Activation Of CD8~+T Lymphocytes Induced By TLR7/8 Agonist R848 Against Lung Cancer

Backgrouds:Toll-like receptor 7(TLR7)is mainly expressed on innate immune cells especially DCs(dendritic cells,DCs),which can recognize corresponding TLR7 ligands or pathogens and initiate adaptive immunity.Therefore,it is widely used for antiviral and anti-tumor therapy.R848(resiquimod)is a new TLR7/8 agonist,selectively acts on TLR7 in mice and has potent Th1 adjuvant activity exhibited by extensive in vivo and in vitro study.Our preliminary research showed that R848 induced strongest CD8~+T immunity compared with other two common TLR ligands,including IFN-γ~+CD8~+T production and CD8~+T proliferation.Despite advances in treatment options such as immune checkpoint inhibitors and targeted drugs for lung cancer,which benefits a number of patients,it remains the leading cause of cancer death.It is documented that R848 achieved remarkable outcome in treatment for breast cancer,advanced pancreatic cancer and melanoma.However,the role of R848 in lung cancer is unclear.Objective:1.We intend to figure out the expression of TLR7 in mice with lung cancer.2.The effect of R848 on tumor growth、metastasis of lung cancer and survival time of tumor-bearing mice were verified.3.Through in vivo and in vitro experiments,we investigate the underlying mechanism of R848/TLR7 against lung cancer.4.We provide other theoretical basis for R848 used in cancer treatment at least in this study.Methods:1.TLR7 expression in mice during tumor growth was detected by flow cytometry,q PCR and immunofluorescence subcutaneous and metastatic models of lung cancer.2.Tumor volume,survival time of mice and metastatic nodules in the lung were examined with R848 injected intraperitoneally or subcutaneously.3.Activation of DCs,NK cells and CD8~+T cells in the peripheral lymphatic organs,cytokines in the blood supernatant of tumor-bearing mice were tested by flow cytometry at different time points after R848 administered through tail vein.Proliferation and activation of CD8~+T cells in the co-culture system of DCs and CD8~+T cells in vitro were tested using flow cytometry.4.Consecutive treatment of tumor-bearing mice with R848 for 10 times,the proportion of immune cells in the tumor microenvironment was detected by flow cytometry.5.In vivo,the tumor volume and survival time were recorded with CD4~+T and NK cells depleted.In vitro,proliferation and activation of CD8~+T cells were detected in the presence or absence of NK cells.Results:1.The expression of TLR7 on DCs in mice increased in the early stage and decreased in the late stage of lung cancer.2.Our results demonstrated that intraperitoneal injection of R848 effectively inhibited the growth of lung tumor,markedly reduced metastasis to lung and prolonged the survival time of tumor-bearing mice.However,In vivo R848 injection had no inhibitory effect on the growth of lung cancer in TLR7 knockout mice,and in vitro R848 had no effect on the proliferation of LLC cells.3.The activation of DCs and CD8~+T lymphocytes induced by R848 in vivo and in vitro,and increasement of cytokines in blood supernatant were detected.4.The treatment of R848 remodeled TME:increased DCs、NK cells and CD8~+T cells,decreased CD4~+T cells and Treg cells.5.In vivo depletion of NK cells we found that the tumor growth was accelerated.In vitro,nk cells added to co-culture system of DCs and CD8~+T cells,the proliferation and activation of CD8~+T cells were enhanced.Conclusions:1.In this study,we confirmed that the expression and trend of TLR7 on dendritic cells in the progression of mice burdened with lung cancer.2.We verified that R848 effectively inhibited lung tumor growth,markedly reduced metastasis to lung and prolonged the survival time of tumor-bearing mice.R848 had no inhibitory effect on lung cancer directly,and its anti-lung cancer role was based on the stimulatory effect of R848 on host TLR7.3.The activation of DCs and CD8~+T lymphocytes induced by R848 was confirmed in vitro and in vivo experiments.4.Treatment of R848 changed the proportion of immune cells in TME.5.The results suggested that NK cells are involved in the anti-tumoral effect of R848 on CD8~+T induction.