(193q) Single-Cell Profiling of Dynamic Cytokine Secretion and the Phenotype of Immune Cells

Quantifying the heterogeneity of immune cells at the single-cell level in a high-throughput manner across multiple biological dimensions — from the genome and transcriptome, to intracellular and extracellular signaling, and to interaction with other kinds of cells — can have a direct impact in improving therapeutic discovery in biotechnology, in diagnosis of diseases, and in facilitating immunotherapy. It is desirable to have a modular method that can quantify and screen for cellular functionality such as motility, interaction with other cells, protein secretion to enable in-depth characterization of individual cells and that is able to integrate these parameters to provide multiplexed molecular profiles of single cells. We have developed and validated an integrated methodology that combines nanowell arrays and bead-based molecular sensors for detecting cytokine secretion dynamically without the need for encapsulation of single natural killer (NK) cells or T cells.

Although NK cells were classically defined as pre-activated effector lymphocytes empowered with innate cytolytic functionality, more recent data suggest that NK cells are 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. 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 (FcγRIIIA, low-affinity Fc receptor). The majority of NK cells in peripheral blood (>90%) are the CD56^dimCD16⁺ phenotype, which is primarily believed to be responsible for cytolytic functionality including antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by CD16. By contrast, the CD56^brightCD16^- phenotype is the minor population in peripheral blood and is described as primarily responsible for secretion of cytokines including interferon gamma (IFN-γ). We utilized our Timelapse Imaging Microscopy In Nanowell Grids (TIMING) platform, which is based on incubation of individual cells within polydimethylsiloxane (PDMS) nanowell arrays, to directly examine the relationship between phenotype and dynamic IFN-γ secretion profile. Despite the fact that PDMS is widely adopted for the fabrication of microfluidic devices, PDMS tends to display a high level of non-specific protein adsorption. We demonstrated the robust passivation of the PDMS-based nanowell arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. Numerical simulations were used to optimize the molecular density of capture antibodies on the surface of the beads to improve the capture efficiency of cytokines by the beads within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells revealed that CD56^dimCD16⁺ NK cells are immediate secretors of IFN-γ upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h); there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Within secretor NK cells population, early-secretor NK cells tended to have a higher level of CD16 surface expression, suggesting the existence of an elite population of CD16⁺ NK cells capable of both lytic functionality and rapid cytokine secretion.

In a second application, we have quantified the dynamic secretion of IFN-γ from T cells using the same platform. Chimeric antigen receptor T cells (CAR⁺ T cells), which are genetically engineered to retarget and program anti-tumor functionality of T cells, are under intense clinical investigation as personalized cancer therapeutics. By utilizing CD19-specific third-generation CAR⁺ T cells, we have explored the relationship between basal motility of CAR⁺ T cells and their ability to secrete IFN-γ.

Collectively, these results establish our methodology as an investigational tool for combining phenotyping, cellular functionality and real-time protein secretion of individual cells in a high-throughput manner.