PBMC Isolation and Spike Protein Stimulation

PBMC were isolated using Ficoll–Paque (Pharmacia, Uppsala, Sweden) gradient centrifugation from citrated peripheral blood samples. PBMC were then resuspended in RPMI-1640 medium (Roswell Park Memorial Institute) from Bio-Whittaker, Verviers, Belgium, containing 10 mM of L-glutamine (Biochrom, Berlin, Germany), 0.1 mg/mL of penicillin and streptomycin (Biochrom), and 10% fetal bovine serum (Biochrom) [18 (link)]. The cell concentration was adjusted to 2 × 10⁶ cells/mL. PBMC were stimulated with a pool of spike peptides (PepTivator S, cat. 130-126-701, PepTivator S1, cat. 130-127-048, PepTivator S+, cat. 130-127-312, Miltenyi Biotec, Bergisch Gladbach, Germany) at the recommended concentrations for 16 h (37 °C, 5% of CO₂), while negative controls were treated with the same amount of vehicle used to dissolve the peptide mix [14 (link),15 (link),19 (link)]. After 2 h of stimulation, samples were treated with 6.5 µL of GolgiStop (554724, BD Biosciences, La Jolla, CA, USA). The staining was carried out as already published [11 (link)], using the reagent list reported in Table S2. Samples were finally acquired by flow cytometry (CytoFLEX, Beckman Coulter, Chaska, MN, USA).