Figure 5

Antitumor immune response in vaccinated animals. (A) Proliferation of splenocytes from animals vaccinated with DCs electroporated with TC-1 RNA (RNA); pulsed with UV-irradiated TC-1 cells (UVB); mock-electroporated DCs (mock), or naïve animals. Splenocytes were stimulated in vitro with gamma-irradiated TC-1 cells for 5 days. Data are expressed as the mean ± SD (n = 5). RNA vs. UVB: p < 0.01; RNA vs. mock: p < 0.001; RNA vs. naive: p < 0.001; UVB vs. mock: p < 0.001; UVB vs. naive: p < 0.001 (ANOVA, Tukey-Kramer multicomparison post-test). (B) Proliferation of splenocytes from animals vaccinated with DCs electroporated with TC-1 cell RNA. Splenocytes were stimulated in vitro for 5 days with gamma-irradiated TC-1 cells, ID8-E6/E7 cells, ID8 cells carrying empty vector or L-929 cells. Data are expressed as the mean ± SD (n = 5). TC-1 vs. ID8-E6/7: non significant (NS); TC-1 vs. ID8: p < 0.001; TC-1 vs. L-929: p < 0.001; ID8-E6/7 vs. ID8: p < 0.001; ID8-E6/7 vs. L-929: p < 0.001. The experiment was repeated two times with similar results. (C) Quantification of IFN-γ producing tumor-reactive splenocytes by ELISPOT from the same experimental groups as in A. Data are expressed as the mean ± SD (n = 5). RNA vs. UVB: p < 0.05; RNA vs. mock: p < 0.005; RNA vs. naive: p < 0.005; UVB vs. mock: p < 0.005; UVB vs. naive: p < 0.005. (D) Quantification of IFN-γ producing tumor-reactive splenocytes by ELISPOT from the same experimental groups as in B. Data are expressed as the mean ± SD (n = 5). TC-1 vs. ID8-E6/7: non significant (NS); TC-1 vs. ID8: p < 0.001; TC-1 vs. L-929: p < 0.001; ID8-E6/7 vs. ID8: p < 0.001; ID8-E6/7 vs. L-929: p < 0.001. (E) IFN-γ producing cells from splenocytes of RNA vaccinated animals upon CD8 immunodepletion. p < 0.01. (F) Cytotoxic assay using TC-1 cells as targets. Tumor cells were incubated at 3:1 effector:target ratio with gradient-purified pooled splenic lymphocytes from animals vaccinated with DCs electroporated with TC-1 cell RNA (RNA) or pulsed with UV-irradiated TC-1 cells (UVB). Some groups were immunodepleted of CD8⁺ cells. Splenocytes were stimulated in vitro with gamma-irradiated TC-1 cells for 5 days. RNA vs. UVB: p < 0.05; Student's t-test. The experiment was repeated two times with similar results. (G) Frequency of anti-E7 CD8⁺ lymphocytes following DC vaccination. E7 tetramer-positive, CD3-gated CD8⁺ precursors were quantified in splenocytes from animals vaccinated with DCs pulsed with UVB-irradiated TC-1 cells (UVB) or DCs electroporated with TC-1 cell RNA (RNA), p < 0.05.