Induction of DKK1- and tumor-specific CD8⁺ CTLs in DKK1-DNA vaccinated mice. Mice (5 per group) were vaccinated thrice with PBS, vector control, DKK1 DNA, or DKK1 DNA plus CpG, followed by challenge with MOPC-21 cells. T cells were isolated from spleens 2 weeks after tumor challenge. (A) Cytotoxicity of T cells against MOPC-21 cells from mice receiving different treatments. (B) Percentages of murine DKK1 peptide-tetramer–positive CD8⁺ T cells from mice receiving different treatments. (A-B) Splenic T cells were restimulated in vitro with irradiated tumor cells for 5 days before assays. (C) Percentages of proliferative T cells analyzed by CSFE dilution assay. Splenic T cells from DKK1-CpG vaccinated mice were labeled with CFSE and restimulated by syngeneic DCs pulsed with or without DKK1 peptides (P11, P15, or P210) or a control influenza–A peptide (HA533) for 7 days. Concentrations of IFN-γ (D) or IL-4 (E) secreted by splenic T cells from DKK1-CpG–vaccinated mice after overnight restimulation by syngeneic DCs pulsed without or with DKK1 peptides P11, P15, or P210. Secreted cytokines in culture supernatants were measured by ELISA. Representative results of 1 of 2 independent experiments performed are shown. *P < .05; **P < .01, compared with controls.