Establishment And Immune Pathogenesis Of A Novel HBV-related Hepatocellular Carcinoma Mouse Model

Chronic HBV infection remains a major public health problem worldwide,with hepatitis,liver fibrosis,cirrhosis and liver cancer caused by chronic HBV infection threatening approximately 260 million people worldwide.Although effective prophylactic vaccines have been developed to effectively reduce the risk of new HBV infections,the prevalence of preventive vaccines in low-income countries remains low.In addition,effective treatments have not been established for patients with chronic HBV.The general nucleoside analogues combined with interferon treatment can effectively inhibit the replication of the virus,but it can not eliminate the virus and has a strong rebound after the drug withdrawn.Patients with chronic HBV infection still face the risk of developing liver cancer.Therefore,it is particularly important to elucidate the mechanism by which chronic HBV infection causes liver cancer.HBV can cause the activation of proto-oncogenes in hepatocytes or the inactivation of tumor suppressor genes by integration into the genome of host cells,leading to malignant transformation of cells.While the body's immune system continues to attack HBV-infected hepatocytes is one of the necessary factors for the malignant transformation of cell carcinoma,so studies on the immuno-pathogenesis of HBV can help reveal the mechanism of HCC caused by HBV infection,but there is no suitable animal model to study this process nowadays.This study mainly established a method to construct a mouse model of HBV by the use of hepatocytes transfer and reconstruction for immune pathogenesis of HBV,and obtained the results as follow:1.Reconstitution of HBsAg+ hepatocytes in the recipient Fah-/-mice.Hepatocytes of HBs transgenic(HBs-Tg)mice and wild-type(C57BL6/J)mice were firstly isolated by use of the liver perfusion;then 1.0×106 hepatocytes were transferred into the 8-12 week old immunocompetent recipient Fah-deficient mice via splenic injection;finally,the NTBC water supply was withdrawn.And the hepatocytes of recipient mouse died,providing space for the growth of donor mouse hepatocytes.After 12 weeks of hepatocyte reconstitution,we successfully constructed HBs-Tg hepatocyte replacement mice(HBs-HepR)and its control B6-HepR.And the expression of HBsAg was detected in the serum and tissues of all HBs-HepR mice.2.Chronic inflammation,fibrosis and HCC development in HBs-HepR miceWhen the reconstitution time of was prolonged,HBs-HepR mice showed an increase in ALT levels at 18 weeks post transfer and liver fibrosis at 26 weeks post transfer,compared with the control.9 months post transfer,tumor nodules appeared in the liver of all HBs-HepR mice.Tumor nodule was HCC which was comfirmed by the gene level and the tissue level identification.The HBs-HepR mice model was different from other existing mouse HCC models,but had the same molecular phenotype as HCC caused by human HBV infection.3.Generation of HBsAg-specific CD8 T cells in HBs-HepR miceBy single-cell sequencing analysis,CD8 T cells were activated in the liver of HBs-HepR mice.More CD8+T cells were transformed from the naive phenotype to the effector phenotype 18 weeks post transfer.And HBsAg-specific CD8 T cells were found in the liver of HBs-HepR mice by flow cytometry and immunefluorescence.CD8 T and mediated apoptosis of hepatocytes.By the use of depletion and genetic deficiency,we demonstrated that the absence of CD8+T cells can significantly reduce the prevalence of HCC.Tumor nodules were not observed in livers at 9 months post transfer of HBsAg+ hepatocytes into HBs-HepR-CD8KO mice.4.B cells,CD4+T cells and NK cells do not play a key role in HBs-HepR model Anti-HBs could not be produced in HBs-HepR mice,and even after 3 weeks post HBsAg-vaccine vaccination,indicating that hepatocyte damage in HBs-HepR mice may not be caused by antibodies produced by B cells.Depletion of CD4 T or NK cells did not affect the HCC development in HBs-HepR mice,indicating that CD4 T cells nor NK cells played no important role on the prevalence of HCC.5.At the stage of tumor formation in HBs-HepR mice,T/NK cells in the liver are functional exhausted.Single-cell sequencing analysis was used to detect decreased expression of functional molecule IFN-y in T cells and NK cells in tumor-forming livers of HBs-HepR mice.At the same time,we also verified this phenomenon by flow cytometry.It is indicated that after the tumor occurs,the tumor microenvironment leads to the functional exhaustion of immune cells in the liver and promotes the escape of the tumor.6.HBV mouse models can also be established by using hepatocytes derived from HBV-transgenic mice.When using 1.0×106 HBV-transgenic mouse derived hepatocytes with C57BL/6J background for model construction,we can also successfully construct a HBV mouse model in which HBcAg-positive hepatocytes can be detected in the liver of HBV-HepR mice,and HBsAg,HBeAg,HBV-DNA can be detected in serum.However,HBV-HepR mice with C57BL/6J background were unable to develop tumors at 12 months post transfer naturally.