| Glioblastoma is the most common primary brain tumor of adu lts,about 40-50%of brain tumor.Because of its invasive nature,it is difficult to remove by conventional surgery and is characterized by poor survival rates.The presence of human cytomegalovirus(HCMV)in glioblastoma multiform(GBM)has sparked considerable interest and controversy.The monoclonal antibody of programmed death factor 1(PD-1)has made some progress in the treatment of glioma.Its ligand 1(PD-L1,CD274)is thought to be involved in the immune escape mechanism of malignant gliomas and is associated with HCMV infection,whereas Toll-like receptor 3(TLR3)can induce antiviral status in pathogen-infected cells or animals.To study the relationship between TLR3 and the expression of PD-L1 in gliomas,we hope to explore new methods of combined immunotherapy to provide new ideas for clinical treatment.Objective: To investigate whether the expression mechanism of PD-L1 is associated with TLR3 in HCMV-infected gliomas.Methods: We used the immunohistochemistry staining to detect the expression of IE2,PD-L1 and TLR3 in clinical glioma specimens.After HCMV was infected with glioma cells,PD-L1 RNA and protein were detected by q-PCR and immunofluorescence staining;TLR3 expression was blocked by cell transfection technique;PD-L1 expression was detected in gliomas with TLR3-blocked after infection with HCMV using WB technique;ELISA was used to detect the secretion of cytokines TGF-β and IL-8 in HCMV-infected glioma cells which were TLR3-blocked or TLR3-unblocked.Results: We found IE2 positive in 70% of grade III and 85% of IV malignant glioma specimens.Compared with IE2-negative specimens,the expression levels of PD-L1 and TLR3 were increased,P<0.05;RNA was extracted at 0,6 and 72 h after the HCMV infected the glioma cells,and the protein was extracted at 0,24 and 72 h.The RNA expression of PD-L1 was increased by more than 20 fold,the protein expression wa s increased by more than 3 fold;after transfection of siRNA,the expression of TLR3 decreased by more than 70% by WB assay,and the blocking was considered successful.After blocking TLR3,the expression of PD-L1 was significantly lower than that of the unblocked group after HCMV infection,which was statistically signifi cant(P<0.05);after blocking TLR3,the secretion of TGF-β in T cells was induced to increase by more than 2 times,but the secretion of IL-8 was inhibited,and it was almost impossible to detect.Conclusion: The expression of PD-L1 in HCMV-infected glioma cells was higher than that in the uninfected group,which may be one of the reasons why HCMV promoted the increase of malignant degree of glioma.After blocking the expression of TLR3,a decrease in the expression of PD-L1 was observed in HCMV-infected tumor cells,and we believe that the expression of PD-L1 can be mediated by TLR3 triggering in glioma cells.Therefore,we propose a novel immunotherapy using a combination of PD-L1 monoclonal antibody and blocking TLR3 triggering against HCMV-infected glioblastoma. |
