Multiplexed, sequential secretion analysis of the same single cells reveals distinct effector response dynamics dependent on the initial basal state

Abstract

The effector response of immune cells dictated by an array of secreted proteins is a highly dynamic process, requiring sequential measurement of all relevant proteins from single cells. Herein we show a microchip-based, 10-plexed, sequential secretion assay on the same single cells and at the scale of ~5000 single cells measured simultaneously over 4 time points. It was applied to investigating the time course of single human macrophage response to Toll-like receptor 4 (TLR4) ligand lipopolysaccharide and revealed four distinct activation modes for different proteins in single cells. In particular, we observed that secreted factors regulated by transcription factor NFO_SCPLOWKC_SCPLOWB (e.g., TNF and CCL2) predominantly show on-off mode over off-on mode. The dynamics of all proteins combined classified the cells into two major activation states, which were found to be dependent on the basal state of each cell. Single-cell RNA-Seq was performed on the same samples at the matched time points and further demonstrated at the transcriptional level the existence of two major activation states, which are enriched for translation vs inflammatory programs, respectively. These results showed a cell-intrinsic heterogeneous response in phenotypically homogeneous cell population. This work demonstrated the longitudinal tracking of protein secretion signature in thousands of single cells at multiple time points, providing dynamic information to better understand how individual immune cells react to pathogenic challenges over time and how they together constitute a population response.