The Effect Of PD-1 Expression Level On The Function Of Tumor-Associated Macrophages And Prognosis Of Patients With Lung Adenocarcinoma

Part 1 Differential expression of macrophages and PD-1 in tumor tissues and adjacent tissues of patients with lung adenocarcinomaObjective:By analyzing the expression of CD8~+T lymphocytes PD-1 and macrophage infiltration in postoperative adenocarcinoma tumor tissues and adjacent tissues of patients with lung adenocarcinoma,the immunosuppression characteristics in adenocarcinoma tumor microenvironment of patients with lung adenocarcinoma were investigated,so as to provide guidance for pathological development and prognosis of patients.Methods:The tumor pathological and adjacent tissues of 115 patients with lung adenocarcinoma were collected.PD-1/CD8 staining and CD86/CD204 staining were performed by immunofluorescence double staining.We fully evaluated the proportion distribution of PD-1 at T cell immune checkpoint,M1 macrophages(CD86+CD204-)and M2 macrophages(CD86-CD204+)and the differential expression in different pathological tissues.We investigated the correlation of expression level of PD-1 and macrophage phenotype with age,tumor stage,clinical subtype and prognosis of lung adenocarcinoma patients.Meanwhile,the correlation between PD-1 expression and M2 macrophages in tumor tissues was analyzed.Results:The expression of CD8~+PD-1~+T lymphocytes and M2 macrophages in lung adenocarcinoma tumor tissues was higher than that in para-cancer tissues(P<0.05),while the expression of M1 macrophages in tumor tissues was significantly lower than that in para-cancer tissues(P<0.05).The expression levels of PD-1 and M2 macrophages in tumor tissues were correlated with TNM stage(P<0.05),but not with patients age,gender and tumor subtype.At the same time,the expression levels of PD-1 and M2 macrophages in tumor tissues were positively correlated,and were negatively correlated with the prognosis of patients.Conclusions:CD8~+PD-1~+T lymphocytes and M2 macrophages are highly expressed in lung adenocarcinoma tumor tissues.PD-1 expression and M2macrophage infiltration degree are positively correlated with TNM stage of lung adenocarcinoma patients.The expression of PD-1 was positively correlated with the infiltration degree of M2 macrophages.The high expression of PD-1 and the high infiltration characteristics of M2 macrophages are correlated with the poor long-term prognosis of patients.Part 2 The PD-1 /B7H1 signaling pathway regulates the phenotype and function of macrophageObjective: To explore the effects of PD-1/B7H1 signaling pathway on macrophage phenotype and function,provide inspiration for clinical immuno-suppression studies in patients with lung adenocarcinoma,and provide new ideas for comprehensive treatment of lung adenocarcinoma.Methods: THP-1 was selected as the research object,and the primary culture was carried out,and the third generation of monolayer cells were selected as the research object.Plasmid vectors of PD-1mimics,PD-1inhibitor and B7H1 inhibitor lentivirus were constructed and transfected into the THP-1 cells respectively.Settiing the following groups: Control group(THP-1 without any treatment),PD-1 mimics group(intracellular transfection of PD-1 mimics),PD-1 inhibitor group(intracellular transfection of PD-1 inhibitor),PD-1-B7H1 Inhibitor group(intracellular transfection with PD-1inhibitor+B7H1 inhibitor).The above groups of THP-1 cells and A549 cells were co-cultured in layers for 72 hours,and then related items were detected and analyzed.We Verified of the targeted regulatory relationship between PD-1 and B7H1,and analyzed the form of THP-1.The mRNA expression of PD-1 and B7H1 in each group were detected by RT-PCR.Flow cytometry was used to detect the purity of macrophages in each group.The concentrations of inflammatory cytokines IL-6,IL-10,TNF-α and IL-12 in macrophages were detected by ELISA.The expression levels of CD86 and CD204 in macrophages of each group were analyzed by RT-PCR/Western-blot.We detected the phagocytosis rate of macrophages in each group.A549 cells after co-cultured with macrophages were collected,and the migration and invasion abilities of tumor cells in each group were detected by scratch assay and Transwell assay.The expression of invasion-related protein MMP-2 and MMP-9 was detected by Western-blot.Results:1)Identification and analysis of PD-1 targeting B7H1: A binding site was predicted between PD-1 and B7H1 mRNA 3 ’non-coding region.Different combinations of wild-type and mutant PGL3-B7H1Y-UTR expression vectors were transfected into THP-1 cells by co-transfection technique.The results showed that the reporter gene activity of wild-type expression vector was significantly higher than that of control group(P<0.05).2)Morphology analysis of macrophages: THP-1 cells grew well,presenting mixed culture state of M1 and M2,and the morphology of macrophages changed in co-culture with A549 cells.3)mRNA expressions of PD-1 and B7H1 genes were detected by RT-PCR/Western-blot analysis.the expression trend of PD-1 in each group was PD-1-B7H1 inhibitor<PD-1 inhibitor<Control<PD-1 mimics(P<0.05).The analysis trend of B7H1 was PD-1-B7H1 inhibitor<PD-1 inhibitor<Control<PD-1 mimics(P<0.05).4)Flow cytometry and Western-blot analysis were used to detect the expression of CD86 and CD204 in each group of cells.The data of CD86 were analyzed as PD-1 mimics<Control<PD-1 inhibitor< PD-1-B7H1 inhibitor(P<0.05).However,The data of CD204 were analyzed as PD-1-B7H1 inhibitor< PD-1 inhibitor<Control <PD-1 mimics(P<0.05).5)Concentrations of THP-1 inflammatory factors IL-6,IL-10,TNF-α and IL-12 were detected by ELISA in each group.The expression of pro-inflammatory factors IL-6,TNF-α and IL-12 showed the same trend.The comparative trend of data in each group was PD-1 mimics<Control<PD-1 inhibitor <PD-1-B7H1 inhibitor(P<0.05).The anti-inflammatory factor IL-10 showed the opposite trend,and the trend was PD-1-B7H1 inhibitor< PD-1 inhibitor< Control< PD-1 mimics(P<0.05).6)Macrophage phagocytic rate detection after co-culture: the phagocytic rate trend of each group was PD-1 mimics<Control<PD-1 inhibitor<PD-1-B7H1 inhibitor(P<0.05).7)Cell migration and invasion ability analysis of A549 cells: the comparative trend of mobility/invasion of A549 cells in each group were PD-1-B7H1 inhibitor< PD-1 inhibitor<Control<PD-1 mimics(P<0.05).The data expression trend of MMP-2 and MMP-9 protein concentration in each group was PD-1-B7H1 inhibitor< PD-1 inhibitor< Control<PD-1 mimics(P<0.05).Conclusions:PD-1 targets the B7H1 signaling pathway to regulate the phenotype of macrophages.When PD-1 is overexpressed,the expression of B7H1 increases,the proportion of M2 macrophages increases,the expression of inflammatory cytokines increases,and the phagocytosis of phagocytes decreases,promoting the pathological development of tumor tissues.When PD-1 was silenced,the opposite trend was observed.PD-1 regulates the migration and invasion of A549 cells through the change of macrophage phenotype.When PD-1 is overexpressed,the proportion of M2-type macrophages increases,and the migration and invasion of tumor cells are enhanced,promoting the occurrence and development of tumor.