Treatment Of Lung Cancer Using The Synergic Therapy Of SiPD-L1 Combined With DC Vaccine Through Reserving T Cell Exhaustion In Vivo

Objectives:The activity of a negative immunonegative molecules,such as the programmed cell death receptor ligand(PD-L1),significantly attenuates dendritic cell(DC)mediated immunotherapy.In this study,we developed a new method using nanomaterials micropoly/PD-L1 that can target DCs,deliver siRNAs and silence the target molecule PD-L1.We also investigated the mechanisms involved in T cell exhaustion through,targeting PD-1/PD-L1 signaling pathways,and activating the effector function of T cells.The therapeutic effect of DC vaccine that has been pre-loaded with tumor antigen and silenced siPD-L1 on LLC lung cancer-bearing mice was investigated in vivo.Methods:1.Flow cytometry was applied to observe the transfection efficiency of micropoly/cy3- siGAPDH to DC.The silencing effect of micropoly/ siPD-L1 on DC was detected by Q-PCR2.Flow cytometry was used to detect changes in DC maturity after gene silencing of PD-L1.3.The DC after gene silencing of siPD-L1 was mixed with T cells of LLC lung cancer-bearing mice.The secretion of cytokines TNF-a and IL-2 were detected by ELISA.The proliferation of T cells was detected by CCK8 method.4.The expression of PD-1,TIM3 and BTLA on T cells was detected by q-PCR.The apoptosis of CD4+ T cells and CD8+ T cells were detected by flow cytometry.5.The PD-L1-silenced DC vaccine was injected into C57BL/6 mice to treat lung cancer,and the therapeutic effect was observed and evaluated.6.CD8+ T cells from tumor bearing mice were sorted by magnetic beads to detect the cutotoxic killing effect on tumor cells.The secretion of cytokines of TNF-a and IL-2 ware detected by ELISA.The proliferative capacity of T cells was detected by CCK8 method.7.T cell apoptosis was detected by Annexin V method.T cell phenotype changes was detected by flow cytometry.Results:1.After transfect micropoly/ siPD-L1 for 6 h,flow cytometry showed that the transfection efficiency of DC was over 70%.The silencing efficacy of micropoly/ siPD-L1 reached to 74% in 24 h after transfect to DC cells.2.Flow cytometry results indicate that silencing the PD-L1 gene can promote DC maturation.3.The DC vaccine after silencing of siPD-L1 can promote the secretion of cytokines including TNF-a and IL-2,and promote the proliferation of T cells.4.The DC vaccine after silencing of siPD-L1 can regulate the expression of inhibitor receptor factors of PD-1,TIM-3 and BTLA in T cells,as cell as inhibit the apoptosis of CD4+ T cells and CD8+ T cells.5.Treatment of micropoly/ siPD-L1 and antigen-loaded DC vaccine to C57BL/6mice can significantly suppress the growth of lung cancer and enhance the anti-tumor immunity of tumor-bearing mice.6.The micropoly/ siPD-L1 therapeutic established in this study can enhance the tumor-specific cytotoxic killing effect of CD8+ T cells,promote the secretion of cytokines such as TNF-a and IL-2,and promote the proliferation of T cells.7.The apoptosis of CD8+ T cells and CD4+ T cells in tumor-bearing mice was reduced,wile the exhaustic phenotype of T cell could be significantly inhibited after PD-L1 silenced DC vaccine treatment.Conclusion:1.This study constructed a micropoly/ siPD-L1 can targetedly deliver siRNA to DC cells and effectively knock down the expression of PD-L1.2.Silencing the gene of PD-L1 on DC vaccine by micropoly/ siPD-L1 can effectively enhance the biological effects of T lymphocytes in tumor-bearing mice.3.DC vaccine with micropoly/ siPD-L1 silencing PD-L1 gene and loaded with tumor antigen can effectively treat lung cancer-bearing mice,can reverse T cell depletion and enhance the anti-tumor ability of tumor-bearing mice.