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，因此在先天性疾病中发挥着重要作用。 主场防守。与细胞毒性T淋巴细胞(CTL)不同，CTL的杀细胞活性是由产生的TCR诱导的 与同源pMHC配体结合，NK细胞的激活受一系列激活和抑制的调节 感受器。这些受体诱导的信号的最终结果决定了NK细胞效应器的程度 干扰素-γ的产生和颗粒介导的细胞毒等活性。NK细胞的主要机制之一-- 介导的细胞杀伤活性依赖于抗体，称为抗体依赖的细胞介导的细胞毒 (ADCC)。γRIIa(CD16)受体不能直接识别靶点上的抗原，可诱导ADCC 细胞，但与靶标表面抗原特异性结合的抗体的Fc片段相互作用 细胞。现有的证据，包括我们的证据，强烈地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 可以通过与靶细胞上的其他膜配体相互作用的抗原共同聚集来进一步调节 NK细胞表面有抑制性受体或黏附分子。我们假设差异 在Fcγ中，CD16RIIIa(CD16RIIa)聚集/共聚集可能对受体介导的 影响ADCC效率的信号转导。因此，这个项目的目标是研究同型性和 异型配体聚集调节配体协同参与CD16抑制作用的能力 受体和黏附分子调节ADCC的效率。我们将使用特征良好的CD16.NK92 我们已经开发的用于模拟NK细胞(7-12)和新鲜激活的人类NK细胞的功能的细胞 研究CD16聚集性/共聚集性与其他受体的作用。为了实现这一目标，我们将利用 可负载CD16、黏附分子和 不同比例和密度的抑制受体，并将检查这些模型的结合动力学 膜簇对NK细胞的作用以及诱导细胞内钙信号的动力学。我们还将 利用呈现相同配体的平面脂质双分子层，或者分散在双分子层中或通过 与二聚化系统进行交联化，并将分析配体的交联会如何影响结构 突触界面和NK细胞脱颗粒的动力学。预期结果将提供 显著提高了对原代和活化的细胞溶解活性控制机制的理解 人类NK细胞将设计新的基于NK细胞的免疫治疗策略，以提高ADCC对 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.