Immunization with rlipo-E7m and CpG ODN elicits antigen-specific CTL responses and robust anti-tumor effects against large tumors. Mice were immunized s.c. with rlipo-E7m (10 μg/mouse) with or without CpG (10 μg/mouse) and were boosted once 14 days after the first immunization. The mice were euthanized 7 days after the second immunization. (a) The isolated spleen cells were re-stimulated with 10 μg/ml of HPV16E749-57 (RAH)-specific peptide (SP) or non-specific peptide (OVA257-264) (NP) for 48 hours, and IFN-γ production was determined using the ELISPOT assay. The data represent the number of IFN-γ spot-forming cells per 106 spleen cells for each duplicate (means + SD). (b) The spleen cells were stained with the RAH/MHC I tetramer and anti-CD8 antibody. (c) Naïve spleen cells were pulsed with SP or NP and then stained with 5 μM CFSEhi or 0.5 μM CFSElo, respectively. The CFSE-labeled cells were i.v. injected into immunized micefor 18 hours, and analyzed by flow cytometry. Specific lysis was determined using the following equation: % specific lysis = [1-(% CFSEhi /% CFSElo)] × 100. (d) Tumor-bearing mice were immunized with rlipo-E7m (10 μg/mouse) and CpG (10 μg/mouse) at 7, 14 or 25 days post-TC-1 cell (2 × 105) implantation, and PBS was used as a control. The tumor volume was calculated using the formula length × width × width/2. (e) The mice were injected with 2 × 105 TC-1 cells intravenously. After 14 days, the mice were injected with PBS, rlipo-E7m, CpG or rlipo-E7m/CpG subcutaneously. (f) Tumor-bearing mice were immunized s.c. with rlipo-E7m in the presence or absence of CpG 25 days following cisplatin (75 μg/mouse) treatment (day 21). Kaplan-Meier analysis was performed on the survival data. (*P < 0.05, **P < 0.01, ***P < 0.001).