Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

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, 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 are shown. It is applied to investigating the time course of single human macrophage response to toll‐like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) and reveals four distinct activation modes for different proteins in single cells. Protein secretion dynamics classifies the cells into two major activation states dependent on the basal state of each cell. Single‐cell RNA sequencing performed on the same samples at the matched time points further demonstrates the existence of two major activation states at the transcriptional level, which are enriched for translation versus inflammatory programs, respectively. These results show a cell‐intrinsic heterogeneous response in a phenotypically homogeneous cell population. This work demonstrates 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.