Abstract
Background: Hepatitis B virus (HBV) constitutes a major global health burden. Previously study has found the expression of programmed cell death 1 (PD-1) is up-regulated during the chronic HBV infection. However, the mechanism of how HBV infection modulates the expression of PD-1 on CD8+T cells are not well understood. In the present study, we aimed to analyzed the role of DNA methylation in regulating the expression of PD-1 on CD8+T cells during HBV infection, we also aimed to evaluate the HBV induced changes of tumor microenvironment (TME).Methods: The methylation microarray was used to assess the profile changes of gene methylation upon chronic HBV infection. CD8+T cells were separated from peripheral blood of health volunteers and HCC patients including HBV related HCC (HBV-HCC) and non-viral HCC. The immune microenvironments of hepatocellular carcinoma (HCC) was interrogated by using Immunofluorescence staining. The PD-1 expression of CD8+T cells from peripheral blood were examined by western blot and flow cytometry. T cells function was determined by cytokine measurement. Sequenom MassARRAY platform was used to detected the DNA methylation status of PD-1 promoter.Results: HBV could drive 413 genes methylated while 3,023 genes including PD-1 demethylated, the reduced PD-1 expression and increased PD-1 demethylation was proved in HBV-HCC tissue. The subsequent analysis indicate that the expression of PD-1 differed by stages of HBV infection, it reduced on the cell membrane of HBV-transfected CD8+T cells upon transient HBV plasmid transfection which might mimic acute HBV infection, while upregulated on the HBV specific CD8+T cells by demethylation during chronic HBV infection. Interferon-gamma increased in the medium of HBV transfected CD8+T cells and interleukin-10 increased in the blood of chronic HBV infection patients. Increased tumor associated macrophage (TAM) cells and T regulatory cells (Tregs) invasion was identified in HBV-HCC tissue when compared with those from non-viral HCC tissue Conclusion: Our data suggested that the expression of PD-1 varies upon the stage of HBV infection, chronic HBV infection could drove the TME more immunosuppressive through PD-1 demethylation related CD8+T cells exhaustion as well as promotion of invaded Treg and TAM cells thereby to attenuating therapy of immune checkpoint inhibitors.
Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma
