Flow cytometry
Stimulated PBMCs were recovered from the culture plates and resuspended
in 100 µL PBS. Cell viability was assessed by staining with
ViobilityTM Fixable Dyes (Miltenyi Biotec, Germany).
Cells were washed, fixed, permeabilized, and then stained with an
antibody cocktail containing Pacific BlueTM anti-human
CD3 (Biolegend, clone: HIT3a), PE/Cyanine7 anti-human CD4 (Biolegend,
clone:A161A1) and PerCP/Cyanine5.5 anti-human CD8 (Biolegend, clone:
SK1) for T cell identification; APC anti-human CD69 (Biolegend, clone:
FN50) and PE anti-human IFN-γ (Biolegend, clone:4S.B3) for the
activation analysis; and FITC anti-human CD14 (Biolegend, clone:HCD14)
and FITC anti-human CD20 (Biolegend, clone:2H7) for the exclusion of
non-specific signals and B cells. Fifty thousand events were analyzed by
a BD LSR-II flow cytometer. The gates applied for the quantification of
the stimulated T cells are illustrated in Fig. S1.