The Differential Regulation Of Tim-3Molecule On The Function Of CD8~+T Cells And Monocytes During The Progression Of Chronic HBV Infection

Background and aims:Chonic hepatitis B virus (HBV) infection is a major threat to global public health. Chronic hepatitis B (CHB) and HBV-related cirrhosis are two sequential stages when chronic HBV infection progresses. CHB is characterized by impaired virus-specific adaptive immune response, while patients with severe HBV-related cirrhosis further represent dissonance of innate immune function. It is not clear about the molecular mechanism underlying the functional alterations of adaptive and innate immune response during the different stages of chronic HBV infection. T cell Immunoglobulin domain and Mucindomain protein-3(Tim-3) was a newly identified immune regulatory molecule. Tim-3plays a comprehensive role in the modulation of immune response, as tt was expressed on the surfaces of both adaptive (CD4+, CD8+T cells and et al) and innate immune cells (monocytes, dendritic cells, mast cells and et al). The study was aimed to investigate the functional status of CD8+T cells and monocytes in CHB and HBV-related cirrhosis patients and the role of Tim-3in the regulation of CD8+T cell and monocyte function, which would deepen our understanding on the immunological characeristics of the progression of chronic HBV infection and shed light on development of novel therapy. Methods:1. Tim-3regulates CD8+T cell function: We collected peripheral blood from125patients with CHB and54cases of healthy volunteers and determined Tim-3expression on on the surface of total CD8T cell by flow cytometry. CD8+T cells were isolated by magnetic separation and Tim-3expression on HBV-specific CD8+T cells were measured by pentamer techonology. The phenotvpe(?)nd cytokine secretion capacity were compared between Tim-3+/-CD8T cells by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with HBV antigen peptide,12-cardamom phorbol acetate acid-13-(phorbol12-myristate13-acetate, PMA)/ionomycin and anti-CD3antibodies respectively in the presence of Tim-3pathway blocking. Cytokines in the supernants were detected by ELISA, intracellular interferon-γ (IFN-γ) secretion was measured by flow cytometry and CD8+T cell proliferation was determined using carboxyflu-orescein diacetate succinimidyl ester (CFSE).2. Tim-3regulates monocyte function:We collected peripheral blood from79patients with HBV-related cirrhosis and31cases of healthy volunteers and determined Tim-3expression on the surface of total CD14+monocytes by flow cytometry. Plasma endotoxin of patients and healthy volunteers was measured using chromogenic substrate method. Monocytes were isolated by magnetic separation and stimulated with lipopolysaccharide (LPS), then dynamic Tim-3expression was determined. The phenotypes were compared between Tim-3+/-CD14+monocytes by flow cytometry. PBMCs were isolated and stimulated with LPS in the presence of Tim-3pathway blocking. Flow cytometry was performed to evaluate phagocytosis capacity, cytokine production and HLA-DR expression and inducible nitric oxide synthase (iNOS) secretion was determined by ELISA.Results:1. Tim-3regulates CD8+T cell function: (1) Tim-3-expression was expressed at significantly higher level on CD8+T cells in CHB patients in relative to health controls. Tim-3-expression on CD8+T cell positively correlated with ALT. Tim-3expression decreased remarkedly after antiviral therapy.(2) Tim-3expression on HBV-specific CD8+T cells was significantly higher than on CMV-specific CD8+T cells in CHB patients.(3) Tim-3+CD8+T cells had higher proportion of terminally differentiation effector memory (TDEM) cells but lower proportion of naive cells than Tim-3-CD8+T cells.(4) CD127was expressed at lower level on Tim-3+than Tim-3"CD8+T cells, while CD57was expressed at higher level. Tim-3+CD8+T cells produced less IFN-γ than Tim-3-CD8+T cells upon antigen challenge.(5) Tim-3signalling blocking significantly restored antiviral cytokine production and proliferation of virus-specific CD8+T cells, but did not improve response to non-specific antigen stimulation.2. Tim-3regulates monocyte function:(1) Tim-3expression was expressed at lower level in patients with severe decompensated HBV-related cirrhosis in relative to health controls.(2) The deccline of Tim-3expression on monocytes was related to endotoxemia in cirrhotic patients. In vitro LPS stimulation could induce decrease of Tim-3expression on monocytes.(3) Tim-3+CD14+monocytes expressed higher CD1a and CD83but lower CD80and CD86compared with Tim-3-cells.(4) Monocytes from patients with severe decompensated HBV-related cirrhosis represented reduced phagocytosis, lower HLA-DR expression but higher iNOS serection, and elevated production of both pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10).(5) Tim-3signalling blocking significantly inhibited monocyte phagocytosis, reduced the level of iNOS secretion, down-regulated TNF-α production and up-regulated the level of IL-10, but have no effect on the expression of HLA-DR. Conclusions:(1) Tim-3pathway negatively reglulated HBV-specific CD8+T cell response in CHB patients.(2) Monocytes displayed immunological dissonance in patients with decompensated HBV-related cirrhosis, the extent to which correlated with the severity of cirrhosis. The decline of Tim-3expression led to the evolution of functional status of monocytes from pro-inflammatory phenotype to anti-inflammatory phenotype.