[Correlations between regulatory T cell and the tumor immune microenvironment in head and neck squamous cell...

Objective: To investigate the role of regulatory T cell (Treg) in the immune microenvironment of head and neck squamous cell carcinoma (HNSCC) and its relationship with human papillomavirus (HPV) status and programmed death-ligand 1 (PD-L1) expression, thereby providing a rationale for differential immunotherapy strategies. Methods: This study collected 132 formalin-fixed, paraffin-embedded HNSCC tissue samples (96 males and 36 females; mean age 60.36±12.58 years) from the Department of Oral Pathology, Shanghai Ninth People's Hospital, between May 2020 and May 2024. Clinical and bioinformatics data from 408 HNSCC cases were obtained from The Cancer Genome Atlas (TCGA) database. FoxP3 immunohistochemical staining was performed on clinical samples to assess Treg infiltration density, and its correlations with clinicopathological characteristics including age, gender, smoking history, drinking history, HPV status and pathological grades were analyzed. Multiplex immunofluorescence and PD-L1 staining were conducted to analyze the correlations among Treg infiltration, immune cells, and PD-L1 expression. Treg infiltration levels in the TCGA cohort were calculated using single-sample gene set enrichment analysis (ssGSEA), and the immune microenvironment was evaluated using ESTIMATE and TME scores. Results: Treg infiltration was significantly higher in HPV⁺HNSCC (2 201 cells/mm²) than in HPV^-HNSCC (545 cells/mm², P<0.001). Among HPV^- HNSCC cases, poorly differentiated tumors exhibited lower Treg infiltration (Grade Ⅰ:611.79 cells/mm²;Grade Ⅱ:308.74 cells/mm²;Grade Ⅲ:183.33 cells/mm², P<0.05). Bioinformatic analysis revealed that Treg infiltration correlated with multiple immune scores, immune cell infiltration densities, and immune checkpoint molecule expression. Differentially expressed genes between high and low Treg infiltration groups were enriched in immune-related pathways such as leukocyte migration and cytokine-cytokine receptor interaction. HPV⁺ HNSCC were specifically enriched in viral protein-cytokine interaction pathways. Treg infiltration was positively correlated with PD-L1 expression (R=0.488, P<0.001), independent of HPV status. Furthermore, Treg infiltration was positively associated with CD4⁺ T cell (R=0.434, P<0.001) and CD68⁺ macrophage infiltration (R=0.303, P<0.001). Conclusions: Treg play a significant role in shaping the immune microenvironment of HNSCC. Their infiltration level is associated with HPV status, with HPV⁺ HNSCC demonstrating higher Treg infiltration and distinct functional pathway enrichment. The positive correlations between Treg, various immune cells, and PD-L1 expression provide a theoretical foundation for combination immunotherapy strategies co-targeting the PD-L1/PD-1 pathway and Treg.