To determine target-specific proliferation, 1 × 105 CAR T cells were labeled with 1 µM CellTraceTM Violet (Life Technology) and co-incubated with either 5 × 105 target cells or 5 × 104 human T-activator CD3/CD28 beads in a final volume of 1 mL RPMI-1640 media supplemented by 10% Hyclone FBS, for 7 days at 37 °C. Cells were then recovered and labeled with anti-human CD3-BV421 (1:100; Biolegend) and anti-V5-FITC (1:200; Invitrogen). After staining, 10 µL of CountBright beads (Thermo Fisher Scientific) were added to the cells in 190 µL volume. The number of cells and beads were calculated following the manufacturer’s protocol.
To measure CD69 expression, cells were pelleted after a 24-h co-culture (E:T = 1:2) for flow cytometry analysis, while supernatants were collected for cytokine analysis. Cells were collected and labeled with anti-human CD69-BV605 (1:100; Biolegend). All flow cytometric analysis was done on an LSRII cytometer (BD Biosciences) using FACSDiva (BD Biosciences) and analyzed with FlowJo software version 10 (Tree Star). Cytokine production was quantified by MSD U-PLEX assays (Mesoscale Discovery) according to the manufacturer’s protocol.
To measure CD69 expression, cells were pelleted after a 24-h co-culture (E:T = 1:2) for flow cytometry analysis, while supernatants were collected for cytokine analysis. Cells were collected and labeled with anti-human CD69-BV605 (1:100; Biolegend). All flow cytometric analysis was done on an LSRII cytometer (BD Biosciences) using FACSDiva (BD Biosciences) and analyzed with FlowJo software version 10 (Tree Star). Cytokine production was quantified by MSD U-PLEX assays (Mesoscale Discovery) according to the manufacturer’s protocol.