Distinct NK Cell Signatures Define Prognosis in HPV-Positive Versus HPV-Negative Head and Neck Cancer

Background/Objectives: HPV status is a key prognostic determinant in head and neck squamous cell carcinoma (HNSCC), yet the immunological mechanisms underlying the survival advantage of HPV-positive (HPV+) over HPV-negative (HPV−) disease remain poorly defined. This study aimed to characterize the tumor-infiltrating natural killer (NK) cell landscape in HPV-stratified HNSCC and identify novel therapeutic targets. Methods: We performed an NK-cell-centric re-analysis of published scRNA-seq data from 28 HNSCC patients (10 HPV+, 18 HPV−; GEO: GSE139324, GSE164690), encompassing NK subset identification, pseudotime trajectory inference, and cell–cell interaction analysis. Key findings were validated by immunohistochemistry (IHC) in an independent cohort of 10 FFPE tissue sections, and prognostic associations were assessed using TCGA-HNSC data. Results: Four transcriptionally distinct NK cell subsets were identified: adaptive, cell-killing, CD56bright, and virus-responsive. A cytotoxic CX3CR1+KLRB1dim NK subset was specifically enriched in HPV+ tumors and independently associated with favorable survival. Conversely, HPV− tumors upregulated CLEC2C and CLEC2D ligands on tumor cell surfaces, engaging the inhibitory receptor KLRB1 on NK cells; this CLEC2–KLRB1 axis correlated with suppressed NK activity and poorer prognosis, and was confirmed at the protein level by IHC. Conclusions: NK cell function in HNSCC is dichotomously regulated by HPV status. The CX3CR1+KLRB1dim subset represents a candidate prognostic biomarker in HPV+ disease, and the CLEC2–KLRB1 axis is a targetable immune evasion mechanism in HPV− HNSCC. These insights support the development of HPV-stratified immunotherapies; however, clinical translation requires validation in large, prospectively designed, subsite-matched cohorts to disentangle HPV-specific effects from anatomical site-dependent immune contextures.