PD-L1 Knock Out Of Irradiation Induced Senescent Tumor Cells,Employed For Pulsing Dendritic Cells(DCs),Strengthens DC Vaccine-induced Antitumor Effect Against Melanom

BackgroundMelanoma is a very aggressive cancer,can easily metastasis via the lymphatics or the blood at early stage,clinical outcome is rather poor.The recent development of new immunotherapies has led to great advances in melanoma treatment,however recurrence and metastasis often occur,this situation allows of no delay to develop new and effective immunotherapeutic approaches.Adequate and activated DCs is the strongest inducer of antitumor immune response,thus DC vaccine is still one of the hottest and most important directions at present.Resent study indicate that senescent tumor cells(STC)can be induced senescent under special conditions,and still maintain its secretion activity,high express and secrete a variety of immune stimulating cytokines and chemokines,that is senescence-associated secretory phenotype(SASP),thus it can persistently attract DC and immune effector cells infiltration,induce DC maturation and activation,then CD4+helper cells and CTLs also migrate to the inoculation site,aggregate and activate more DC precursors and macrophages,release higher levels of IFN-y,TNF-a and other cytokines,stimulate systemic immune response.Therefore senescent tumor cells can be used for loading DC,may be possible to induce a higher level of DC activation as a novel DC vaccine.PD-1/PD-L1 can restrain T lymphocyte proliferation,activation and function,induce antigen-specific T cell apoptosis,promot the differentiation of CD4+T cells into Foxp3 regulatory T cells,thus,it plays an important role in maintaining tumor suppressive immune microenvironment.Blocking the interaction between PD-L1 and PD-1 may enhance the anti-tumor effect of functional T cell.We try to knockout the PD-L1 gene of mouse malignant melanoma cell line B16F10,and employ X ray irradiation to induce senescence,then loading DCs with these senescent tumor cells to study its ability to stimulate DC activation,and then inoculate these activated DCs into mice and study its anti melanoma effect,may develop a novel and effective DC vaccine.Object and methods1.Knockout the PD-L1 gene:employ CRISPR/Cas9 lentivirus to knockout the PD-L1 gene of B16F10 cells,detect the PD-L1 expression level of the cells cultrue in vitro and cells from tumor tissue by Western Blot and Flow Cytometry,to determine the effect of knockout.2.Induce senescent tumor cells and detect SASP:employ 6Gy,9Gy and 12Gy X ray irradiation treatment then culture for 3 days,5 days and 7days to induce B16F10 cells into senecence,detect senescence associated beta galactosidase and observe the proportion of senescent cells to determine the best inducing irradiation dose and cultrue time.Detect the SASP of B16F10 cells by RT-qPCR and observe its characteristics.3.Loading DC:divided into three groups to operate,experimental group(DC loading with PD-L1 knockout senecent B16F10 cells),NC group(DC loading with senecent B16F10 cells)and control group(pure DCs),detect DC phenotype by Flow Cytometry and cytokine expression level by RT-qPCR to determine the extent of DC activation.4.Mouse experiment:curatively and prophylacticly inoculate mice with the above mentioned three group DCs,tumor volume and survival time of mice were observed,and compare the therapeutic and prophylactic effects of these three vaccines.Results1.Fluorescence expression of experimental group and NC group under fluorescence microscope both were>90%.Western Blot ting and Flow Cytometry indicated that as compared to control group,PD-L1 expression level of cells cultrue in vitro and tumor tissue from experimental group were much lower.As compared to cells culture in vitro,PD-L1 expression level of tumor tissue from experimental group and control group were much higher.2.Compared to control group and other experimental group,cells of 7days after 12Gy irradiation group were more irregular in morphology,bigger in cell volume,observed long spindle cells and large round cells,the cells became flat,the particles increased,karyoplasmic ratio decreased,the number of cells was not significantly increased,showed the highest SA-β-gal positive rate,the relative mRNA expression of p16,p21,IL-12,TNF-α,TNF-β,IFN-γ,CCL5 and CXCL9 were much higher,the relative mRNA expression of IL-10 was much lower,the difference was statistically significant(P<0.05).3.Flow Cytometry indicated that,as compared to control group,the expression of CD11c,CD80,CD86 and I-A/I-E of the experimental group and NC group were significantly increased.RT-qPCR indicated that as compared to NC group,the mRNA expression of IL-6,IL-12p40 and IFN-γ of the experimental group was significantly increased,TGF-β expression significantly decreased(P<0.05).4.In curative and prophylactic vaccination setting,as compare to NC group and control group,experimental group showed smallest tumor volume.In prophylactic vaccination setting,median survival time of experimental group was much longer(P<0.05).Conclusion1.CRISPR/Cas9 lentivirus in our study effectively knockout the PD-L1 gene of mouse malignant melanoma cell line B16F10,B16F10 cells culture in vitro low expressed PD-L1,tumor microenvironment in vivo can induce the expression of PD-L1.2.X ray irradiation can effectively induce B16F10 cells into senecence,senecenct B16F10 cells high expressed immune stimulating cytokines and chemokines.3.DC loading with PD-L1 knockout senecent B16F10 cells can stimulate DC mature,induce DC activation and potentiate DC function.4.DC loading with PD-L1 knockout senecent B16F10 cells can potentiate mice antitumor immune response,prolong the survival time of mice,can be used to prevent and treat malignant melanoma.