An Xingyue, Sendra Victor G, Liadi Ivan, Ramesh Balakrishnan, Romain Gabrielle, Haymaker Cara, Martinez-Paniagua Melisa, Lu Yanbin, Radvanyi Laszlo G, Roysam Badrinath, Varadarajan Navin
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, United States of America.
Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
PLoS One. 2017 Aug 24;12(8):e0181904. doi: 10.1371/journal.pone.0181904. eCollection 2017.
Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.
自然杀伤（NK）细胞是先天性淋巴细胞的高度异质群体，构成了我们抵御多种肿瘤和微生物感染的第一道防线。了解这些淋巴细胞的异质性需要将其潜在表型与动态功能行为相结合的能力。我们开发并验证了一种单细胞方法，该方法基于纳米孔阵列和基于珠子的分子生物传感器整合细胞表型分析和动态细胞因子分泌。我们展示了用聚乙二醇（PEG）对基于聚二甲基硅氧烷（PDMS）的纳米孔阵列进行的稳健钝化，并通过与酶联免疫斑点（ELISPOT）测定法比较验证了我们的测定。我们使用数值模拟来优化珠子表面抗体的分子密度，作为开放孔系统中细胞因子捕获效率的函数。对数百个离体分析的个体人类外周血NK细胞的分析表明，CD56dimCD16+NK细胞在被佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和离子霉素激活后（<3小时）立即分泌干扰素γ（IFN-γ），并且没有证据表明NK细胞之间存在协同激活或更快IFN-γ分泌的合作。此外，我们观察到单个NK细胞分泌IFN-γ的量和速率均依赖于供体。总的来说，这些结果将我们的方法确立为一种以高通量方式结合单个细胞表型分析和实时蛋白质分泌的研究工具。
