Fig. 1

Sulforaphane (SFN) enhanced the antitumor function of meso CAR-T cells in vitro by downregulating PD-1 expression. A Schematics of meso CAR constructs. B Transduction efficacy of meso CAR-T cells. Transduction efficacy was examined by green fluorescent protein (GFP) expression on day 5. C Analysis of specific lysis of tumor cells. H322 cells were incubated with meso CAR-T cells at various effector to target (E:T) ratios (1:1, 5:1, and 10:1) for 6 h, and lysis of H322 cells was analyzed by flow cytometry. D H322 cells were incubated with meso CAR-T cells at a 10:1 ratio for 6 h. Expression of CD107a on CAR-T cells was tested by flow cytometry. E–G After co-culturing with H322 cells at a 1:1 ratio for 24 h, the secretion of IFN-γ (E), perforin (F), and granzyme B (G) by CD8⁺ meso CAR-T cells was investigated by flow cytometry. H, I PD-1 expression on CD8⁺ meso CAR-T cells with or without SFN treatment was analyzed by flow cytometry (H) and western blot (I). J PD-1 expression on CD8⁺ meso CAR-T cells was tested by flow cytometry, with or without treatment with SFN and PI3K/AKT inhibitor PF-04691502 (10 μM). K Western blot analysis of phosphorylated and total AKT, mTOR, and S6 proteins, as well as PD-1 and β-actin levels, in meso CAR-T cells with or without SFN and PF-04691502 treatment. Results are representative of five independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t test)