The Expression Status And Biological Function Of PD-L2 In Head And Neck Squamous Cell Carcinoma

Objective:The tumor microenvironment(TME)of head and neck squamous cell carcinoma(HNSCC)had a high level of immunosuppression.The PD-1 monoclonal antibody was recommended by the new NCCN guidelines for the treatment of recurrent and metastatic(R/M)HNSCC,without local therapy possibility.The promising clinical efficacy of PD-1 monoclonal antibody in a subset of advanced HNSCC patients underscores the need for a better understanding of the immunobiology of HNSCC.Although HNSCC patients with high expression of PD-L1 in tumor cells have a relatively poor prognosis,the expression level of PD-L1 is not completely consistent with the efficacy of PD-1 monoclonal antibodies.PD-L2 is another ligand of PD-1,and its expression status is also an important predictor of HNSCC anti-PD-1 therapy.However,the PD-L2 based immune signature in HNSCC is still unclear.The relationship between potential predictive pathological biomarkers such as PD-L1,PD-L2,and PD-1,as well as the relationship between these biomarkers and clinical features,and the relationship between PD-L2 and other immune factors is still unclear,especially in Chinese HNSCC cohort.We sought to identify PD-L2 based immune signature as a prognostic biomarker for HNSCC and to investigate the prognostic value of tumor infiltrating lymphocytes(TILs:CD3~+,CD8~+,and FOXP3~+)and PD-L2expression in HNSCC.Methods:First,the expression levels of PD-1,PD-L1,PD-L2,CD3,CD8,FOXP3 and IL-6in tumor tissues of 59 patients with HNSCC were detected by Immunohistochemistry staining.Then statistical methods such as Chi-Square test or Mc Nemar test,Wilcoxon paired t test or Mann-Whitney U test and analysis of variance were used to analyze the correlation between the above factors and the clinicopathological characteristics of the patients.Kaplan-Meier curve was used to analyze the differences in survival time among patients with different expression levels of the above markers.Proportional Hazards Model analyzed the correlation between the above markers and the survival time of patients and constructed a STA model based on it.Second,mice HNSCC-derived SCC7 cells were injected into C3H mice to establish a subcutaneous model.After receiving PD-1,PD-L1 and PD-L2 antibody treatment,the effects of the above mono antibodies on the C3H immunoactivity mouse model were detected.Third,single cell sequencing was performed on two HNSCC specimen.PCA was performed based on highly variable genes.Dimensional reduction analysis using t-SNE revealed the diversity of tumor and non-malignant cell types.The effect of IL-6 on the expression of PD-L2 was analyzed by PCR and flow cytometry.Result:The positive rate of PD-L2 in the current HNSCC cohort is 62.71%,while the positive rate of PD-L1 was only 25.42%.In patients without PD-L1 expression,the positive rate of PD-L2 was 61.36%.In tumor cells,the expression of PD-L2 was positively correlated with PD-1,CD3,CD8 and FOXP3(P<0.0001,P=0.0289,P=0.0004,P=0.0063),while in stromal cells,the expression of PD-L2 was not significantly correlated with CD3,CD8 and FOXP3(P=0.1611,P=0.8585,P=0.2577).In tumor cells,the expressions of CD3,CD8 and FOXP3 were positively correlated with each other,and their expressions were significantly lower than those in stromal cells.Sta showed that high PD-L2 expression was an independent predictor of poor overall survival.The median overall survival(OS)was 72 months and the PI was-3.95(95%CI,-5.14 to-2.76),while the lowest median OS was 10 months and the PI was 1.43(95%CI,0.56 to 2.30).In the SCC7 derived mice subcutaneous tumor model,PD-L2 m Ab treatment inhibits immune escape inhibits tumor growth in vivo.Single cell RNA sequence analysis of two HNSCC patients showed that PD-L2expression was associated with IL-6.We confirmed that IL-6 increased the level of PD-L2,suggesting that inflammatory environment may contribute to the expression of PD-L2.Conclusion:Our study indicates that PD-1/PD-L2 axis plays a robust role in immune escape of HNSCC than PD-1/PD-L1 axis.Therefore,PD-L2 can be used as a potential target for immunotherapy of HNSCC,and Anti-IL-6 therapy in adjuvant therapy may be beneficial to HNSCC patients with high level of PD-L2 expression.