Figure 7.

Conversion to NK-like poly-functional T cells required both IL12 and CD3ζ CAR endodomain

(A) Schematic representation of the IL12-CAR constructs with deletions of the CD3ζ and/or CD28-CD3ζ endodomains. TM, transmembrane domain. (B) CAR T cells were cultured for 4 days, flow sorted, and co-cultured for 48 h in 96-well micro-test plates with CEA⁺ LS174T or CEA⁻ Skov3 tumor cells (each 2 × 10⁴ cells/well). Viability of tumor cells was determined by the XTT assay and specific cytotoxicity was calculated. IFN-γ in the supernatant was determined by ELISA. Numbers represent mean values of technical replicates ±SD. Statistical differences were determined by Student's t test (∗∗∗p < 0.005; ∗∗p < 0.01; ∗p < 0.05; n.s., not significant). Representative data of two independent experiments are shown. (C) Acquisition of NK-like cytotoxicity by CAR T cells requires both IL12 and CAR signaling. T cells were engineered with the conventional CAR and IL12-CAR, respectively, and co-cultivated for 48 h with CEA⁻ Skov3 or CEA⁺ LS174T tumor cells (each 2 × 10⁴ cells/well) in presence or absence of the CAR-specific anti-idiotypic antibody BW2064 (4 μg/mL), which serves as a CAR-specific antigen, and added IL12 (5 ng/mL), respectively. Viability of tumor cells was determined by the XTT assay and specific cytotoxicity was calculated. Numbers represent mean values of technical replicates ±SD. Statistical differences were determined by Student's t test. Representative data of two independent experiments are shown. (D) CAR CD3ζ endodomain is required for upregulating CD94 in IL12-CAR T cells. T cells were engineered with the conventional CAR, IL12-CAR, and the CAR variants with truncated endodomains, respectively. After transduction, T cells were cultured for 2 days and analyzed for CAR and CD94 expression by flow cytometry. Numbers represent mean values of seven or eight different donors ±SD. Significant differences were calculated by Student's t test. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05.