PD-1 Expression And Significance In Immune Tolerant Phase And Immune Clearance Phase Of HBV Infection

Objective: In accordance with immune characteristics, chronic hepatitis B virus (HBV) infection can be clinically categorized into three periods, namely immune tolerant phase, immune clearance phase, and immune stable phase or inactive carrier phase. The mechanisms responsible for the persistent HBV infection are not clear. The impairment of HBV-specific T cell function may be one of the important factors. It is widely accepted that negative co-stimulatory signal plays a very important role in T cell function defect. Recent data has shown that the programmed death 1 (PD-1) and its ligand 1 (PD-L1) is an important negative co-stimulatory signal, they correlated with CD8~+T cells inactivation and viral infection persistence. Recent studies have shown that in patients with HBV infection, expression levels of PD-1 on the virus specific CD8~+T cells increased. Blocking PD-1/PD-L1 pathway can improve T cell function. Although reports are available on the changes in expression levels of PD-1 and T-cell responses in patients with HBV infection, the change pattern of PD-1 expression in the immune tolerant phase and immune clearance phase of chronic HBV infection has not yet been presented. This study aimed to investigate PD-1 expression and significance in immune tolerant phase and immune clearance phase of HBV infection.Methods: All of 105 chronic HBV infection subjects including 55 patients in immune clearance phase and 50 patients in immune tolerant phase were enrolled in this study. All patients underwent liver biopsy, inflammation graded as G0~G4, fibrosis was divided into S0~S4. 15 healthy persons were enrolled in this study.1 Fresh peripheral blood were collected from all of the subjects. PBMC were isolated by separating medium of lymphocytic cell. The PBMC was stained with anti-PD-1 FITC and all these cells analysised with flow cytometry. And the flow cytometry was also used to detect the HLA typing.2 PBMC were isolated from 10 HLA-A2-positive normal subjects, 30 patients in immune clearance phase and 30 patients in immune tolerant phase by separating medium of lymphocytic cell. The PBMC was stained with pentamer-PE, anti-CD3 ECD, anti-CD8 PE-Cy5, anti-PD-1 FITC and all these cells analysised with flow cytometry. The amounts of HBV-specific CD8~+T cells and their PD-1 expression were detected.Results:1 The change of PD-1 expression on PBMC and HBV-specific CD8~+T cells in different stages of chronic HBV infection patients: The PD-1 expression levels on PBMC and HBV-specific CD8~+T cells were significantly higher in the immune tolerant phase than in normal control group and in the immune clearance phase of chronic HBV infection patients (P<0.05 or P<0.01).2 The change of the frequency of HBV-specific CD8~+T cells in different stages of chronic HBV infection patients: Compared to the immune tolerant phase and normal control group, there was a higher frequency of HBV- specific CD8~+T cells in the immune clearance phase of chronic HBV infection patients (P<0.01).3 The correlation between HBV-specific CD8~+T cell levels and PD-1 expression levels was analyzed, which showed that the HBV-specific CD8~+T cell level was inversely correlated with PD-1 (r=-0.463, P<0.01).4 The correlation between PD-1 expression on HBV-specific CD8~+T cells and HBV viral load: The patients of chronic HBV infection were divided into three groups (<3 log10, 3-6 log10 and >6 log10) according to their HBV DNA levels. PD-1 expression on HBV-specific CD8~+T cells in >6 log10 group was significant higher than those in <3 log10 group and 3-6 log10 group (F=82.94, P<0.01). In this study, there was a significant positive correlation between HBV viral load and PD-1 expression on HBV-specific CD8~+T cells of chronic HBV infection patients (r=0.882, P<0.01).5 The correlation between PD-1 expression on HBV-specific CD8~+T cells and serum ALT levels in immune clearance phase: The patients in immune clearance phase were divided into three groups according to their ALT levels. We used 3 times and 10 times the upper limit of normal ALT level (3×ULT, 120 IU/L, 10×ULT, 400 IU/L) as the cut-off value in dividing the patients. No significant difference among the three groups (P>0.05). Further analysis showed that there was no correlation between PD-1 expression levels on HBV-specific CD8+T cells and serum ALT levels in the study patients (r=0.085, P>0.05).Conclusion:1 Compared to the immune clearance phase, there was a lower frequency of HBV-specific CD8+T cells in the immune tolerant phase of chronic HBV infection patients. It means that HBV-specific CD8+T cells play an important role in HBV clearance.2 The expression of PD-1 on HBV-specific CD8+T cells in the immune tolerant phase was higher than those of the immune clearance phase of chronic HBV infection patients. It means that the up-regulation of PD-1 may affects the activation of HBV-specific CD8+T cells.3 The HBV-specific CD8+T cell level was inversely correlated with PD-1. It means that PD-1 influence the progression of the disease by inhibiting the activation of HBV-specific CD8+T cells. Up-regulation of PD-1 leads the inactivation of T cells may be one of the mechanisms of immune tolerance in chronic hepatitis B.