BackgroundOropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV-positive) is associated with better clinical outcomes than HPV-negative OPSCC. However, the clinical benefits of immunotherapy in patients with HPV-positive OPSCC remain unclear.MethodsTo identify the cellular and molecular factors that limited the benefits associated with HPV in OPSCC immunotherapy, we performed single-cell RNA (n=20) and T-cell receptor sequencing (n=10) analyses of tonsil or base of tongue tumor biopsies prior to immunotherapy. Primary findings from our single-cell analysis were confirmed through immunofluorescence experiments, and secondary validation analysis were performed via publicly available transcriptomics data sets.ResultsWe found significantly higher transcriptional diversity of malignant cells among non-responders to immunotherapy, regardless of HPV infection status. We also observed a significantly larger proportion of CD4⁺follicular helper T cells (Tfh) in HPV-positive tumors, potentially due to enhanced Tfh differentiation. Most importantly, CD8⁺resident memory T cells (Trm) with elevatedKLRB1(encoding CD161) expression showed an association with dampened antitumor activity in patients with HPV-positive OPSCC, which may explain their heterogeneous clinical outcomes. Notably, all HPV-positive patients, whose Trm presented elevatedKLRB1levels, showed low expression ofCLEC2D(encoding the CD161 ligand) in B cells, which may reduce tertiary lymphoid structure activity. Immunofluorescence of HPV-positive tumors treated with immune checkpoint blockade showed an inverse correlation between the density of CD161⁺Trm and changes in tumor size.ConclusionsWe found that CD161⁺Trm counteracts clinical benefits associated with HPV in OPSCC immunotherapy. This suggests that targeted inhibition of CD161 in Trm could enhance the efficacy of immunotherapy in HPV-positive oropharyngeal cancers.Trial registration numberNCT03737968.