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. 2019 Jul 16:10:1644.
doi: 10.3389/fimmu.2019.01644. eCollection 2019.

TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

Paramita Baruah  1   2 , Jessica Bullenkamp  2 , Philip O G Wilson  3 , Michael Lee  1 , Juan Carlos Kaski  2 , Ingrid E Dumitriu  2
Affiliations

TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

Paramita Baruah et al. Front Immunol. .

Abstract

Background: The co-inhibitory receptor PD-1 is expressed in many tumors including head and neck squamous cell carcinoma (HNSCC) and is an important immunotherapy target. However, the role of PD-1 ligands, PD-L1, and particularly PD-L2, in the tumor-stromal cell interactions that cause a tumor-permissive environment in HNSCC is not completely understood and is the focus of our study. Methods: Expression of PD-L1 and PD-L2 was analyzed by immunohistochemistry in situ in HNSCC tumor tissue. Co-cultures were established between stromal cells (fibroblasts and macrophages) and human papilloma virus (HPV)-positive and HPV-negative HNSCC cell lines (HNSCCs) and PD-1 ligands expression was analyzed using flow cytometry. Results: PD-L1 and PD-L2 were expressed both in tumor cells and stroma in HNSCC tissue in situ. In vitro, basal expression of PD-L1 and PD-L2 was low in HNSCCs and high on fibroblasts and macrophages. Interestingly, HPV-positive but not HPV-negative HNSCCs increased the expression of both PD-1 ligands on fibroblasts upon co-culture. This effect was not observed with macrophages. Conversely, both fibroblasts and macrophages increased PD-1 ligands on HPV-positive HNSCCs, whilst this was not observed in HPV-negative HNSCCs. Crucially, we demonstrate that up-regulation of PD-L1 and PD-L2 on fibroblasts by HPV-positive HNSCCs is mediated via TLR9. Conclusions: This work demonstrates in an in vitro model that HPV-positive HNSCCs regulate PD-L1/2 expression on fibroblasts via TLR9. This may open novel avenues to modulate immune checkpoint regulator PD-1 and its ligands by targeting TLR9.

Keywords: HNSCC; HPV; PD-1; PD-L1; PD-L2; TLR9; fibroblasts.

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Figures

Figure 1
Figure 1
PD-L1, PD-L2, PD-1, CD8, and CD4 expression in p16-positive and p16-negative HNSCC. PD-L1, PD-L2, PD-1, CD8, and CD4 expression was assessed in tumor biopsy tissue from five p16-positive and four p16-negative HNSCC patients using immuno-histochemistry (details in Methods). Representative staining (scale bars, 100 μm) and cumulative data of marker expression (grading scale PD-L1/PD-L2: 1, low; 2, moderate; 3 high expression; grading scale PD-1, CD8, CD4: 1, <50 cells/field; 2, 50–150 cells/field; 3, >150 cells/field).
Figure 2
Figure 2
CD68, CD163, and iNOS expression in p16-positive and p16-negative HNSCC. Macrophage marker expression was assessed in tumor biopsy tissue from five p16-positive and four p16-negative HNSCC patients using immuno-histochemistry. Representative staining (scale bars, 100 μm) and cumulative data of marker expression (grading scale: 1, <50 cells/field; 2, 50–100 cells/field; 3, >100 cells/field).
Figure 3
Figure 3
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.
Figure 4
Figure 4
PD-L1 and PD-L2 expression in co-cultures of macrophages and HPV-positive or HPV-negative HNSCCs. Macrophages were cultured alone (w/o) or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCC cell lines (HNSCCs). Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone (A; black histograms). Macrophages were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 (B; red histograms) or HPV-negative SCC099 (C; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on macrophages cultured alone (w/o) or co-cultured directly with HNSCCs (D). HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with macrophages. 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 macrophages (E; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on HPV-positive SCC154 cultured alone or co-cultured directly with macrophages (F). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-negative SCC099 cultured alone (black histograms) or co-cultured directly with macrophages (G; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on HPV-negative SCC099 cultured alone or co-cultured directly with macrophages (H). *p < 0.05; **p < 0.01; ns, not significant (D: one-way ANOVA with Bonferroni correction for multiple comparisons; F,H: unpaired two-tailed Student's t-test) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.
Figure 5
Figure 5
Conditioned medium from HPV-positive HNSCCs up-regulates PD-L1 and PD-L2 on fibroblasts. Fibroblasts were cultured alone or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured with conditioned medium from HPV-positive SCC154 (A; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 13) on fibroblasts cultured alone (w/o), co-cultured directly with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM) (B). HPV-positive (SCC154) HNSCCs were cultured alone or co-cultured in direct contact with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured with conditioned medium from fibroblasts (C; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on HPV-positive SCC154 cultured alone (w/o), co-cultured directly with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM) (D). Macrophages were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on macrophages for the indicated conditions (E). HPV-positive SCC154 HNSCCs were cultured alone (w/o) or co-cultured in direct contact with macrophages (MFs direct) or with conditioned medium from macrophages (MFs CM). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 2) on HPV-positive SCC154 HNSCCs for the indicated conditions (F). **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.
Figure 6
Figure 6
Blockade of IFN-γ, TNF-α, or CD81 does not affect PD-L1 and PD-L2 up-regulation by HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) as indicated. Graphs (A) show IFN-γ and TNF-α levels in culture supernatants (mean ± SEM; n = 4). The dashed red line indicates the lowest value (15.6 pg/ml) of the dynamic range for the ELISA assays used. Neutralizing antibodies anti-IFN-γ (B,C), anti-TNF-α (D,E), or anti-CD81 (F,G) were added to the cultures as indicated. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 or with conditioned medium from HPV-positive SCC154 alone (red histograms) or in the presence of blocking antibodies (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on fibroblasts for the indicated treatments. 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.
Figure 7
Figure 7
The TLR9 antagonists ODN TTAGGG and chloroquine inhibit PD-1 ligands up-regulation on fibroblasts co-cultured with HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) in the presence or absence of the TLR9 antagonists ODN TTAGGG (ODN) or chloroquine (CHQ). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 (red histograms) or co-cultured directly with HPV-positive SCC154 in the presence of ODN (A) or CHQ (H) (green histograms). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), cultured with conditioned medium from HPV-positive SCC154 (red histograms) or with conditioned medium from HPV-positive SCC154 in the presence of ODN (B) or CHQ (I) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 6) on fibroblasts for the indicated treatments (C,J). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or in the presence of ODN (D) or CHQ (K) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on fibroblasts for the indicated treatments (E,L). HPV-positive SCC154 were cultured alone or co-cultured in direct contact (direct) with fibroblasts in the presence or absence of TLR9 inhibitor ODN TTAGGG (ODN) or chloroquine (CHQ). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms), co-cultured directly with fibroblasts alone (red histograms), or co-cultured directly with fibroblasts in the presence of ODN (F) or CHQ (M) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 6) on HPV-positive SCC154 cultured alone or for the indicated co-culture conditions (G,N). *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.
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