The incubation of soluble CAR antigen did not increase the functional activity and tumor-killing capacity of B7H3–CAR-T cells. (A and B) Flow cytometry analysis for functional activity of CAR-T cells incubated with tumor antigen B7H3 protein (0.5 µg/ml) for indicated time from 1 to 4 h. The functional activity of CAR-T cells was measured by the expression of IFNγ, CD107a, and granzyme B (n = 3 biological replicates). (C) Immunoblotting analysis for CAR-mediated proximal signaling. The phosphorylation of ZAP70, LAT, and PLCγ of CAR-T were measured after exposure to B7H3 protein (0.5 µg/ml) at indicated times from 0 to 30 min. A representative experiment is shown, repeated twice. (D) Ca²⁺ influx was measured by Fluo-4 fluorescence probe in CAR-T cells. CAR-T cells were incubated with soluble B7H3 protein (0.5 µg/ml), and then the MFI of Fluo-4 was measured by flow cytometry in real time. A representative experiment is shown, repeated twice. (E–G) Cytotoxic activities of CAR-T cells against GSC-1 (E), U87 (F), and LN229 (G) cells (n = 3 biological replicates). CAR-T cells preincubated with 0.5 µg/ml soluble B7H3 protein for 1 h, and then U87-B7H3-KO cells were cocultured with preincubated CAR-T cells at the indicated ratio from 0.1:1 to 3:1 (CAR-T:Tumor) for 24 h. Data represent the mean ± SD from three independent experiments. Statistics by unpaired two-tailed Student’s t test (B) or two-way ANOVA (E–G). *, P < 0.05; **, P < 0.01; N.S., no significance. Source data are available for this figure: SourceData FS2.