Flow Cytometry

The phenotype of T cells was assessed at different time points of the culture by flow cytometry. To determine antigen specificity, HLA-A0201/LMP2_426-434 dextramer (Immudex) or H2-Db/gp33 tetramer staining was performed (the H2-Db/gp33-41 biotinylated monomers were obtained through the NIH Tetramer Core Facility, and tetramers were generated using ExtrAvidin®-PE (Thermo Fisher Scientific). Cell viability was assessed by Zombie Aqua fixable viability dye. Cells were surface stained with human monoclonal antibodies against: CD3, CD4, CD8, CD45RO, CD45RA, CD62L, CCR7, PD-1, TIM3, 2B4, CD38, CD27, CD28, NGFR (BioLegend) or mouse monoclonal antibodies against: CD4, CD8, CD62L, CD69, CD44, PD-1, (BD Biosciences), before acquisition on a LSRII or FortessaX-20 instrument (BD Biosciences). For SA-β-Gal evaluation, cells were stained with the Quantitative Cellular Senescence Assay kit (Cell Biolabs). The Vybrant DyeCycle Green Stain (ThermoFisher) was used for cell cycle analysis. For T-cell function, cells were incubated with 7.5µg/mL brefeldin A (Sigma-Aldrich) for 4 hours in the presence or not of phorbol 12-myristate 13-acetate (PMA; 50ng/ml) and ionomycin (500ng/ml) (Sigma-Aldrich). Cells were permeabilized with Foxp3 staining buffer set (eBiosciences) before intracellular staining with antibodies against IFNγ and TNFα (BioLegend). For DNA damage and proliferation determination, cells were also permeabilized with Foxp3 staining buffer set prior to intracellular staining with anti-γH2AX and anti-Ki67 antibodies, respectively (BioLegend). Data were analyzed using FlowJo software (BD Biosciences).