HPV-driven DNA methylation plasticity reshapes immune regulation in head and neck cancer

The incidence of HPV-related HNC has exponentially increased over recent decades in head and neck cancer (HNC). Although HPV infection is known to reprogram the host epigenetic landscape, the cellular and functional consequences of HPV-mediated DNA methylation changes remain poorly defined. Here we demonstrated that HPV-driven epigenetic reprogramming reshapes immune regulation within the HNC tumor microenvironment (TME), generating distinct methylation landscapes associated with viral status. Genome-wide methylation analysis identified differentially methylated loci enriched in immune-related pathways, supporting a central role for epigenetic remodeling in shaping HPV-specific tumor–immune interactions. To link epigenetic differences to cellular organization and function within the TME, we characterized immune and stromal compartments, cell–cell interactions, and spatial architecture using high-dimensional single-cell imaging mass cytometry (IMC). Key findings were independently validated in an external single-cell RNA sequencing cohort. We identified 770 differentially methylated positions (P < 0.05, |logFC| <0.20) mapping to 386 genes that distinguish HPV-positive from HPV-negative tumors, with significant enrichment in immune-related pathways. Single-cell high-dimension IMC revealed distinct immune architectures between tumor types. In HPV-negative tumors, immune exclusion was primarily associated with fibroblast-dominated spatial organization that physically and functionally insulated tumor cells from immune surveillance. In contrast, T cells in HPV-positive tumors exhibited an intrinsic autocrine regulatory program characterized by robust type I and type II interferon signaling, consistent with enhanced tumor recognition and immune engagement. These findings demonstrate that HPV-associated DNA methylation reprogramming shapes the spatial and functional organization of the TME in HNC, promoting divergent mechanisms of immune regulation and tumor immune evasion.