Stimulation-induced cytokine polyfunctionality as a dynamic concept

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. Firstly, we observed simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Secondly, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide better understanding for various immune dynamics in therapy and disease.

Methods
The data here was generated using peripheral blood mononuclear cells (PBMCs) that were extracted from a buffy coat (from the Zurich blood bank, anonymous donor).The buffy coat was processed as described in Portmann et al., Cell Reports Methods, 2024, and the cells were stored frozen in liquid nitrogen until use.

On the day of the experiment, the cells were thawed at 37°C in pre-warmed cell buffer, washed two times and stained using 5 μM CellTrace violet (ThermoFisher, analyzed in the DAPI channel). Afterward, the cells were FcR blocked, washed and counted. Thereafter, the cells were diluted and different stimulants were used to induce or control for cytokine secretion. Namely, the cells were either stimulated with

Cell buffer alone (control, Data ‘Media_control’),

1 µg/ml lipopolysaccharide (Data ‘LPS’),

50 ng/ml Phorbol-myristate-acetate and 1 µg/ml Ionomycin (Data ‘PMA_Iono’),

100 µg/ml zymosan (Data ‘Zymosan’),

10 µg/ml phytohemagglutinin-L (Data ‘PHA’),

or 5 µg/ml anti-CD3 and anti-CD28 (Data ‘CD3_CD28’)

for 1 and 24 hours using ultra-low binding plates (as indicated in the excel sheets). Afterward, the cells were encapsulated into microfluidic droplets for analysis as described in Portmann et al., eLife, 2024. The cells were analyzed for their secretion using three different panels, termed panel 1 (IL-6, TNF-α, IFN-γ), panel 2 (IL-2, IL-8, MIP-1α), and panel 3 (IL-6, TNF-α, IL-1b, as indicated in the excel sheets). The order of the cytokines above corresponds to the signal in FITC, TRITC and Cy5 channels that were analyzed and indicated in the data provided. The provided Excel files are the raw output data files from the analyzed images. For the publications, these files were further analyzed as described in the corresponding literature.