Cd27 bv605 o323

The whole blood was washed in PBS 1X with 0.4 g/l human albumin. Samples were stained with master mix of antibodies for analysis on CantoII or LSRFortessa flow cytometers (BD) before lysis of red cells and fixation (BD FacsTM Lysing solution 10X). Data were analyzed using Flow Jo software (Tree Star).The anti-human antibodies used were CD8b PC5 (2ST8.5H7; Beckman Coulter), TCR γδ FITC (11F2), (BD Biosciences), CD45RA PC7 (HI100 eBiosciences), CD4 PE TX (S3.5 Invitrogen), CD3 Alexa700 (UCHT1), CCR7 BV421 (G043H7), CD27 BV605 (O323), and CD127 BV650 (A019D5)—all from Biolegend.

Probes for delineating SARS-CoV-2 S-specific B cells within cryopreserved human PBMC were generated by sequential addition of streptavidin-PE (Thermofisher) to trimeric S protein biotinylated using recombinant Bir-A (Avidity). SARS-CoV-2 RBD protein was directly labelled to APC using an APC Conjugation Lightning-link kit (Abcam). Cells were stained with Aqua viability dye (Thermofisher). Monoclonal antibodies for surface staining included: CD19-ECD (J3-119) (Beckman Coulter), CD20 Alexa700 (2H7), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (O323) (Biolegend), IgA-Vio450 (clone) (Miltenyi). Cells were washed, fixed with 1% formaldehyde (Polysciences) and acquired on a BD LSR Fortessa or BD Aria II. Gating is shown in Supplementary Fig. 4.