<p>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 KRT15^high 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 KRT15^high 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 KRT15^high 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.</p>