Role of immune receptor clustering in controlling efficacy of antibody-dependent FcγRIIIa-mediated cytotoxicity by NK cells

项目摘要 NK细胞能够杀死病毒感染的细胞和癌细胞，因此，在先天免疫调节中发挥重要作用。 宿主防御与细胞毒性T淋巴细胞（CTL）不同，其细胞溶解活性由生产性TCR诱导 与同源pMHC配体的接合，NK细胞的活化由一组活化和抑制性调节。 受体。由这些受体诱导的信号的净结果决定了NK细胞效应物的程度。 活性如INF-γ产生和颗粒介导的细胞毒性。NK细胞的主要机制之一- 介导的细胞溶解活性依赖于抗体，称为抗体依赖性细胞介导的细胞毒性 （ADCC）。ADCC由FcγRIIIa（CD 16）受体诱导，这些受体不直接识别靶点上的抗原 细胞，但与特异性结合靶细胞表面抗原的IgG抗体的Fc片段相互作用 细胞现有的证据，包括我们的证据，强烈表明配体介导的激活聚集， 细胞毒性淋巴细胞上的受体调节细胞内信号传导的动力学，形成高度有序的 突触界面、颗粒递送动力学和靶细胞裂解效率（1-6）。受体聚集 可以通过抗原与靶细胞上的其他膜配体共聚集来进一步调节， 与NK细胞表面的抑制性受体或粘附分子结合。我们假设， 在FcγRIIIa（CD 16）中，簇集/共簇集对受体介导的 影响ADCC效率的信令。因此，该项目的目标是研究如何同型和 异型配体簇集调节配体在CD 16、抑制性CD 18和CD 19的接合中合作的能力。 受体和粘附分子调节ADCC的效率。我们将使用充分表征的CD16.NK92 我们已经开发了NK细胞（7-12）和新鲜活化的人NK细胞的功能模型， 研究CD 16与其他受体聚集/共聚集的作用。为了实现这一目标，我们将利用 荧光纳米颗粒（NiNLP），其可以装载有用于CD 16、粘附分子和 抑制受体在不同的比例和密度，并将检查这些模型的结合动力学 膜簇与NK细胞的关系以及诱导的细胞内Ca 2+信号传导的动力学。我们还将 利用呈现相同配体的平面脂质双层，所述配体分散在双层中或通过 与iDimerize系统交联，并将分析配体的交联如何影响结构 的突触界面和NK细胞脱粒的动力学。预期结果将提供 显著提高了对控制原代和活化细胞溶解活性的机制的理解， 设计新的基于NK细胞的免疫策略，以提高ADCC的效率， 治疗病毒感染、自身免疫性疾病和癌症。

Project Summary NK cells are capable of killing virus-infected and cancer cells and, therefore, play an important role in innate host defense. Unlike cytotoxic T lymphocytes (CTL), whose cytolytic activity is induced by productive TCR engagement with cognate pMHC ligands, activation of NK cells is regulated by a set of activating and inhibitory receptors. The net result of the signals induced by these receptors determines the extent of NK cell effector activities such as INF-γ production and granule-mediated cytotoxicity. One of the main mechanisms of NK cell- mediated cytolytic activity depends on antibodies and is termed antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is induced by FcγRIIIa (CD16) receptors that do not directly recognize antigens on the target cells, but interact with Fc fragments of IgG antibodies specifically bound to the antigens on the surface of target cells. Available evidence, including ours, strongly suggests that ligand-mediated clustering of activating receptors on cytotoxic lymphocytes regulates kinetics of intracellular signaling, formation of a highly ordered synaptic interface, kinetics of granule delivery, and efficiency of target cell lysis (1-6). The receptor clustering can be further modulated by antigen co-clustering with other membrane ligands on target cells, which interact with inhibitory receptors or adhesion molecules on the surface of NK cells. We hypothesize that differences in FcγRIIIa (CD16) clustering/co-clustering could have significant impact on receptor-mediated signaling influencing efficiency of ADCC. Thus, the goal of this project is to study how homotypic and heterotypic ligand clustering regulates ability of the ligands to cooperate in the engagement of CD16, inhibitory receptors and adhesion molecules to modulate efficiency of ADCC. We will use well-characterized CD16.NK92 cells that we have developed to model functions of NK cells (7-12) and freshly activated human NK cells to investigate the role of CD16 clustering/co-clustering with other receptors. To achieve this goal, we will exploit fluorescent nanoparticles (NiNLPs) that can be loaded with ligands for CD16, adhesion molecules, and inhibitory receptors at various ratios and densities, and will examine the binding kinetics of these model membrane clusters to NK cells as well as the kinetics of induced intracellular Ca2+ signaling. We will also utilize planar lipid bilayers presenting the same ligands, either dispersed in the bilayers or co-localized by cross-linking with the iDimerize system, and will analyze how cross-linking of the ligands will influence structure of the synaptic interface and the kinetics of NK cell degranulation. The expected results will provide significantly improved understanding of the mechanisms controlling cytolytic activity of primary and activated human NK cells to design new NK cell-based immunotherapeutic strategies to improve efficiency of ADCC to treat viral infections, autoimmune diseases, and cancer.