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 ..., 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-..., , # Data set 'Stimulation-induced cytokine polyfunctionality as a dynamic concept'

[https://doi.org/10.5061/dryad.612jm64c2](https://doi.org/10.5061/dryad.612jm64c2)

The data sets contain raw Excel output files from analyzed images from blood peripheral mononuclear cells from anonymized healthy donors that were frozen, thawed and stimulated with different stimulants (different files) at different times using two different cytokine panels (sheets) to assess frequencies of secreting cells, their secretion rates and corresponding secretion dynamics as well as polyfunctionality.

If preferred, the raw images can be received from the corresponding author upon reasonable request.

## Description of the data and file structure

The following conditions are present in the excel files:

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

1 µg/ml lipopolysaccharide (Data ‘LPS and Data 'LPS_3rd panel'),

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

100 µg/ml zymos...