Kadivar, M.; Snejbjerg, D. B.; Viuff, M. C.; Nos, G.; Larsen, S. K.; Bonde, J.; Kirschner, B.; Kjaer, S. K.; Jochumsen, K. M.; Hadrup, S. R.

Human papillomavirus (HPV) remains the leading cause of cervical cancer, yet the mechanisms underlying its immune evasion remain poorly defined. We performed an integrated analysis of HPV-specific CD8 T cell responses and the immune microenvironment in cervical cancer, high-grade intraepithelial neoplasia (CIN3), and healthy controls using flow cytometry, transcriptomics, and DNA-barcoded peptide-MHC multimer screening across cervical biopsies, peripheral blood, and liquid-based cytology (LBC). Cervical cancer tissues exhibited a profoundly immunosuppressive milieu, with enrichment of exhausted CD8 and CD4 T cells, increased regulatory T cells, and PD-L1 expressing myeloid subsets. Conventional dendritic cells and macrophages showed reduced frequencies, while plasmacytoid dendritic cells and intermediate monocytes were elevated. Interestingly, LBC samples reliably reflected T cell exhaustion signatures observed in biopsies, supporting their use as a minimally invasive tool for T cell immune monitoring; however, they were not suitable for detailed myeloid profiling. Transcriptomic analysis revealed distinct gene expression profiles in tumor tissues with elevated signatures of immune checkpoints and regulatory immune cell infiltration. Importantly, HPV-specific CD8 T cell responses were significantly reduced in cancer patients compared to CIN3 and controls, with decreased breadth and frequency of HPV peptides recognition. HPV peptides screening identified HPV-specific CD8 T cell responses toward 109 unique peptide-MHC complexes, including 37 novel HPV-derived peptide from E2, E6, and E7 proteins. These findings reveal impaired HPV immune recognition and a suppressive tumor microenvironment in cervical cancer, underscoring the need to enhance HPV-specific T cell responses and target immune suppression in therapeutic strategies.