Characterization of persistent HPV-specific activated T cells in head and neck squamous cell carcinoma

Background Human papillomavirus–positive oropharyngeal squamous cell carcinoma (HPV + OPSCC) generally has favorable outcomes yet remains prone to late recurrence. Durable control likely depends on persistent tumor-specific CD8+ T cells, but their persistence and function in metastasis are poorly understood.Methods We integrated bulk RNA sequencing (n = 56) with single-cell RNA and paired T-cell receptor (TCR) sequencing of tumor-infiltrating CD8+ T cells (n = 4), including a longitudinal primary–lung metastasis pair obtained three years after curative therapy. HPV genotyping, HLA typing, epitope prediction, and NFAT-reporter Jurkat-luciferase assays were used to identify and functionally validate HPV16 E6/E7-specific TCRs. Repertoire tracking and single-cell/bulk transcriptomics were evaluated.Results HPV + tumors showed higher inferred CD8+ T cell infiltration and more favorable prognosis than HPV– tumors. Single-cell analysis revealed oligoclonally expanded exhausted clusters enriched in HPV tumor-specific CD8+ T cells. We isolated and functionally validated nine patient-derived HPV16 E6/E7-specific TCRs. High-avidity receptors recognizing an alternatively spliced region in E6 (aa 49–110; E6*) persisted in metastatic lesions, whereas lower-avidity clones, including an E7_11–19-specific TCR currently under clinical investigation, were lost. Compared with the primary tumor, metastatic lesions showed reduced stem-like/TCF7-associated CD8+ T-cell populations and enrichment of HPV-specific CD8+ T cells expressing KLRB1 and ZNF683, consistent with a tissue-adapted TRM-like transcriptional state with inhibitory signaling features.Conclusions High-avidity, KLRB1 + HPV-specific CD8+ T cells represent a persistent effector subset with prognostic and therapeutic relevance to HPV + OPSCC. Their persistence in metastatic lesions and association with reduced recurrence risk support further investigation of KLRB1-associated HPV-reactive T-cell states as biomarkers and immunotherapeutic targets.