Characterizing CD4+ T Cell Responses to SARS-CoV-2 Variants

T cell lines were plated at a concentration of 25 × 10⁴ cells/well in 100 μL AIM-V medium (ThermoFisher, Waltham, MA, USA) with 2% human serum in 96-wells plate, and restimulated with either “WT CD4⁺ pool” or “Omicron BA.4/BA.5 CD4⁺ pool” (1 μM/peptide) for 6 h at 37 °C, 5% CO₂. Cells were washed and stained as previously described [22 (link)] with minor modifications as indicated.
Cells were stained intracellularly for anti-CD154 (clone 24–31; Biolegend, San Diego, CA, USA), and anti-IFN-ɣ (clone 4S.B3; BD Bioscience, Allschwil, Switzerland), anti-IL-2 (clone MQ1-17H12; Biolegend), anti-TNF-α (clone Mab11; Biolegend, San Diego, CA, USA), anti-IL-5 (clone TRFK5) and anti-IL-13 (clone JES10-5A2; Both BD Biosciences, Allschwil, Switzerland). Approximately 133,000 events were acquired on a FACS Symphony A3 analyzer (BD Biosciences, Allschwil, Switzerland). FlowJo (version 10, Tree Star, Ashland, OR, USA) was used for flow cytometry data analysis.