Fig 1: HPV-positive HNSCCs increase PD-L1 and PD-L2 on fibroblasts. PD-L1 and PD-L2 expression on primary BJ human fibroblasts, HPV-positive (SCC154), and HPV-negative (SCC099) HNSCC cell lines (HNSCCs) was detected by flow cytometry. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts (black histograms), HPV-positive (red histograms), or HPV-negative (blue histograms) HNSCCs (A). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 5) in fibroblasts, HPV-positive, and HPV-negative HNSCCs (B). Fibroblasts were cultured alone or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs. Fibroblasts were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 (C; red histograms) or HPV-negative SCC099 (D; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 5) on fibroblasts cultured alone (w/o) or co-cultured directly with HNSCC cells (E). HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with fibroblasts. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with fibroblasts (F; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on HPV-positive SCC154 cultured alone or co-cultured directly with fibroblasts (G). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-negative SCC099 cultured alone (black histograms) or co-cultured directly with fibroblasts (H; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on HPV-negative SCC099 cultured alone or co-cultured directly with fibroblasts (I). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.