Comprehensive Analysis Of ULBP3/4 And PD-L1 Status In Head And Neck Cancer And Co-Regulation Of Immune Evasion In Nasopharyngeal Carcinoma

Purpose1.To study the relationship between expression of Programmed Death Ligand-1(PD-L1),UL16 Binding Proteins 3(ULBP3),UL16 Binding Proteins 4(ULBP4)in head and neck cancer(HNC)and their clinicopathological characteristics.2.To explore the co-regulatory mechanism of ULBP3,ULBP4 and PD-L1 in the cytotoxicity of specific T cells on nasopharyngeal carcinoma.Method1.The clinicopathological information and RNA sequencing(RNA seq)dataset was downloaded from The Cancer Genome Atlas(TCGA),which involved totally 500 cases that was diagnosed with head or neck cancer from 1992 to 2013 by pathology.Pearson correlation analysis was used to analyze the correlation between CD8,IFNgamma,PD-L1,ULBP3 and ULBP4.The chi-square test was used to analyze the relationship between the clinicopathological characteristics and the expression of CD8,IFN-gamma,PD-L1,ULBP3 and ULBP4.Comparison of the expression levels of CD8,IFN-gamma,PD-L1,ULBP3 and ULBP4 in cancer tissues and normal tissues was performed using paired sample t test.2.Peripheral blood CD8+ T cells were isolated,and CD8+ T cells were purified by flow cytometry.Specific CD8+ T lymphocytes were induced by dendritic cells(DC).The ULBP3 and ULBP4 genes of the 5-8F nasopharyngeal carcinoma cell line were knocked out by CRISPR/Cas9,and the PD-L1 overexpression vector was transfected.Gene sequencing and flow cytometry were used to detect the expression of ULBP3 and ULBP4 after gene knockout.Flow cytometry and Western-blot were used to identify the expression of PD-L1 after transfection of the expression vector.The CFSE-PI-flow cytometry was performed to reflect the cytotoxic activity of specific T cells on different 5-8F nasopharyngeal carcinoma cell lines.Result Part11.Pearson correlation analysis indicated that the expression of IFN-gamma was positively correlated with CD8 and PD-L1(R=0.765,P<0.0001;R=0.453,P<0.0001),and IFN-gamma is negatively correlated with ULBP3(R=-0.239,P<0.0001).There was a positive correlation between CD8 expression and PD-L1 expression(R=0.337,P<0.0001),a negative correlation between CD8 expression and ULBP3 expression(R=-0.224,P<0.0001),and a weak negative correlation between the expression of PDL1 and ULBP3(R=-0.113,P=0.0115).2.The clinicopathological characteristics analysis showed the presence of low expression of ULBP3,ULBP4 and high expression of CD8 expression in the high histological grade(P=0.001,P=0.000,P=0.015).Compared to female patients,males showed lower ULBP4 expression level(P=0.000).CD8 and IFN-Gamma had a high level of expression in older patients(P=0.032).3.The expression of CD8,IFN-gamma,PD-L1 and ULBP3 in cancer tissue is higher than normal tissues(P=0.017,P=0.001,P=0.000,P=0.000),and expression of ULBP4 in cancer tissues is lower than that in normal tissues(P=0.022).Part 21.The isolated PBMCs were amplified,then sorted and purified through flow cytometry,which increased the proportion of CD8+ T cells from 49.13% to 92.57%.After CRISPR/Cas9 knockout,gene sequencing and flow cytometry shows that ULBP3 and ULBP4 have been successfully knocked out.After transfection of the expression vector,both Western-blot and flow cytometry confirmed that PD-L1 was overexpressed.2.As the effector to target ratio increased,the cytotoxic activity of CD8+ T cells to 5-8F increased significantly.At different effector to target ratio,PD-L1oe-5-8F,ULBP3ko-5-8F,ULBP4ko-5-8F,ULBP3ko-PD-L1oe-5-8F and ULBP4ko-PD-L1oe-5-8F can all cause the functional deficiency of CD8+T cell.Moreover,CD8+T cell has lowest cytotoxic effect on ULBP3ko-PD-L1oe-5-8F and ULBP4ko-PD-L1oe-5-8F.Conclusion1.IFN-gamma was positively correlated with CD8 and PD-L1,and IFN-gamma was negatively correlated with ULBP3,which indicates that as IFN-gamma up-regulate the expression of PD-L1,it may take part in down-regulating the expression of ULBP3 in head and neck cancer.2.Head and neck tumors with high histological grade had low expression of ULBP3,ULBP4 and high expression of CD8.The expression of CD8,IFN-gamma,PD-L1 and ULBP3 in tumor tissue was higher than that in normal tissue,which suggest that these proteins may be involved in tumorigenesis,tumor development and immune evasion.Whereas,the expression of ULBP4 in tumor tissue was lower than that in normal tissue.3.ULBP3-4/NKG2 D activation pathway and PD-1/PD-L1 inhibitory pathway influence the lethal effect of CD8+ T cells.Down-regulation of ULBP3 and ULBP4 may be involved in the resistance of PD-1 inhibitors in head and neck cancer.