PD-1/PD-L Costimulatory Pathway In The Pathogenesis Of Chronic Hepatitis B Virus Infection

Viral hepatitis B is a common and serious infectious liver disease caused by the hepatitis B virus (HBV). Approximately 400 million patients infected chronically and develop chronic liver inflammation leads to cirrhosis and hepatocellular carcinoma. Mechanism of virus persistent remains disputable.It is now widely accepted that CD8+ CTL immune response mediates clearance of HBV. But most chronic HBV infected patients show a severe impairment of HBV-specific T-cell function presented by low levels of antiviral cytokines and an impaired cytotoxic T lymphocytes (CTLs) activity. The mechanisms responsible for the T-cell dysfunction in chronic HBV infection are not completely understood.Co-signalling molecules are essential for the communication of a T cell with virtually all other host cells. Programmed death 1 (PD-1) is a co-inhibitors of the CD28 family and its two ligands, PD-L1 and PD-L2, show distinct roles in regulating the immune responses. PD-1 ligation down-regulates TCR-mediated signaling and it is well documented that PD-1/PD-L1 pathway plays a negative role in regulation of T cell activation, proliferation, and cytokine production. In 2006, Barber reported that PD-1 was upregulated in"exhausted"T cells during the chronic phase of lymphocytic choriomeningitis virus (LCMV) infection in mice models. Blocking the interaction between PD-1 and its ligand could reactivate these T cells and significantly reduce virus load. So far, many evidences demonstrated an important role of the PD-1 pathway in inhibiting virus-specific CD8+ T-cell function in chronic viral infections including HIV, HCV and HBV.But a good interpretation of PD-1/PD-L pathway needs an in depth understanding of the detailed expression pattern of PD-1 on T cells and an overall message of PD-L express. It is still unclear in chronic HBV infection. In this regard, we analyzed PD-1 expression on HBV specific CTL in relation to cellular activation, differentiation and function. And we also assessed PD-L expression on PBMC and in liver of chronic HBV infected individuals.By FACS analysis and pentamer staining, we found that PD-1 expression on HBV specifc CTL is significantly higher than on CMV specific CTL and on total CD8+T cells from the same chronic hepatitis B (CHB) infected individual. Then, we assessed the relationship between PD-1 expression, plasma viral load, and ALT levels. There was no correlation between PD-1 expression and plasma ALT levels. In contrast, significantly positive correlation was found between PD-1 expression and plasma viral load. These data indicate that a large amount of viral antigen in chronic HBV infection increase the expression of PD-1 on HBV specific CTL.To clarify whether PD-1 expression could effect the activation, differention and antivirus potency of HBV specific CTL, PD-1 expression (as percentage of PD-1 positive cells) was therefore assessed on 5 differentiation subsets clarified by CD27, CD45RA and CCR7. We observed significant differences in PD-1 expression between the distinct CTL subsets along the differentiation pathway. While CCR7+ CD27+CD45RA+ naive CD8+ T cells exhibit no or little PD-1, it is up-regulated after antigenic priming (CD45RA-) and expressed particularly on CD8+ T cells at intermediate stage of differentiation (CCR7- CD27+CD45RA-). However, its expression decreases as cells reach late stages of differentiation (CCR7-CD27- CD45RA+). This suggests a direct link between the level of differentiation and the expression of PD-1 on CTL. HBV specific CTL are usually found in the mid-half or midanaphase of the differentiation pathway and exhibit high PD-1 expression, thus receive strong inhibit from PD-L.We also found PD-1+CTL expressed high levels of activation markers such as CD38 and HLA-DR in CHB patients. ICS staining shows that HBV specific CTL express higher levels of Granzyme B,but low levels of perforin and IFN-γ, Which are effective antivirus molecules in CTL. These results indicate an activation but functional exhaustion status of specific CTL in CHB patients.Not only the amount of PD-1 expression but also the extent of engagement of PD-1 by its ligands regulates the threshold for T cell activation and quantities of cytokines produced. Thereby, we measured PD-L1 and PD-L2 expression by flow cytometry on CD3+, CD14+ or CD19+ cells in CHB patients and health controls. Data shows that PD-L1 expression is significant increased on CD14+ monocyte in CHB patients and there is significiant positive correlation between PD-L1 expression on CD14+ monocyte and plasma ALT levels.Which indicate that PD-L1 expression is upregulate by inflammation, maybe to prevent immuno- pathogenic damage.Whether PD-L1 expression on monocytes would inhibit HBV specific CTL in CHB patients or not? And would PD-1/PD-L1 pathway blockade rescue specific CTL? We block PD-1/PD-L1 interaction with a blocking antibody to PD-L1. Data by CFSE proliferation assay shows that PD-1/ PD-L1 blockade promote specific CTL clonal expansion. And a significiant increase in IFN-γ-producing specific CTL was seen in PBMC incubated with anti-PD-L1 compared to PBMC incubate with isotype control antibody. This data indicates that PD-1/PD-L1 pathway play a crucial role in specific CTL dysfunction in peripheral blood in CHB infection and blockade of this pathway will reactivate specific CTL.Liver is the sufferer organ of HBV infection. Hepatic tolerance may also contribute to the ineffectiveness of immune responses against HBV and result in viral persistence. Thus we analyzed PD-1/PD-L pathway in CHB infected liver. By immunohistochemical staining, we found that PD-1 was positively stained on inflammatory cells infiltrating the portal area and the sinusoidal area of CHB infected liver, while no significant expression in normal liver. PD-1 was found to express not only on T cells but also on B cells by immunofluorescence staining. PD-L1 positive signal was frequently observed not only on infiltrating cells but also in the sinusoidal area. And PD-L1 significantly expressed on Kupffer's cells, dendritic cells, and liver sinusoidal epithelial cells (LSEC), being especially significant on sinusoidal epithelial cells. Furthermore, the level of PD-L1 expression on liver residential antigen presentation cells were significantly correlated to the extent of inflammation damage and the number of infiltrating cells in CHB liver. On the other hand, we found no significant increase in PD-L2 expression in CHB liver compared to normal.We found that PD-1 is significant upregulate on HBV specific CTL while its ligand PD-L1 is also highly expressed on peripheral blood and liver residantal antigen presenting cells, and blockade of PD-1/PD-L1 pathway will reactivate specific CTL. We hope such findings could one day lead to the clinical manipulation of PD-1/PD-L1 pathway for the treatment of chronic HBV infection.