Figure 3.

Advantage of intra-cheek vaccinations for inducing local and loco-regional antigen-specific CD8⁺ T-cell responses in tumor-bearing mice. (A) IFNγ ELISpot assay performed at day 10 using cell suspensions from draining lymph nodes (dLNs), spleen and non-draining lymph nodes (ndLNs), C57BL/6 mice being immunized at days 0–2–4 with pVLP-E7 using intra-dermal (ID), intra-cheek (IC) or intranasal (IN) routes of vaccination. Data were obtained from three separate experiments using 4–6 mice per group. (B) IFNγ ELISpot assay performed at day 10 using cell suspensions from dLNs, C57BL/6 mice (4–5 mice) being immunized at days 0–2–4 with pVLP-E7 or E7 polypeptide (+ CpG-ODN) using the ID or IC route. (C) and (D) C57BL/6 mice were injected in the cheek with TC-1-Luc cells, and then ID or IC immunized with pVLP-E7 at days 7–9–11 following tumor challenge. (C) Detection of E749-57-specific CD8⁺ T cells: cell suspensions from tumors were pooled from either 5 mice (Experiment 1) or 6 mice (Experiment 2) and stained with E7-tetramers at day 18. Data obtained from Experiment 1 (5 mice pooled) and Experiment 2 (6 mice pooled). (D) IFNγ ELISpot assay performed using cell suspensions from tumor draining lymph nodes (TdLNs) cells at day 18. Presented data are pooled from two separate experiments (5 and 6 mice per group). (E) Kaplan–Meier curves showing tumor-free survival rates. For ELISpot assays, cells were loaded and stimulated with E749-57 peptide. Background (always ≤ 100 spots per 10⁶ cells) obtained with not pulsed cells was subtracted due to some variability between the different tissues tested. NS, non-statistical difference = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.