T-cell response
The T-cell response was assessed as previously described . Briefly, peripheral blood mononuclear cells (PBMCs) were stimulated with 4 µL of BD Fast ImmuneTM CD28/CD49d (BD Biosciences, San Jose, CA, USA) and PepMixTM SARS-CoV-2 S-RBD/ NCAP (JPT Peptide Technologies, Berlin, Germany) or anti-human CD3 low endotoxin as a positive control. After incubation, PBMCs were stained with antibody-fluorochrome conjugates for FACS. Subsequently, cells were stained for viability with LIVE/DEAD Violet Viability Dye (Invitrogen, Waltham, MA, USA). Finally, samples were measured on a BD LSR II flow cytometer to detect intracellular production of the cytokines interleukin 2 (IL-2), interferon α (IFNγ), and tumor necrosis actor α (TNFα) in CD4+ T cells, and data were analyzed using FlowJo software (version 10.6.1; BD Biosciences).
Only patients who responded to non-specific anti-CD3 stimulation were considered for further analysis of antigen-specific responses. A positive response required a >1.5-fold increase above the non-stimulated controls and detection of >20 responding cells, as previously described for sensitive CMV-specific T-cell detection . All combinations of IL-2, IFNγ, and TNFα production upon stimulation were analyzed for the total CD4+ T-cell response. The gating strategy is illustrated in Supplementary Fig 1.