Chronic HBV Infection Induces The Exhaustion Of Hepatic CD8~+T Cells And Related Mechanisms

ObjectiveThe liver is the largest internal organ in the body and undertakes the function of metabolism and detoxification.The liver also serves as a hematopoietic organ in embryo.Meanwhile,liver is also a special immune organ.It is rich in natural immune cells and adaptive immune cells,including medullary cells such as Kupffer cells,neutrophils and macrophages as well as lymphocytes such as NK,CD4+T and CD8+T cells,which are widely involved in natural and adaptive immunity.As an immune organ,the liver has the characteristic of immune tolerance.The immune tolerance of liver is related to its physiological structure and cell component.Firstly,the liver has a rich blood supply,and the blood volume entering the liver through the portal vein every minute accounts for about 30%of the whole body.It is rich in a large number of food components and intestinal pathogenic microorganisms,which continuously stimulate the immune cells in the liver.Secondly,the structure of hepatic sinus is narrow,the blood flow is abundant but slow,which provides the basis for cell interaction.Finally,liver aggregates a variety of APC that induce tolerance.Maintaining normal tolerance state of the liver is not only important for the physiological function of liver,but also for the overall health.Immune exhaustion refers to the low function of immune cell to remove pathogens effectively.It is a common phenomenon in tumor microenvironment and chronic virus infection.Exhausted immune cells are functionally impaired and highly express inhibitory receptors,which are not conducive to the recognition and clearance of tumor cells and virus.HBV,as a hepatophile virus,mainly invade hepatocytes after entering the body.Most adults can produce effective antiviral immunity and form self-limited acute infection.Infants,young children and some adults will gradually form chronic infection.It has been reported the HBV-specific CD8+T cells are exhausted.Chronic HBV infection continuously stimulates liver immune cells,aggravating liver immune tolerance and even systemic immune tolerance.Understanding the functional status of immune cells in the liver and whether immune exhaustion occurs is very important for reversing the impact of chronic HBV infection on liver immune tolerance.Regulation mechanisms of immune exhaustion are diverse and complex.At present,in the LCMV chronic infection model,it has been found that the mechanisms regulating exhaustion include transcription factors,inhibitory cytokines,metabolism and so on.Comparatively,the regulation mechanisms of exhaustion are not very clear in HBV chronic infection.Understanding the mechanism that induces immune exhaustion is crucial for reversing liver tolerance caused by chronic HBV infection and restoring liver immune cell effector function to clear viral infection.For a more comprehensive understanding whether immune exhaustion happened of liver immune cells during chronic HBV infection and the regulatory mechanism of immune exhaustion,this article carried out research from the following aspects.Firstly,chronic HBV infection mouse model was constructed to detect the effector function of liver CD8+T,CD4+T and NK cells in the liver of mice.It was found that CD8+T cells were significantly exhausted,and the effector function of CD4+T and NK cells in the liver was also inhibited.Secondly,it was found from the regulatory mechanism that chronic HBV infection inhibited the glycolysis level of CD8+T cells in the liver,and further found that mTOR signaling activity,which plays an important regulatory role in glycolysis,was impaired.On the other hand,impaired mTOR activity up-regulated FoxO1,a transcription factor related to the expression of inhibitory receptors,further promoting the exhaustion of CD8+T cells in the liver.Finally,we observed that mTOR agonist treatment in vitro could restore the effector function of exhausted CD8+T cells to some extent.Our results preliminarily revealed part of the regulatory mechanism of CD8+T cell exhaustion in liver caused by chronic HBV infection.Methods1.Chronic HBV infected mouse model was constructed by tail vein hydrodynamic injection with HBV plasmids and screened out through testing serum HBsAg level.2.Flow cytometry was used to analyze IFN-y and TNF-a secretion of liver CD8+T,CD4+T and NK cells from WT and HBV chronic infected mouse.3.Flow cytometry was used to analyze the expression of inhibitory receptors on liver CD8+T,CD4+T and NK cells from WT and HBV chronic infected mouse.4.Seahorse XFe96 was used to analyze the glycolysis of liver CD8 T cells.5.Phosphorylation of S6 ribosomal protein(Ser235/236)of liver CD8 T cells was measured by intracellular flow cytometry to evaluate mTOR activity.6.qPCR was used to analyze the mRNA level of transcription factors associated with immune exhaustion in liver CD8 T cells.Results1.Compared with WT mice,the ability of liver CD8+T,CD4+T and NK cells in chronic HBV infected mice to secrete IFN-y was impaired.2.Compared with WT mice,CD8 T,CD4 T and NK cells in the liver of chronic HBV infected mice highly expressed a variety of inhibitory receptors,among which CD8 T cells increased most obviously.3.Chronic HBV infection promoted CD8+T cells in liver to co-express PD-1,lag-3,CD 160 and 2B4.4.Compared with WT mice,the glycolysis of liver CD8 T cells from chronic HBV infected mice was inhibited.5.The phosphorylation of S6 ribosomal protein was low,and the activity of mTOR signal was inhibited.6.mRNA level of FoxO1 of liver CD8+T from chronic HBV infected mice was significantly higher than that of WT mice.7.The activity of mTOR signal was recovered,and the secretion of IFN-γ was increased of liver CD8 T cells after agitation with MHY1485 in vitro.8.mRNA level of FoxOl was decreased after agitation with MHY1485 in vitro.Conclusions1.CD8 T cells in the liver of chronic HBV infected mice were exhausted and characterized as impaired ability to secrete IFN-γ and high/co-expression of PD-1,Lag-3,CD 160 and 2B4.2.Chronic HBV infection inhibited Glycolysis of CD8 T cells in liver,resulting in abnormal energy supply and mTOR signal was inhibited.3.Chronic HBV infection up-regulated FoxO1 expression in CD8 T cells in liver.4.Exhaustion of CD8 T cells was reversed after agitation with MHY1485 in vitro.