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Data from: Stimulation-induced cytokine polyfunctionality as a dynamic concept

Cite this dataset

Portmann, Kevin; Linder, Aline; Eyer, Klaus (2024). Data from: Stimulation-induced cytokine polyfunctionality as a dynamic concept [Dataset]. Dryad. https://doi.org/10.5061/dryad.612jm64c2

Abstract

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.

README: Data set 'Stimulation-induced cytokine polyfunctionality as a dynamic concept'

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:

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.

Funding

Personalized Health and Related Technologies (PHRT), Strategic Focus Area

Swiss National Science Foundation

Olga-Mayenfisch Stiftung

Novartis Foundation