Figure 5 Generation of tumor-specific CTLs ex vivo. Splenic CD3+ T cells were isolated from B6 mice with MACS. T cells were primed with MAGE-1-modified DCs as described in Materials and Methods. DC-Ad-LacZ and untreated DCs were used as controls. Primed T cells (effector cells) were titrated by serial dilution, then mixed with MFC or B16F10 target cells, and their lytic activity was assayed. Results are given as means ± SD from three independent experiments. A therapeutic effect mediated by DC-Ad-MAGE-1 in vivo Therapeutic

potential of DC-Ad-MAGE-1 was further explored with an JNK-IN-8 purchase established tumor model. 5 × 105 MFC or B16F10 tumor cells were implanted s.c in B6 mice, and tumor-bearing mice were injected with different modified or unmodified DCs on days 5 and 12. Fig. 6A shows that tumor growth was significantly inhibited in mice vaccinated with DC-Ad-MAGE-1. For example, tumor volumes on day 27 were as follows: untreated DC control 14.98 ± 1.81 cm3, DC-Ad-LacZ control 15.44 ± 1.99 cm3, DC-MFC Ag control 7.79 ± 1.55 cm3, DC-Ad-MAGE-1 3.46 ± 1.12 cm3, DC-Ad-MAGE-1 vs. the other control groups (P < 0.05). Half of the tumor-bearing mice immunized with DC-Ad-MAGE-1 survived in a period of over 60 days. By contrast, only AC220 order 10% of the tumor-bearing

mice immunized with DC-MFC Ag survived; all mice from the other control groups succumbed to growing tumors within 25 days, thus providing no therapeutic effect (Fig. 6B). The differences between the DC-Ad-MAGE-1 group and all control groups were statistically significant (P < 0.05).

Figure 6 Inhibition of tumor growth in tumor-bearing mice by immunization with MAGE-1-modified, CCL3 and CCL20-recruited DC vaccine. (A), Each of 10 mice in a group was challenged s.c. with 1 × 105 viable MFC tumor cells. Mice were subsequently injected s.c. with DC-Ad-MAGE-1 5 filipin days later. As controls, tumor-bearing mice were injected with DC-Ad-LacZ, DC-MFC Ag, or untreated DCs. Survival was observed over time after immunization of mice harboring preexisting tumors. Survival rate was compared with a long-rank test of Kaplan-Meier curves. (B), Tumor growth was measured every 2~3 days after the second immunization. Data are given as means ± SD of 10 mice per group from three independent experiments. To confirm that tumor-specific CTLs had indeed been generated in the immunized mice, the following evaluation was performed. Spleen T cells from mice immunized s.c with DC-Ad-MAGE-1, and thus rendered tumor-free after MFC tumor challenge, were restimulated ex vivo with irradiated tumor cells and tested for cytolytic activity. As shown in Fig. 7A, these effector cells efficiently lysed MFC, but not B16F10 tumor cells. Control spleen T cells from naive mice stimulated with irradiated MFC tumor cells failed to demonstrate CTL activity. Furthermore, splenic CD3+ T cells derived from those mice that survived MFC challenge produced high levels of IFN-γ, but not when stimulated with B16F10 cells (Fig. 7B).