Figure 7.

Antigen-redirected and antigen-independent target cell killing is mediated by CAR⁺ CD56⁺ and CD56⁻ CIK cells with similar efficiency. (a) CD8⁺CD56⁻and CD8⁺CD56⁺ CIK cells express NKG2D with similar frequency. CIK cells were sorted into CD8⁺ and CD8⁻ cells and analyzed for NKG2D and CD56 expression, respectively. (b) Purified CD28-ζ CAR CIK cells were cultivated for 14 days on anti-idiotypic monoclonal antibody-coated plates and sorted into CD4⁺, CD8⁺CDC56⁻, and CD8⁺CD56⁺ CIK cells by flow cytometry-based procedures. (c) Fractionated CAR⁺ CIK cells were cocultivated at an effector-to-tumor (E:T) cell ratio of 1:5 with CEA⁺ LS174T or CEA⁻ Colo 320 tumor cells (each 2.5 x 10⁴ cells/well) or at an E:T ratio of 1:1 with carboxyfluorescein diacetate succinamidyl ester-labeled Daudi cells (2.5 x 10⁴ cells/well) for 24 hours. Cytolysis of LS174T and Colo 320 cells was determined by an XTT-based colorimetric assay. To determine Daudi cell killing cells were stained with 7-aminoactinomycin D (0.5 µg/ml) and the number of life and dead cells was determined by flow cytometry. The assay was performed in triplicate. Numbers represent mean values ± SD. CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; CIK, cytokine-induced killer.