Cytokine Flow Cytometry Analysis
Fresh whole blood was incubated with SARS-CoV-2-specific peptides for 9 hours and immune cells were stained for surface markers. Cells were stained with fluorochrome conjugated antibodies to CD3+ (FITC), CD4+ (PerCP Cy5.5), CD8+ (V450), CD45+ (V500) and CD56+ (PE-CF594) (BD Bioscience, CA). After erythrocytes were lysed by permeabilization, intracellular cytokines were stained with fluorochrome conjugated antibodies to IL-2 (APC), IFN-γ (PE), and TNF-α (PE-Cy7) (BD Bioscience, CA). The CD4+ (IL-2/TNF-α)+ cells and CD8+ (TNF-α/IFN-γ)+ stimulated with S, B.1.1.7 S, VME, NCAP, AP3A, NS7A were enumerated and defined as CoV-2-specific T-cells after deducting the background levels in blood only conditions. Dual cytokines % in CD4+ or CD8+ cells ≥ 0.05% were considered positive.