Cd27 apc ef780

The following antibodies were used: cluster of differentiation (CD)4 BV605, CD8 BV650, CCR7 PE-CF594 (Becton Dickinson), CD45RA PECy7, CD28 BV421, CD127 BV711, PD-1 PerCP-Cy5.5, CD95 APC, CD62L BV785 (BioLegend, Dedham, MA), CD3 AF700, CD45RO FITC, CD27 APC-ef780 (eBioscience, San Diego, CA). The dead cell exclusion stain (Live/Dead Aqua) was purchased from Invitrogen (Carlsbad, CA). To detect NY-ESO-1^c259TCR-expressing cells, purified anti-phycoerythrin-conjugated dextramer reagents specific for the HLA-A*02:01 SLLMWITQC complex (Immudex) were used at the manufacturer’s recommended concentrations.

PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.

Leukocyte subsets were identified using an antibody panel containing CD45-PO (Life Technologies) for lymphocytes, CD3-AF 700 (BioLegend) for T cells, CD14-PeCy7 (BioLegend) for monocytes, CD19-BV711 (BioLegend) for B cells, and CD16/CD56-APC (eBioscience, BD Biosciences) for NK cells. T cell subsets were distinguished by CD3-AF 700 (BioLegend), CD4-BV711 (BioLegend), CD8-PeCy7 (BD), CD27-APC-eF780 (eBioscience, San Diego, CA, USA), and CD45RO-PB (BioLegend). Each sample was also stained with isotype-matched control monoclonal antibodies for spectral compensation and to correct for background fluorescence. Because of the extensive antibody panel, some antibodies were measured in the same fluorescence channel, e.g., CD4 and CD19, CD8 and CD14, and CD16 and CD56. A representative example of the gating strategy to identify the different lymphocyte subsets is provided in Figure S1 in Supplementary Material.