Fluorescent beads

Tetramer staining was carried out as previously described (16 (link), 18 ). In brief, 30 to 90 million CD3 or CD4 enriched T cells were stained at room temperature for 1 hour using 5 ug of each tetramer in 50ul reaction. Tetramer tagged cells were enriched by adding anti-PE and/or anti-APC magnetic beads and passing the mixture through a magnetized column (Miltenyi). The tetramer-enriched samples were stained with live/dead dyes, exclusion markers (anti-CD19 and anti-CD11b, BioLegend), and other surface markers (Table S5) for 30 minutes at 4°C. Samples were acquired by flow cytometry using LSRII (BD) or sorted on FACS Aria (BD). Frequency calculation was obtained by mixing 1/10^th of sample with 200,000 fluorescent beads (Spherotech) for normalization (16 (link)). Non-zero populations were included in the analyses performed using FlowJo (BD). Spectral flow cytometric analyses were performed with following modifications: 2ug of tetramers loaded with each of the twelve SARS-CoV2 peptides used in this study were tagged to the same fluorochrome and combined in the staining reaction. Tetramer enriched cells were stained with live/dead dyes, exclusion markers, and a panel of additional surface antibodies (Table S5) for 1h at 4°C followed by fixation with 2% paraformaldehyde. Samples were acquired on spectral flow Cytex AURORA (ARC 1207i).