Abstract: PURPOSE: To explore the expression of hub genes, pathways and inhibitory immune checkpoints of CD8⁺ T cells in metastatic lymph nodes of head and neck squamous cell carcinoma(HNSCC) in C3H/He mice. METHODS: A popliteal lymph node metastasis model of immunocompetent C3H/He mice was constructed with SCC-7 cell line of HNSCC, and the lymph node metastasis status was determined by immunofluorescence. CD8⁺ T cells in normal and metastatic lymph nodes were sorted by flow cytometry, and transcriptome sequencing analysis was performed to screen out differentially expressed genes. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed sequentially. Flow cytometry and multiplex immunohistochemical were used to detect the expression of four immune checkpoints of CD8⁺ T cells in metastatic lymph nodes of tumor-bearing mice. SPSS 26.0 software package was used for statistical analysis. RESULTS: After 4 weeks of foot pad injection of SCC-7 cells, C3H/He mice displayed obvious metastasis in popliteal drainage lymph node. A total of 912 differentially expressed genes were screened out by transcriptome sequencing analysis of CD8⁺ T cells in normal and metastatic lymph nodes, including three inhibitory immune checkpoint-related genes which were upregulated(Pdcd1, Lag3 and Tigit). Eight tumor-related signaling pathways were screened out by KEGG network analysis, including Toll-like receptor signaling pathway, NF Kappa-B signaling pathway, TNF signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, PD-L1 and PD-1 immune checkpoint pathway in tumors and p53 signaling pathway. Flow cytometry showed high expression level of PD-1 and TIM-3 in CD8⁺ T cells in mouse metastatic lymph nodes. It was further confirmed by multiplex immunohistochemical that PD-1 was highly expressed in CD8⁺ T cells in metastatic lymph nodes of patients with HNSCC. CONCLUSIONS: Tumor-related signaling pathway of CD8⁺ T cells in mouse metastatic lymph nodes is significantly activated. The PD-1 expression level of CD8⁺ T in metastatic lymph nodes of mice and patients with HNSCC is markedly increased. Immunotherapy targeting CD8⁺ T cells may become a new strategy for the prevention and cure for lymph node metastasis of HNSCC.

Key words: Head and neck squamous cell carcinoma, Lymph node metastasis, C3H/He mice, CD8+ T cell, PD-1