Figure 6.

Chimeric antigen receptor (CAR) signaling improves CAR- and HLA-independent lysis of Daudi tumor cells. (a) Cytokine-induced killer (CIK) cells were engineered with the CD28-ζ and CD28-ζ–OX40 CAR, and CAR expressing cells were isolated by cell sorting. CAR CIK cells were cultivated on plates coated with the CAR-specific anti-idiotypic monoclonal antibody (mAb) BW2064 (1 µg/ml coating concentration). After 5 and 14 days of culture, cells were stained for CD4, CD8, and CD56 expression and analyzed by flow cytometry. Numbers represent the mean fluorescence intensity. (b) Cells that were cultivated for 14 days on anti-idiotypic mAb-coated surfaces were recovered and cocultivated with carboxyfluorescein diacetate succinamidyl ester-labeled Daudi cells (2 × 10⁴ cells/well) for 24 hours. Cells were stained with 7-aminoactinomycin D (0.5 µg/ml) and the numbers of life and dead cells were determined by flow cytometry. Numbers represent mean values ± SD. P values were determined using the Student's t-test.