Cd69 fitc fn50

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.

PBMC and BALF samples were incubated with Fc-receptor blocking reagent (BD Biosciences) at room temperature for 15 min and surface stained with the following fluorochrome-conjugated antibodies at 4°C for 40 min: CD45-APC (HI30, BioLegend), CD3-APC-cy7 (UCHT1, BioLegend), CD8-BV510 (SK1, BioLegend), CD4-BV421 (RPA-T4, BD Biosciences), CD161-PE (HP-3G10, BioLegend), CD161-BV421 (HP-3G10, BioLegend), Vα7.2-PE-cy7 (3C10, BioLegend), CD69-FITC (FN50, BioLegend), CD69-PE (FN50, BioLegend), and PD-1-BV421 (EH12.1, BD Biosciences). MAIT cells were identified as CD3⁺CD161^high Vα7.2⁺ cells.
For intracellular cytokine staining, PBMCs were cultured in RPMI 1640 supplemented with 10% FBS in the presence of phorbol 12-myristate 13-acetate (PMA) (25 ng/ml) and ionomycin (500 ng/ml) for 40 min followed by a 3.5-h incubation with brefeldin A in a 5% CO₂ incubator at 37°C. IL-17A- and/or IFN-γ-producing cells were identified by intracellular staining with anti-IL-17A-FITC (BL168, BioLegend) and anti-IFN-γ-FITC (4S.B3, BioLegend) for 45 min after surface staining and fixation/permeabilization. Fluorescence Minus One (FMO) was used as negative control; the gating strategy for surface markers and intracellular cytokines is shown in Supplemental Figure 1. Cells were acquired by flow cytometry using a FACSVerse (BD Biosciences) and analyzed with FlowJo software (TreeStar).