Fig. 2: IT injection of an L1-derived MHC-I-restricted peptide with poly I:C but not HPV-vax confers tumor control, induces epitope spreading, and reprograms the tumor microenvironment.

Experimental design: C57BL/6 mice were prime-boost immunized intramuscularly with the HPV vaccine or unimmunized prior to TC-1 tumor subcutaneous challenge one week after boost. When tumor reached a volume between 50 and 100 m³, mice were treated six times with saline, HPV-vax alone (1/25^th of the human dose) or L1_165-173 peptide (0.5 μg) admixed with polyI:C LMW (50 μg). Mice were monitored twice a week for (A, C) tumor growth and (B, D) survival. A, C Tumor growth is shown as the mean of tumor volume for each group and SE (n = 6–9 per group). Statistical analysis with Dunn’s test was performed for multiple comparison of tumor growth. B, D Survival comparisons were assessed by Mantel–Cox test (****P < 0.0001, ***P < 0.001, **P < 0.01 *P < 0.05 n.s.: not significant). E IFN-γ production was assessed after IT treatment by intracellular staining of circulating CD8⁺ T cells after in vitro restimulation with L1_165-173 peptide. Data are shown as individual values and mean of cytokine production of CD44⁺CD8⁺T cells with SE. F Circulating anti-tumor E7-specific CD8⁺ T cell responses were measured by dextramer staining after IT treatment. Data are shown as individual percentage and mean of H2-D^b/E7_49-57⁺CD44⁺CD8⁺ T cells. G anti-HPV16 L1 VLP antibody responses were measured by ELISA in plasma samples. Data are shown as individual and geometric mean of IgG EC50 titer and 95% confidence interval. H Cytokine and chemokine production was measured using a bead-based multiplex immunoassay in tumor lysates. Heatmap shows the average Z-score of the amount of each analyte per 50 μg of protein. I–L The cellular infiltrate was assessed by flow cytometry. Data are shown as individual values and mean percentage within CD45⁺ cells of (I) activated CD8⁺ T cells defined as CD8⁺CD44⁺CD62L^-PD1⁺, (J) myeloid cells defined as CD11b⁺, and percent within CD11b⁺ cells of (K) neutrophils defined as CD11b⁺Ly6G⁺Ly6C^int and (L) macrophages defined as CD11b⁺F480⁺Ly6G^-Ly6C^int. Data are shown as individual values and mean percentage of cells within indicated gated population and SE. Statistical analysis (*P < 0.05 or numeric values) was performed using Dunn’s test for multiple comparison between groups (n = 4–5 per group).