Figure 4
TetCAR activity can be fine-tuned in vitro with a dose-dependent response to minocycline. (a) Killing of SupT1-CD19-GFP after 24 h of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600 nM were added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, ± SD. n = 4 donors from 1 experiment. (b) IFN-γ and (c) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at various minocycline doses. Data shows mean ± SD, n = 4 donors from 1 experiment. (d) IL-2 secretion from FMC63-BBz or 28BB-Fab-Tet-z CARs 1–5 h after co-culture with SupT1-CD19-GFP at a 2:1 E:T ratio. 100 nM minocycline was added to separate wells every hour. Data shows the mean (± SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. Color coded bars indicate the number of hours that the co-cultures were exposed to minocycline for. n = 4 donors from 2 independent experiments. (e) Cytotoxicity or (f) IL-2 secretion by 28BB-Fab-Tet-z CARs after coculture with SupT1-CD19 at a 1:1 E:T ratio. Inhibition by minocycline was removed by washing cells with complete media at 48, 24 and 2 h before addition of SupT1-CD19 targets. Wash steps are indicated by “[W]”. Data shows mean (± SD) % of live targets relative to NT T cells (e) or mean (± SD) IL-2 secretion (f) after 24 h. n = 3 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with multiple comparisons between the 28BB-Fab-Tet-z CAR under different conditions. P values for cytotoxicity (e) were: 48 h wash versus no wash (*, 0.0118), 24 h wash versus no wash (**, 0.0050) and no drug versus no wash (**, 0.0044). P values for IL-2 secretion (f) were: 48 h wash versus no wash (**, 0.0015), no drug versus no wash (**, 0.0044), no drug versus 2 h wash (*, 0.0103) and 48 h wash versus 2 h wash (**, 0.0033).
