Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Immune cell effector responses involve dynamic protein secretion.
• Measuring secreted proteins from single cells sequentially is crucial for understanding these dynamics.
• Existing methods often lack the ability to perform longitudinal measurements on the same single cells.

Purpose of the Study:

• To develop and apply a microchip-based assay for simultaneous, multi-time point, sequential protein secretion measurement from thousands of single immune cells.
• To investigate the dynamic response of human macrophages to lipopolysaccharide (LPS) stimulation.
• To characterize cell-intrinsic heterogeneity in immune cell responses.

Main Methods:

• Development of a 10-plexed, sequential secretion assay on a microchip.
• Simultaneous measurement of ≈5000 single human macrophages over 4 time points.
• Application of single-cell RNA sequencing at matched time points to correlate protein secretion with transcriptional states.

Main Results:

• Identified four distinct activation modes for protein secretion in single macrophages upon toll-like receptor 4 (TLR4) ligand stimulation.
• Demonstrated that protein secretion dynamics classify cells into two major activation states, dependent on their basal state.
• Single-cell RNA sequencing confirmed two major transcriptional states (translation vs. inflammatory programs) corresponding to the observed protein secretion states.
• Revealed cell-intrinsic heterogeneity within a phenotypically homogeneous macrophage population.

Conclusions:

• The developed microchip assay enables longitudinal tracking of protein secretion signatures in thousands of single cells over multiple time points.
• This approach provides dynamic insights into individual immune cell responses to pathogenic challenges.
• The findings highlight the heterogeneous nature of immune cell responses at both protein secretion and transcriptional levels, contributing to a better understanding of population-level immune responses.