Fig. 6.

Vaccination with HPV E6/E7 peptides in combination with α4-1BB leads to regression of intravaginally implanted tumors. Mice were challenged intravaginally with 2 × 10⁴ TC-1 tumor cells expressing firefly luciferase and then immunized intranasally twice (days 5 and 11) with HPV peptides and αGalCer either alone or in combination with αCTLA-4 or α4-1BB therapy (days 5, 8, and 11). Control animals received either PBS or monotherapy alone. Tumor growth and survival of the mice was monitored over time. (A) Representative images of luciferase⁺ tumor progression over time in mice assigned to different treatment groups. (B) Survival curve (Left) and average tumor growth (Right) as measured by average radiance of luciferase activity of mice treated with peptide vaccine alone or in combination with immunotherapy. Tumor measurements ceased upon the first tumor-related mortality in each group. Survival and tumor growth are from a representative experiment of two independent experiments with three to five mice per group. (C) In a separate experiment, the percent of E7 antigen-specific CD8 T cells infiltrating into vaginal tumors and the associated genital tract was analyzed by isolating TILs and staining with fluorescently labeled E7^49-57/K^b tetramer and antibodies to CD8. (D) The percent of ThEO/TcEO cells capable of infiltrating intravaginally implanted TC-1 tumors was measured by gating on Eomes⁺KLRG1⁺ CD4 Teff or CD8 T cells, respectively. (E) CD8 T cells from the draining lymph node were restimulated with E6/E7 peptide-pulsed DCs and their cytokine production measured by cytometric bead array (BD Biosciences). Experiments were performed in duplicate on at least three mice per group except cytokine production, which was performed with quintuplicate wells derived from five pooled LNs per group. Bars represent mean ± SD. Statistical significance was calculated using a one-way ANOVA. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.