Wang, K.; Deng, N.; Tao, Y.; Yang, X.; Yuan, M.; Gao, L.; Jiang, S.; Shang, W.; Deng, J.; Wang, L.

HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) harbors dense lymphocytic infiltration yet responds poorly to immune checkpoint blockade, a paradox whose mechanistic basis remains unresolved. Here we constructed an integrated single-cell and spatial transcriptomic atlas of HPV OPSCC (23,119 cells, 24 cell types), complemented by CRISPR-Cas9 functional validation in two HPV cell lines and multiplex immunofluorescence across independent cases. Pseudotime trajectory analysis identified TOXCD8 T cells, rather than canonically defined exhausted cells, as the true terminal exhaustion endpoint, and revealed a previously uncharacterized IEGCD8 effector memory population at a fate-decision juncture between activation and irreversible exhaustion, representing a candidate cellular window for therapeutic rescue. Actively cycling Tregs with comprehensive co-stimulatory and co-inhibitory receptor expression underwent in situ immunosuppressive differentiation. Compartmentalized expression of LDHA/MCT4 in cancer cells versus LDHB/MCT1 in T cells defined a directional lactate flux model, and spatial deconvolution confirmed that immune cells were restricted to the tumor periphery, establishing a metabolic barrier underlying immune exclusion. Most notably, we discovered a KMT2D-KLF7-PD-L1 regulatory axis: KMT2D maintains KLF7 transcription through H3K4me1-dependent enhancer activation; KLF7 simultaneously drives neural crest differentiation facilitating perineural invasion and upregulates PD-L1 enabling immune evasion, thereby linking tumor-intrinsic epigenetic remodeling to both neural programming and immune checkpoint expression within a single regulatory cascade. These findings reveal that ICB resistance in HPV OPSCC arises from converging multi-scale mechanisms, and nominate the KMT2D-KLF7-PD-L1 axis as a combinatorial therapeutic target pairing PARP inhibitors with immune checkpoint blockade.