Time is displayed as hh: mm and movie is sped up 1800
Time is displayed as hh: mm and movie is sped up 1800.(MP4) pone.0181904.s005.mp4 MPH1 (1.7M) GUID:?8D34A28C-FA28-42EF-BF7F-436A8858FC8E Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of gamma-secretase modulator 2 defense against several types of tumors and microbial infections. displayed as hh: mm and movie is sped up 1800.(MP4) pone.0181904.s005.mp4 (1.7M) GUID:?8D34A28C-FA28-42EF-BF7F-436A8858FC8E Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Natural killer (NK) cells are a highly heterogeneous populace 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 strong 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 revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-) 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- secretion. Furthermore, we observed that both the amount and rate of IFN- secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational gamma-secretase modulator 2 tool for combining phenotyping and real-time protein secretion of individual cells in gamma-secretase modulator 2 a high-throughput manner. Introduction Although natural killer (NK) cells were classically defined as pre-activated effector lymphocytes empowered with innate cytolytic functionality, more recent data suggest that NK cells are also endowed with complex functionalities including cytokine secretion and activation of antigen presenting cells, and can thus act as a bridge between innate and adaptive immunity [1]. NK cells are of pivotal importance in the execution of antiviral and anti-tumor responses [2]. Human NK cells are identified as CD3-CD56+ cells and are typically classified into different subsets based on the relative expression of the cell surface markers CD56 (adhesion marker) and CD16 (FcRIIIA, low-affinity Fc receptor) [3, 4]. The majority of NK cells in peripheral blood ( 90%) are the CD56dimCD16+ phenotype, which is usually primarily believed to be responsible for cytolytic functionality including antibody-dependent cell mediated cytotoxicity (ADCC) mediated by CD16. By contrast, the CD56brightCD16- phenotype is the minor populace in peripheral blood and is described as primarily responsible for secretion of cytokines like interferon gamma (IFN-) [3, 4]. The secretion of the pro-inflammatory cytokine IFN- is an important mechanism of defense mediated by lymphocytes. Unlike cytotoxicity that only influences the target cell that is directly conjugated to the lymphocyte, IFN- secretion has a more profound influence on all cells within the microenvironment via multiple mechanisms including elevated expression of HLA-class I molecules [5], induction of chemokines that can promote immune cell infiltration [6], mediation of angiostasis [7], and prevention of the outgrowth of antigen-loss variants [8]. From a clinical perspective, the secretion of IFN- by immune cells is likely an important contributor to the efficacy of immunotherapies including treatment with antibodies against PD-1 and CTLA-4 [9, 10]. Direct measurement of NK cell (or lymphocyte) functions at the single-cell level requires the simultaneous gamma-secretase modulator 2 monitoring of multiple parameters including the cells phenotype, its migration and conversation with other cells, secretion of proteins, and its survival. These challenges have been tackled by measuring just a subset of these effector functions and relying on correlative studies to establish links among cellular.