Cd45ra pe cy7 hi100

EDTA-anticoagulated whole blood samples were stained within 6 h of collection. Briefly, 100 μl of whole blood was stained with CD8 PerCP (SK1), CD4 FITC (SK3), CD3 APC-H7 (SK7), CD20 APC (2H7), PD-1 BV786 (EH12.1), CCR6 BV711 (11A9), inducible costimulator (ICOS) BV650 (DX29), CD21 BV421 (B-ly4), CD45RA PE-Cy7 (HI100), CD27 PE-CF594 (M-T271) (BD Biosciences, CA), CXCR3 BV510 (G025H7) (Biolegend, San Diego, CA), and CXCR5 PE (MU5UBEE) (eBioscience, San Diego, CA) for 15 min, after which red blood cells were lysed with FACS lysing solution (BD Biosciences), washed and resuspended in FACSflow and acquired on a four laser BD LSRFortessa™ X-20 Special Order Research Product (BD Biosciences, San Jose, CA) within 4 h. CS&T beads and mid-range Rainbow Fluorescent Particles (both BD Biosciences) were run before sample acquisition. Compensation was performed for each experiment using BD™ CompBeads (BD Biosciences). Samples were analyzed using FlowJo software version 9.9.6.

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.