Study Of The Relationship Between LAG-3Inhibitory Costimulatory Molecules And Chronic HBV Infection

Background and AimsLymphocyte activation gene-3(LAG-3) is an independent negative immune regulatory molecule, having function in homeostasis maintenance and immune regulation. It’s closely related with chronic viral infection caused by depletion of CD8+T lymphocyte. The main pathogenesis of chronic hepatitis B is a pathological immune response. Persistent infection with HBV is related to HBV-specific CD8+T lymphocyte responses dysfunction. This paper mainly investigate the relationship between LAG-3and the disease progression of chronic hepatitis B, providing new ideas for seeking new antiviral treatment strategies. Material and Methods1MaterialA total of90patients with chronic HBV infection were enrolled during Feb.2012to Jun.2013in the First Affiliated Hospital of Medical College, Zhejiang University. All patients were divided into two groups by hepatitis B clinical diagnostic standard as chronic hepatitis and chronic asymptomatic HBV carriers, and18healthy individuals as control.2Methods1) Separation of peripheral blood mononuclear cells (PBMC):PBMCs were separated from whole blood samples of fasting patients.using Ficoll.2) Detection of the CD223on CD8+T lymphocyte CD223(LAG-3) expression on CD8+lymphocyte was detected by flow cytometry3) Detection of the intracellular IFN-y the different secretion levels of the intracellular IFN-yinCD8+Tlymphocytes with or without LAG-3were identified by flow cytometry, after rupture of membranes and staining.4) The effect ion of LAG-3antibody blocking to the secretion of IFN-y on lymphocyte. ELSPOT5) Virological assessments:HBsAg,anti-HBs,anti-HBc,HBeAg and anti-HBe were tested by using chemiluminescent microparticle immunoassay kits(Abbot Laboratories,USA). Serum HBV-DNA was quantified by fluorescent quantify assay with DNA detection limit of103copies/ml.6) ALT assay:Serum ALT was tested using HITACHI7600-110auto biochemical analysis system. 7) Statistic alanalysis:SPSS13.0statistic software and GraphPad Prism5software.Results1) The frequency distribution of CD223on CD8+lymphocyteof chronic hepatitis B group was significantly higher than the normal control group and chronic HBV carriers group(P<0.01).2) The frequency distributions of CD223on CD8+T lymphocyte among chronic hepatitis B patients with high ALT low DNA and high ALT high DNA were both significantly higher than the normal control group(P<0.05); But The frequency distribution of CD223on CD8+T lymphocyte among chronic hepatitis B patients with low ALT low DNA and low ALT high DNA had no statistical significance compared to normal control group.3) The frequency distribution of CD223on CD8+lymphocyte with the group of chronic hepatitis B and chronic HBV carriers had obvious positive correlation with the level of corresponding ALT(P=0.03, r=0.23).4) Among the patients with chronic hepatitis B in PBMC, the frequency distribution of IFN-γ+in CD8+CD223-cells was significantly higher than that in CD8+CD223+cells.(P=0.02)5) Among the PBMCs of patients with chronic hepatitis B, the spot forming cells which were stimulated by HBcAg with CD223antibody was significantly higher than that stimulated by HBcAg(p<0.01), HBcAg with IgG(P<0.01) and HBcAg with PD-Ll(p=0.01). Conclusion1) LAG-3expression level was significantly increased on CD8+T cells in peripheral blood of chronic hepatitis B patients, and it had positive correlated with the degree of liver inflammation and irrelevant with HBV DNA load2) The function of CD8+T cells in chronic hepatitis B patients with high expression of LAG-3was impaired, characterized by the level of the secretion of cytokine IFN-γ decline.3) The function of CD8+T cells in chronic hepatitis B patients was recovered by blocked with LAG-3antibody,and blockade of CD223and PD-1seemed to be superior to blockade of PD-1alone for the recovery of the CD8+T cells function.