Abstract
Tertiary lymphoid structures (TLS) are associated with an improved response to Immune checkpoint therapy (ICT) in head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) infection constitutes a high-risk factor for HNSCC carcinogenesis. However, its role in TLS formation has yet to be elucidated. Herein, immunohistochemical (IHC) analysis from 59 HNSCC patients revealed a higher prevalence of mature TLS in HPV-positive (HPV+) HNSCC compared to HPV-negative (HPV-) cases. Furthermore, integrated analysis of single-cell RNA sequencing, spatial transcriptomics, and RNA-seq data indicated that TLS-positive tumors were characterized by an expanded population of KRT15high tumor cells in HNSCC. IHC and cytological experiments confirmed upregulation of KRT15 in HPV+HNSCC tumor cells, which also showed high expression of cancer stem cell marker genes. These KRT15high stem-like tumor cells specifically secreted CCL20, which was related to the infiltration of TLS-associated immune cells in HPV+HNSCC. Murine models confirmed that CCL20 treatment promoted TLS formation and enhanced the efficacy of anti-PD-1 therapy. Multiplex immunofluorescence showed that TLS provided specialized microenvironments that supported the proliferation of CD39+PD-1+CD8+T cells. Collectively, our findings proposed that CCL20 secreted by HPV-infected KRT15high tumor cells promoted TLS formation, thereby enhancing anti-PD-1 therapy responses in HPV+HNSCC. This study provides mechanistic insights into HPV-mediated TLS development and supports precision immunotherapeutic strategies for HNSCC.
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Data availability
The scRNA-seq data analyzed in this study were obtained from the Gene Expression Omnibus (GEO) at GSE139324 and GSE164690. RNA-seq data were obtained from The Cancer Genome Atlas (TCGA). The ST-seq data generated in this study are available upon request from the corresponding author.
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Acknowledgements
We would like to thank the Center for Computational Science and Engineering at Southern University of Science and Technology for technical support.
Funding
This study was supported by the following funding sources: the Shenzhen Science and Technology Program (JCYJ20240813101805007 and JCYJ20240813101802004); the National Natural Science Foundation of China (No. 82473326); and the Guangdong Province Basic and Applied Basic Research Fund Project (2023A1515220104).
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SZ and YJ were responsible for the writing and revision of the manuscript. SZ also contributed to the overall bioinformatics analysis. HL performed animal model generation and associated experimental procedures. XL was responsible for pathological area classification and paraffin tissue embedding. LT and TL contributed to immunohistochemistry experiments. RL and ZL were responsible for scRNA-seq data analysis. MW, JS, and ZH contributed to the collection of HNSCC tissues and clinical information. HM and LW were responsible for the overall study design, coordination of the research, and establishment of the research objectives. LW also contributed to the revision of the manuscript.
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The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Third People’s Hospital in Shenzhen [No. 2025-016]. All animals’ experimental procedures received ethical approval for the animal research from the Ethics Committee in the Third People’s Hospital in Shenzhen [2025-017-01].
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Zhang, S., Liu, H., Li, X. et al. CCL20 secreted by KRT15high tumor Cells promotes tertiary lymphoid structure formation and enhances anti-PD-1 therapy response in HPV+HNSCC. Cell Death Dis (2025). https://doi.org/10.1038/s41419-025-08359-5
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DOI: https://doi.org/10.1038/s41419-025-08359-5
