Tumor vaccines using dendritic cells (DCs) have been shown to induce antitumor CTL responses. The choice of the tumor antigen preparation used for DC loading is still an unresolved issue. We compared DCs pulsed with cell lysates, whole apoptotic tumor cells or their supernatants of the HLA-A2(+) human pancreatic carcinoma cell line Panc-1 for their capacity to activate T cells. Monocyte-derived DCs from HLA-A2(+) donors were pulsed with tumor antigen, matured subsequently, and cocultured with autologeous peripheral blood mononuclear cells. After three weekly restimulations with DCs, T-cell activation was assessed by intracellular IFN-gamma staining and cytotoxicity assays. Compared with lysate, pulsing DCs with the supernatant of apoptotic tumor cells induced a higher frequency of activated CTLs and T-helper cells, as well as an enhanced MHC class I-restricted tumor cell lysis. No activation of natural killer (NK) or gammadelta T cells was detected. Pulsing DCs with whole apoptotic tumor cells induced an even more pronounced lytic effect. However, in this case, MHC class-I blocking was only partially effective, and unrelated cell lines were also killed. IFN-gamma staining revealed activation of CTLs and T-helper cells, as well as NK and gammadelta T cells. Trans-well cultures of NK cells, apoptotic tumor cells, and DCs showed that NK cell activation was dependent on direct cell-to-cell contact with tumor cells and the presence of interleukin-12 produced by DCs. These results indicate that the choice of antigen preparation is a critical determinant in the induction of antitumor immunity. Tumor vaccines consisting of DCs and apoptotic tumor cells may be able to activate CTLs, as well as effector cells of the innate immune system.