Figure 1.

Representative example of cytokine production by CD4+ and CD8+ T cells from a Covid‐19 patient with severe pneumonia after in vitro stimulation after in vitro stimulation with anti‐CD3/CD28 (1ug/mL) for 16 h in the presence of anti‐CD107a‐PE (Biolegend, San Diego, CA).14, 15 PBMC were stained with viability marker (AQUA Live Dead, ThermoFisher) and anti‐CD4‐AF700 and CD8‐APC‐Cy7 (Biolegend). Cells were fixed and permeabilized with Cytofix/Cytoperm (Becton Dickinson, San Josè, CA) according to manufacturer protocols. Finally, cells were stained with anti‐IFN‐γ‐FITC, anti‐TNF‐α‐BV605, anti‐IL‐17A‐PE‐Cy7, anti‐IL‐2‐APC, and anti‐Granzyme B‐BV421 (all from Biolegend). Data were acquired by using attune NxT acoustic flow cytometer. (A) Intracellular staining of different cytokines in previously gated living CD3+,CD4+ in a healthy donor (upper plots) and in a patient (lower panels); (B) intracellular staining of different cytokines in previously gated living CD3+,CD8+ in a healthy donor (upper plots) and in a patient (lower panels); (C) analysis of the polyfunctionality of CD8+ T cells by using “Simplified Presentation of Incredibly Complex Experiments (SPICE),”, kindly provided by Dr. Mario Roederer (NIH, Bethesda, MD).16 Arcs represent the total production of each cytokine, pie slices the polyfunctional capacity of cells. For the functional analysis of CD8+ T cells, that in theory can provide 64 populations of cells producing different combination of cytokines, a threshold of 0.5% was set on the basis of the distribution of negative values generated after background subtraction. Note that, as expected, in patient and control no CD8+ T cell was able to produce IL‐2. [Color figure can be viewed at wileyonlinelibrary.com]