微镜标记裂解颗粒，我们表明NK细胞使用两种不同的模式用于裂解颗粒的胞吐作用：完全融合，其特征是颗粒含量的损失和植物膜上FAS配体的快速扩散。和不完整的融合，其特征是临时融合孔的打开和FAS配体在融合部位的保留。因此，我们得出的结论是，NK细胞中的裂解颗粒与质膜都完全且不完全融合，并提出不完整的融合可能会在释放细胞毒性效应分子后促进裂解颗粒膜的有效回收。