Figure 4.

ChAdOx1/MVA P1A vaccination promotes CD8⁺ T-cell infiltration into the TME. (A) DBA/2 mice were implanted with 1x10⁶ 15V4T3 cells via s.c. injection and treated with PBS control, ChAdOx-Ii-P1A (10⁷ IU) /MVA-P1A (10⁶ PFU), anti-PD-1 or combination treatment. Mice were sacrificed following treatment, and tumors surgically excised for analysis of immune cell infiltrate by flow cytometry. (B) Representative flow cytometry gating plots of CD8⁺ TILs. (C) Percentage and (D) total numbers of CD8⁺ TILs in 15V4T3 tumors, as quantified by flow cytometry. (E) Representative flow cytometry gating plots of P1A_35-43 tet⁺ CD8⁺ TILs. (F) Percentage and (G) total numbers of P1A_35-43 tet⁺ CD8⁺ TILs in 15V4T3 tumors, as quantified by flow cytometry. (H) RNA-sequencing analysis of tumor mRNA from 3 mice per group. Heatmaps showing log-CPM expression values of T-cell inflamed and IFN-γ gene expression signatures across all samples. Gene expression level has been scaled by Z-score, indicated by the heatmap color key. (I) Expression of PD-1, LAG-3 and TIM-3 receptors on tumor CD8⁺ T cells from vaccine ± anti-PD-1 groups was evaluated by flow cytometry. Pie charts show the mean relative proportion of P1A-specific CD8⁺ T cells expressing combinations of the receptors. (J) PD-1 mean fluorescence intensity (MFI) of tumor PD-1⁺ CD8⁺ TILs. (K) PD-1 MFI of PD-1⁺ P1A_35-43 tet⁺ CD8⁺ TILs (L) LAG-3 MFI of LAG-3⁺ P1A_35-43 tet⁺ CD8⁺ TILs. (M) TIM-3 MFI of TIM-3⁺ P1A_35-43 tet⁺ CD8⁺ TILs. Tumor immune cell infiltrate data are shown as mean ± SEM. Each symbol represents an individual mouse, with 7-10 mice per group, pooled from 2-3 independent experiments. Statistically significant differences between multiple groups were determined by a Kruskal-Wallis test with Dunn’s multiple comparisons test, between only two groups with a Mann-Whitney U test. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, ****, p ≤ 0.0001.