T cell stimulation assay and SARS-CoV-2 peptide pools
In vitro T cell stimulation assays were carried out with spike (S), membrane (M), and nuclear (N) structural proteins. Briefly, viable cell numbers were determined in the thawed PBMCs by staining with crystal violet and counting with a hemocytometer. For the assays, 106 cells were resuspended in 100 µL RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. The SARS-CoV-2 peptide pools (Miltenyi Biotec, Germany) were prepared according to the manufacturer’s recommendations. Next, 1 µg of peptide/mL (0.6 nmol) separately or in a mixture was introduced to the T cells. Along with the peptide pools, 0.1 µg/mL purified anti-human CD28 (Miltenyi Biotec, Clone: REA612) and 0.1 µg/mL purified anti-human CD49d (Miltenyi Biotec, Clone: MZ18-24A9) as coactivators of T cells were also added to the wells for the entire stimulation period. The T cells and peptide mixtures were incubated at 37oC in 5% CO2 for 16 hours. Brefeldin A (Biolegend, San Diego, CA) at a concentration of 0.1 µg/mL was added to the culture medium in the last 4 hours to enhance intracellular cytokine staining signals. The negative control was 10% DMSO and the positive control was an activation cocktail (Biolegend)) containing 8.1 nM phorbol-12-myristate (PMA) and 1.3 mM ionomycin.