Fig. 2: Base editing generates CAR T-cells resistant to adenosine-mediated suppression.
a Flow cytometry histogram plots (a) and MFI quantification (b) of phosphorylated CREB (pCREB) in untransduced (UTD) T cells, unedited (CAR) and A2AR-edited CAR T-cells (A2AR-KO) treated with 2-chloroadenosine (cADO, blue) compared to an untreated control (DMSO), (Kruskal–Wallis, n = 4 independent biological donors, mean ± SD. P*** = 0.0005, n.s. = 0.212). c–f IFN-γ (c), IL-2 (d), TNF-α (e) and GM-CSF (f) production by UTD T cells (white), unedited (gray) or A2AR-KO (red) CAR T-cells measured by ELISA 48 h post-stimulation with H226 tumor cells in the presence (+) or absence (−) of cADO (Kruskal–Wallis test, n = 4 independent biological donors, mean ± SD. P*** = 0.00013, n.s. = 0.21, P** = 0.0014, n.s. = 0.383, P* = 0.014, n.s. = 0.083 P** = 0.0013, n.s. = 0.28). g Magnitude of GM-CSF, IFN-γ, IL2, TNF-α secretion by unedited (gray) and A2AR-KO (red) CAR T-cells in the presence of cADO normalized to respective untreated CAR T-cells (Two-sided Mann–Whitney t-test, n = 4 independent biological donors, mean ± SD. P*** = 0.0007, P*** = 0.0004, P*** = 0.0003, P** = 0.0037). h Incucyte live imaging tumor spheroid cytotoxicity of UTD T cells (white), unedited (gray) and A2AR-KO (red) CAR T-cells in the presence of cADO. Cytotoxicity was measured as a decrease in cumulative tumor GFP+ area over time (n = 3 average of technical replicates, mean ± SD). For all data, symbols and error bars reflect mean ± SD of individual biological replicates, except (h) where symbols represent technical replicates. b–g Kruskal–Wallis test performed to calculate statistical significance, g Mann–Whitney t-test performed to calculate statistical significance, ****P < 0.0001, ***P < 0.001, **P < 0.01.
