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Published on: May 2, 2025
TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma
Paramita Baruah1,2, Jessica Bullenkamp2, Philip O G Wilson3
1Department of Ears, Nose and Throat (ENT), St. George's University Hospitals NHS Foundation Trust, London, United Kingdom.
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.
Insights
Human papillomavirus-positive head and neck squamous cell carcinoma (HNSCC) upregulates PD-1 ligands on fibroblasts via TLR9. This finding offers new strategies for targeting immune checkpoints in HNSCC treatment.
Area of Science:
- Immunology
- Oncology
- Cell Biology
Background:
- Programmed cell death protein 1 (PD-1) is a key immunotherapy target expressed in head and neck squamous cell carcinoma (HNSCC).
- The roles of PD-1 ligands, PD-L1 and PD-L2, in tumor-stromal interactions within HNSCC remain incompletely understood.
- This study investigates the intricate relationship between HNSCC, stromal cells, and PD-1 ligand expression.
Purpose of the Study:
- To elucidate the expression patterns of PD-L1 and PD-L2 in HNSCC tumor tissues and their regulation by tumor-stromal cell interactions.
- To determine the influence of human papillomavirus (HPV) status on PD-1 ligand expression in HNSCC.
- To identify the molecular mechanisms, specifically Toll-like receptor 9 (TLR9), mediating these interactions.
Main Methods:
- Immunohistochemistry was employed to analyze PD-L1 and PD-L2 expression in HNSCC tumor samples.
- Co-culture models were established using HNSCC cell lines (HPV-positive and HPV-negative) and stromal cells (fibroblasts, macrophages).
- Flow cytometry was utilized to quantify PD-1 ligand expression, and TLR9 involvement was investigated.
Main Results:
- PD-L1 and PD-L2 were detected in both tumor cells and stromal components of HNSCC tissues.
- In vitro, HPV-positive HNSCCs significantly increased PD-L1 and PD-L2 expression on fibroblasts, but not macrophages.
- HPV-positive HNSCCs also enhanced PD-1 ligand expression on both fibroblasts and macrophages, an effect not seen in HPV-negative HNSCCs.
- Crucially, HPV-positive HNSCC-induced upregulation of PD-L1/2 on fibroblasts was mediated by TLR9.
Conclusions:
- HPV-positive HNSCCs actively modulate PD-L1 and PD-L2 expression on fibroblasts through the TLR9 signaling pathway.
- Targeting TLR9 presents a potential novel therapeutic strategy for modulating PD-1 immune checkpoint regulation in HNSCC.
- These findings contribute to understanding tumor-immune microenvironment dynamics and offer new avenues for immunotherapy in HNSCC.
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