Fig 1.
Tumor growth in B16F10-tumor bearing mice.
(A) Tumor growth of B16F10-OVA tumors. Error bars indicate SEM. * statistically significant difference compared to the naïve group. (B) Tumor volume at day 15 post tumor induction. Statistically significant difference compared to naïve group: *(p<0.05), **(p<0.01), ***(p<0.001). (C) Kaplan-Meier survival curves of naïve and treated mice. * statistically significant difference compared to the naïve group. N = 6 mice per experimental group.
Fig 2.
Antigen-specific immune response in B16F10-OVA tumor-bearing mice.
(A) Time-line of the experiment with exact time points for the administration of DNA vaccine or immune checkpoint inhibitors. (B) In vivo killing assay; (1) Percentage of the killing of target cells. *statistically significant difference compared to the naïve group, ** statistically significant difference compared to all other groups. (2) Flow cytometry analysis presenting 2 separated peaks for low CFSE concentration and high CFSE concentration. In naïve mice, which were unable to eliminate target cells labeled with CFSE (FITC), both peaks are present. In mice immunized with pOVA vaccine, which completely eliminated target cells previously labeled with CFSE, the peak for high CFSE is absent. (C) IgG titers in serum samples; (1) Total anti-OVA IgG titer. (2) Anti-OVA IgG1 titer in immunized mice. (3) Anti-OVA IgG2A titer in immunized mice. *statistically significant difference between both marked experimental groups. (D) A number of tetramer-positive CD8+ T cells in tumors normalized to tumor volume. *statistically significant difference compared to the naïve group. Error bars indicate SEM. N = 7 mice per experimental group.
Fig 3.
Effect of therapy combining therapeutic DNA vaccine coding for gp100 and dual CTLA-4/PD-1 blockade on the immune response.
(A) Time-line of the experiment with exact time points for the administration of DNA vaccine or immune checkpoint inhibitors. (B) Infiltration of immune cells in spleen; (1) Percentage of CD4+ T cells in the spleen. CD4+ T cells were additionally divided to CD4+/FOXP3- T helper cells (white column) and CD4+/FOXP3+ T regulatory cells (gray column with percentage marked with numbers inside the column). (2) Percentage of CD8+ T cells in the spleen. (C) Infiltration of immune cells in tumors. (1) A total number of CD4+ positive cells in tumors, normalized to tumor volume. (2) A total number of CD8+ positive cells in tumors, normalized to tumor volume. * statistically significant difference compared to the naïve group. ** statistically significant difference between combined group and vaccinated group. N = 8 mice per experimental group.
Fig 4.
Long-term antitumor effect of combined treatment in B16F10 tumor-bearing mice.
(A) Time-line of the experiment with exact time points for the administration of DNA vaccine or immune checkpoint inhibitors. (B) Tumor growth of B16F10 tumors. Error bars indicate SEM. * statistically significant difference compared to the naïve group. (C) Kaplan-Meier survival curves of mice after tumor challenge. * statistically significant difference compared to the naïve group. n.s. = no statistically significant difference. N = 8 mice per experimental group.