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Cells were stained overnight at 4°C with anti‐IFNγ (4S.B3), anti‐IL‐6 (MQ2‐13A5), anti‐IL17a (N49‐653), anti‐RORγt (Q21‐559), anti‐IL‐2 (MQ1‐17H12), anti‐IL‐10 (JES3‐9D7), anti‐T‐bet (4B10), anti‐CTLA‐4 (BNI3), anti‐GATA3 (L50‐823) (all from BD Biosciences); anti‐IL‐4 (MP4‐25D2), anti‐TNFα (Mab11), anti‐FOXP3 (206D) (all from BioLegend). Data were collected on BD Symphony (BD Biosciences). A maximum of 5 × 105 events were acquired for each sample. tSNE, FlowSOM and heatmap analysis were performed in R (version 3.6.2). Raw.fcs data were compensated using AutoSpill, 22 and dead cells and debris were gated out prior to file export. The complete set of FCS files used for the COVID‐19 cytokine immune phenotyping has been deposited on FlowRepository and annotated in accordance with the MIFlowCyt standard. These files may be downloaded for further analysis from https://flowrepository.org/experiments/2713 . The concatenated data set was analysed through successive FlowSOM clustering and tSNE representation after exporting similar event numbers for each sample per condition group and then subsampling equal event numbers per condition. First, lineage markers (CD3, CD4, CD8, FOXP3, CD19, CD14) were used to separate leukocyte subsets. Second, activation markers (4‐1BB, CCR7, CD25, CD45RA, CTLA‐4, HLA‐DR, IFNγ, IL‐2, IL‐4, IL‐6, IL‐10, IL‐17a, PD‐1, RORγt, T‐bet, TNFα) were used to distinguish phenotypic clusters of each leukocyte subset, again using FlowSOM and tSNE. The characteristics of each identified cluster were assessed by means of histograms and heatmaps.

https://onlinelibrary.wiley.com/doi/full/10.1002/cti2.1204

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