Fig. 5: Tumor stress response and CD8+ T cell infiltration.
Experimental design: C57BL/6 mice were prime-boost immunized intramuscularly with the VZV and HPV vaccines in opposite quadriceps prior to TC-1 tumor subcutaneous challenge one week after boost. When tumor reached a volume between 50 and 100 m3, mice were treated four times with saline, AS01B, VZV-vax alone (1/40th of the human dose) alone or admixed with L1165-173 peptide (0.5 g). A Tumor RNA was extracted 24 h after the fourth injection and analyzed with the Nanostring Immune Exhaustion panel (n = 5). Heatmap shows the global significance score of each pathway measured in each group relative to saline-treated (A). B Volcano plot representation of the differential gene expression of each group compared with saline. Adjusted (adj.) P values were generated using the Benjamini–Yekutieli procedure and are shown as gray plain (P < 0.01), dashed gray (P < 0.05), dotted (P < 0.1) and dashdot (P < 0.5) lines. The plain red line indicates non-adjusted P value (P < 0.01). C The heatmap shows the Z-score of selected differentially expressed genes (DEG) selected from the saline-treated versus VZV-vax + L1165-173 peptide-treated groups. D The tumor cell stress response was measured by flow cytometry on CD45- tumor cells. Data are shown as individual values and mean fluorescence intensity for each marker (MHC-I, Fas, calreticulin, PD-L1 and RAE-1γ) on live tumor cells with SE (n = 5 per group). E CD39 and PD1 expression by tumor infiltrating CD8+ T cells was measured by flow cytometry. Representative flow cytometry plot overlay showing CD39 and PD1 expression in saline (blue) and VZV-vax + L1165-173 peptide-treated (red) (n = 5). D, E Statistical analysis (*P < 0.05, n.s.: not significant) was performed using Dunn’s test for multiple comparison between groups (n = 5 per group). F Representative immunofluorescence image of tumor tissue sections from tumors treated with saline or VZV-vax + L1165-173 peptide. Sections were stained with CD8α (red) and CXCL9 (green) antibodies and nuclei were stained with DAPI (blue).
