Figure 1.
Figure 1. Dasatinib is a potent, rapid, and reversible inhibitor of CAR-T cell function. Mock (untransduced), CD19.28ζ, and CD19.BBζ CAR-T cells were cocultured with CD19+ Nalm6-GL at 1:1 and/or 1:8 effector:target (E:T) ratio in the presence of the concentrations of dasatinib noted. (A) CD69 and CD107a expression following 6 hours of coculture at 1:1 E:T (representative histograms, similar results observed using n = 3 donors). (B) Cocultures at a 1:1 and 1:8 E:T were analyzed in an IncuCyte to assess CAR-T cell cytotoxicity. Tumor GFP fluorescence intensity was normalized to the first time point (triplicate wells; representative donor, top and middle). The fold change in tumor fluorescence intensity from t = 0 to t = 72 hours for 1:8 E:T cultures across 3 donors is shown with each dot representing results from 1 donor (bottom). (C) IL-2 and interferon-γ secretion after a 24-hour coculture (top, triplicate wells from 1 representative donor; bottom, normalized data from 3 independent donors, with each dot representing results from 1 donor). (D) CAR-T cells were preloaded with Cell Trace Violet (CTV) cytosolic dye and cocultured at a 1:1 E:T in 1 μM dasatinib or vehicle for 72 hours. CAR-T cell proliferation was assessed via flow cytometry (representative histograms of n = 3 donors). (E) CAR-T cells were cocultured in 1 μM dasatinib or vehicle and analyzed in an IncuCyte as described previously. At t = 20 hours, dasatinib was either removed (top) or added (bottom) (triplicate wells; result is representative of n = 3 donors). (F) CAR-T cells cultured in the presence of 1 μM dasatinib or vehicle were stimulated with 5 μg/mL anti-FMC63 idiotype + 5 μg/mL goat anti-mouse secondary for 5 minutes. Cells were lysed; phosphoprotein and total protein levels were subsequently assessed via western blot (representative blots, n = 3 donors). Representative plots display the mean ± standard deviation of 3 technical replicates; dot plots display the mean ± standard error of the mean of 3 independent donors. *P < .05; **P < .01; ****P < .0001; not significant (ns), P > .05. IFN-γ, interferon-γ; ND, not detectable.

Dasatinib is a potent, rapid, and reversible inhibitor of CAR-T cell function. Mock (untransduced), CD19.28ζ, and CD19.BBζ CAR-T cells were cocultured with CD19+ Nalm6-GL at 1:1 and/or 1:8 effector:target (E:T) ratio in the presence of the concentrations of dasatinib noted. (A) CD69 and CD107a expression following 6 hours of coculture at 1:1 E:T (representative histograms, similar results observed using n = 3 donors). (B) Cocultures at a 1:1 and 1:8 E:T were analyzed in an IncuCyte to assess CAR-T cell cytotoxicity. Tumor GFP fluorescence intensity was normalized to the first time point (triplicate wells; representative donor, top and middle). The fold change in tumor fluorescence intensity from t = 0 to t = 72 hours for 1:8 E:T cultures across 3 donors is shown with each dot representing results from 1 donor (bottom). (C) IL-2 and interferon-γ secretion after a 24-hour coculture (top, triplicate wells from 1 representative donor; bottom, normalized data from 3 independent donors, with each dot representing results from 1 donor). (D) CAR-T cells were preloaded with Cell Trace Violet (CTV) cytosolic dye and cocultured at a 1:1 E:T in 1 μM dasatinib or vehicle for 72 hours. CAR-T cell proliferation was assessed via flow cytometry (representative histograms of n = 3 donors). (E) CAR-T cells were cocultured in 1 μM dasatinib or vehicle and analyzed in an IncuCyte as described previously. At t = 20 hours, dasatinib was either removed (top) or added (bottom) (triplicate wells; result is representative of n = 3 donors). (F) CAR-T cells cultured in the presence of 1 μM dasatinib or vehicle were stimulated with 5 μg/mL anti-FMC63 idiotype + 5 μg/mL goat anti-mouse secondary for 5 minutes. Cells were lysed; phosphoprotein and total protein levels were subsequently assessed via western blot (representative blots, n = 3 donors). Representative plots display the mean ± standard deviation of 3 technical replicates; dot plots display the mean ± standard error of the mean of 3 independent donors. *P < .05; **P < .01; ****P < .0001; not significant (ns), P > .05. IFN-γ, interferon-γ; ND, not detectable.

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