Nir fixable viability dye

Cryopreserved PMBCs were thawed in RPMI1640 medium (Gibco) and washed. 1 × 10⁶ cells were cultured in R10 medium (RPMI1640 supplemented with 10% heat-inactivated fetal calf serum, 1% HEPES (Gibco) and 1% penicillin/streptomycin). The cells were cultured for 18 h with 20 µg/mL of PE-labelled anti-CD107a (clone H4A3, BioLegend) and a peptide pool of 10 non-spike 15-mer peptides (Table S2) [30 (link)] at 10 µg/mL at 37 °C and 5% CO₂. After 1h, Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) was added in a final concentration of 5 µg/mL. Under the same conditions, SEB (1 µg/mL) served as a positive control, and solely R10 medium as a negative control. Subsequently, the cells were washed and stained with a 1:150 dilution Zombie NIR fixable viability dye (BioLegend) and monoclonal antibodies targeting surface antigens (Table S3) for 30 min at room temperature. The cells were fixed and permeabilized with the Foxp3 transcription factor staining buffer kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for 45 min at 4 °C, and stained for intracellular cytokines (Table S3) with monoclonal antibodies.

Cryopreserved PBMC were thawed in RPMI1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and washed. A PE-labelled DRB1*11:01 MHC class II Tetramer loaded with a 20-mer sequence of the SARS-CoV-2 membrane protein (aa145–164; LRGHLRIAGHHLGRCDIKDL) was added at a final concentration of 2.5 µg/mL to the cell suspension of 1 × 10⁷ cells/mL for 30 min at room temperature [20 (link)]. Subsequently, the cells were washed and stained with a 1:150 dilution Zombie NIR fixable viability dye (BioLegend, San Diego, CA, USA) and monoclonal antibodies, as indicated in Table S1, for 30 min at room temperature, and fixed with 4% paraformaldehyde for 45 min at 4 °C.