Integrated single-cell and spatial transcriptomic analysis of T cell exhaustion and immunometabolic remodeling in HPV-positive oropharyngeal squamous cell carcinoma

ABSTRACT HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) harbors dense lymphocytic infiltration yet responds poorly to immune checkpoint blockade (ICB), 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 placed TOX⁺CD8⁺ T cells, rather than canonically defined exhausted cells, at the terminal exhaustion endpoint, and uncovered a previously uncharacterized IEG⁺CD8⁺ effector memory population at a fate-decision juncture between activation and irreversible exhaustion. Actively cycling Tregs with broad co-stimulatory and co-inhibitory receptor expression underwent intratumoral immunosuppressive differentiation. Compartmentalized LDHA/MCT4 expression in cancer cells versus LDHB/MCT1 in T cells established a directional lactate flux model, and spatial deconvolution confirmed peripheral immune exclusion. We identified a KMT2D-KLF7-PD-L1 regulatory axis: KMT2D sustains KLF7 transcription through H3K4me1-dependent enhancer activation; KLF7 concurrently drives neural crest differentiation and upregulates PD-L1, thereby linking epigenetic remodeling to both neural programming and immune checkpoint expression. These findings define converging multi-scale mechanisms underlying ICB resistance in HPV⁺ OPSCC and nominate the KMT2D-KLF7-PD-L1 axis as a combinatorial therapeutic target.