SARS-CoV-2 Spike Peptide-Specific T-cell Assay

Cryopreserved human PBMC were thawed and rested for 4 h at 37 °C. Cells were cultured in 96-well plates at 1 × 10⁶ cells/well and stimulated for 20 h with 2 μg/peptide/mL of peptide pools (15mer, overlapping by 11) covering the S1 or S2 domains of SARS-CoV-2. Selected donors were also stimulated with SEB (1 μg/mL) as a positive control, or individual peptides at 2 ug/mL: NCTFEYVSQPFLMDL (S1 epitope; previously described in ref. ^{45 (link)}); LPIGINITRFQTLLA (S1 epitope); GWTFGAGAALQIPFA (S2 epitope); ALQIPFAMQMAYRFN (S2 epitope); LLQYGSFCTQLNRAL (S2 epitope;^{19 (link),45 (link)}); QALNTLVKQLSSNFG (S2 epitope). Following stimulation, cells were washed, stained with Live/dead Blue viability dye (ThermoFisher), and a cocktail of monoclonal antibodies: CD27 BUV737 (L128), CD45RA PeCy7 (HI100), CD20 BUV805 (2H7), (BD Biosciences), CD3-BV510 (SK7), CD4 BV605 (RPA-T4), CD8 BV650 (RPA-T8), CD25 APC (BC96), OX-40 PerCP-Cy5.5 (ACT35), CD69 FITC (FN50), CD137 BV421 (4B4-1) (Biolegend), and CXCR5 PE (MU5UBEE, ThermoFisher). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSR Fortessa using BD FACS Diva. Gating is shown in Supplementary Fig. 7.