Abstract

Tumor spatial architecture significantly influences immune responses, but the impact of regional variations on T cell exhaustion and clonality in human papillomavirus (HPV)-related cervical squamous cell carcinoma (CESC) remains poorly understood. Using single-cell RNA sequencing (scRNA-seq, n = 13) and TCR sequencing (scTCR-seq, n = 9), this study profiled T cells from paired tumor core and edge samples of patients with CESC. This was supplemented by bulk RNA-seq data ( n = 41), TCGA datasets from three cancer types (CESC, head and neck squamous cell carcinoma, and lung squamous cell carcinoma), and scRNA-seq data from colorectal cancer for broader validation. We found that CD8 + T cells, natural killer T (NKT) cells, and γδ T cells in the tumor core exhibited higher inhibitory and cytotoxicity scores, while CD4 + T cells showed increased regulatory T (Treg) and cytotoxic features in the tumor core. Bulk RNA-seq data confirmed elevated inhibitory and cytotoxic scores in the tumor core relative to the edge. Additionally, four subsets of exhausted CD8 + T cells (CD8 + Tex) were identified, including a stress-associated subset characterized by heat shock protein (HSP) family gene expression, which was validated by immunofluorescence and inversely correlated with survival in patients with CESC undergoing radiotherapy. TCR analysis revealed clonal expansion and reduced clonal diversity in the tumor core. Notably, CD8 + Teff-CD160 cells displayed substantial clonal sharing across regions and were associated with a favorable prognosis. Overall, our findings highlight distinct T cell states between the tumor core and edge in HPV-related CESC, offering insights into prognostic biomarkers and potential therapeutic targets.