SARS-CoV-2 Antigen-Specific T-Cell Assay

The polyclonal antigen-specific cellular assay was performed on thawed PBMC samples grown in supplemented RPMI1640 (R10), containing 1 µg/mL of SARS-CoV-2 PepMix™ (JPT Peptides, Berlin, Germany), for 48 h in a 37 °C, 5% CO₂ chamber. After initial incubation, cells were harvested, washed with FACS buffer, centrifuged, and stained using a viability dye (Live/Dead Fixable Blue, Thermo Fisher Scientific, Waltham, MA, USA) followed by surface staining using human antibodies: CD3 APC-Cy7, CD4 BV421, CD8 BV605, CD38 PE-Cy7, CD19 BB700, CD27 BB515, CD134 (OX40) BV650, CD197 (CCR7) BV510, and CD45RA APC. All human antibodies were purchased from BD Biosciences, San Jose, CA, USA. Two types of compensation beads were used as compensation controls (UltraCompBeads, Invitrogen, Waltham, MA, USA; ArCTM Armine Reactive Compensation Bead kit, Invitrogen, Waltham, MA, USA) [32 (link)].
The samples were acquired using LSRFortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software v.10.8 (BD Biosciences, San Jose, CA, USA). The gating strategy used for clustering memory B and T cells is represented in Supplementary Figure S1. PBMCs grown in unsupplemented R10 were used as a negative control and as a measure of baseline fluorescence. The minimum percentage considered for final analysis was 1% in the gate of activated memory cells.