Fig. 3

T-box domain of T-bet is not required for enhanced efficacy of CAR T cells against B7H6-expressing tumors. a B7H6-specific CAR/T-bet variants were constructed to remove the T-box domain from the mouse T-bet. The B7H6-specific CAR/T-bet (STOP) was created by inserting a stop codon before the T-box domain. The (∆TBOX) variant was created by deleting the T-box domain, leaving the transactivation domain intact. b Expression of mouse CD4⁺ T cells transduced with B7H6-specific CAR/T-bet variants. CD4⁺ T cells transduced with vector only (Mock) was used as a negative control. Transduced T cells were stained for T-bet or for the CAR. Specific antibody binding is shown in dark lines and fluorescence minus one (for CAR staining) or isotype control (for T-bet staining) are shown in light lines. Values indicate the mean fluorescence intensity or percent of cells above staining controls. Data shown are representative of four independent experiments. MFI and the total mean from four independent experiments are shown (right). c IFN-γ production by Mock CD4⁺ T cells (1 × 10⁵) or CD4⁺ T cells expressing the B7H6-specific CAR alone or with T-bet, T-bet (STOP), or T-bet (∆TBOX) co-cultured with RMA tumors, RMA-B7H6, B16F10 tumors, or B16F10-B7H6 for 24 h. ANOVA Dunnett’s test (*p < 0.05, **p < 0.01, ***p < 0.001). d Survival of tumor cells was assessed for CD4⁺ CAR T cells co-cultured with tumor cell lines at E:T ratios of 0.2:1, 1:1, and 5:1 for 24 h. The RLU values are shown ± SD. ANOVA Dunnett’s test (*p < 0.01, ^∇p < 0.01 B7H6-specific CAR/TBET (∆TBOX) vs Mock, ^φp ≤ 0.01 B7H6-specific CAR/TBET/B7H6-specific CAR/TBET (∆TBOX) vs Mock). The data shown are representative of three independent experiments