DOI: 10.5061/dryad.612jm64c2
title: Data from: Stimulation-induced cytokine polyfunctionality as a dynamic concept

description: 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.
description: 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.
description: # 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 zymosan (Data ‘Zymosan’), 10 µg/ml phytohemagglutinin-L (Data ‘PHA’), or 5 µg/ml anti-CD3 and anti-CD28 (Data ‘CD3_CD28’) Within each Excel file, the sheets represent the data sorted by cytokine panel (panels 1, 2 or 3 as described in the methods herein), and timepoint that describes the duration of incubation, either 1 and 24 h. Within each sheet, the DropIdx gives a unique identity to each analyzed droplet, whereas * TrueCentroid_x gives x and y coordinates over time * DAPI_WD_Mean_t gives the average of the whole droplet fluorescence in the DAPI channel over time * DAPI_WD_Mode_t indicates the mode of the whole droplet fluorescence in the DAPI channel over time * DAPI_WD_Std_t gives the standard deviation of the whole droplet fluorescence in the DAPI channel over time * DAPI_WD_PosPxlPercent_t describes the percentage of positive pixels above a threshold of the whole droplet fluorescence in the DAPI channel over time * FITC_BL_Mean_t is the average fluorescence measured in the droplet in FITC over time * FITC_BL_Max_t describes the maximal fluorescence measured in the vertical direction within the droplet (i.e., value on the beadline) in FITC over time * FITC_BL_Ratio_t gives the ratio of maximal fluorescence divided by whole droplet fluorescence over time - TRITC and Cy5 channels are organized in a similar manner as the FITC channel. * TrackingMove indicates the distance that the droplet has been tracked over time * DiameterMicrons indicates the size of the droplet For the meaning of the channels, i.e., cell detection and cytokines measures, please refer to the Methods section in this Dryad repository as panel 1, 2 and 3 refer to different cytokine panels that were measured. The files contain all information for all detected droplets, and were further analyzed as described within the article to derive the data displayed in Portmann et al., eLife, 2024 and Portmann et al., Cell Reports Methods, 2023. ## Sharing/Access information Data was derived from the following sources: * [https://doi.org/10.7554/eLife.89781.2](https://doi.org/10.7554/eLife.89781.2)