Cytolytic function and IL-12p40 release by CIK cells generated with TG. Panel A: The gating strategy for the analysis of CIK-mediated cytotoxicity is shown in a representative experiment. After co-culture with CIK cells, K562 targets were identified and gated based on CAM expression. The percentage of lysed K562 cells was then calculated on a CAM/7-AAD contour plot. Panel B: After 21 days of culture in the presence of either lowαCD3 mAb or different concentrations of TG, cells were harvested and co-cultured with NK-sensitive tumor cell targets (K562 cells) for 4 hours at the indicated effector-to-target (E:T) ratio. K562 cells were pre-labeled with CAM, a fluorescent probe. The percent specific lysis was calculated as detailed in Materials and Methods. * denotes a p value < 0.01 when compared with cultures containing intTG, lowTG and lowαCD3 mAb. Panel C: IL-12p40 release was measured at the end of culture (21 days) in the presence of escalating concentrations of either αCD3 mAb or TG. Bars depict median values recorded in 3 independent ELISA run in duplicate with supernatants from 3 different PBMC preparations. * denotes a p value < 0.01 when compared with cultures containing int/lowTG, int/lowαCD3 mAb and hiαCD3.