Anti human cd19 pc7 clone j3 119

For B-cell count and B-cell subpopulations determination, 200,000 PBMC were transferred into 96 U-well plate (Greiner Bio-One, Germany). After PBS was added up to volume of 200 µl, the plate was centrifuged (360 × g, 6 min, RT). Cells were stained with 50 µl of mixture of anti-human CD19-Pc7 (clone J3-119; Beckman Coulter, USA), anti-human CD27-BV421 (clone M-T271; Becton Dickinson, USA), anti-human IgM-PerCP Cy5.5 (clone MHM-88; BioLegend, USA) and PBS with 0.5% FCS for 30 min, 4 °C, dark. Afterwards 150 µl PBS with 0.5% FCS was added and the samples were centrifuged (360;× g, 6 min, RT), supernatant discarded. The cells were resuspend in 100 µl of PBS, followed by adding 100 μl of 4% PFA (paraformaldehyde) (Sigma-Aldrich Chemie GmbH, Germany) and the suspension was well mixed. The samples were kept in fridge (4 °C) overnight. Next day, the samples were washed before the measurement and cells were resuspended in PBS with 0.5% FCS. Samples were measured on a BD LSRII SORP flow cytometer with BD FACS Diva software version 8.0.1 (Becton Dickinson, USA) and analyzed with Kaluza Analysis Software (Beckman Coulter, USA). The gating strategy is shown in Supplementary Fig. 8.

Before staining, untouched B cells were enriched from PBMC using the EasySep human B-cell enrichment kit (StemCell Technologies, Canada). TT-specific B cells in buffy coat PBMC were then characterized with an antibody panel consisting of anti-human CD19-Pc7 (clone J3-119; Beckman Coulter, USA), anti-human CD27-BV711 (clone O323; BioLegend, USA), anti-human IgM-BV605 (clone MHM-88; BioLegend, USA), anti-human IgA (clone REA1014; Miltenyi, Germany) and with TT purchased from AJ vaccines, Denmark, biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, UK) and fluorescently labeled with streptavidin-BV421 (BioLegend, USA). Samples were measured on a FACS Aria™ Fusion (Beckman Coulter, USA). The gating strategy is depicted in Supplementary Fig. 2c.