HBV-specific CD8 T cell responses are critical in viral clearance and immune-mediated liver injury (3
). CD8 T cell responses are stronger in acute HBV infection than in chronic HBV infection, and unregulated CD8 T cell response to HBV may lead to fulminant hepatitis (6
). Despite the associations between strength of CD8 T cell responses and outcome of HBV infection, it has been unclear if HBV proteins affect CD8 T cell response. In this study, we found that expression in primary hepatocytes of HBx, one of four HBV proteins, limits CD8 T cell activity via enhanced apoptosis of CD8+
T cells and reduces the production of IFN-γ an important cytokine for suppression of HBV replication in hepatocytes. Several viral proteins have been reported to suppress CD8 T cell function. Expression of structural proteins of hepatitis C virus (HCV) in hepatocytes increases apoptosis of CD8 T cells in mice (28
). Dendritic cells infected with human immunodeficiency virus (HIV) that contain viral protein R (Vpr) dysregulate CD8 T cell proliferation and induce apoptosis (29
). Our results indicate a role of HBx in immune evasion of HBV through suppression of CD8 T cell function.
Recently, CD8 T cell exhaustion has been observed in several chronic viral infections such as those caused by HIV, HCV, and HBV (9
). The phenotype of exhausted CD8 T cells is characterized by impaired proliferation and IFN-γ production, and by increased apoptosis. Presently, CD8 T cells cultured under HBx expressing conditions showed an increased frequency of apoptosis and reduced production of IFN-γ but displayed no difference in division number compared to CD8 T cells cultured with hepatocytes infected with control viruses. Also, production of IFN-γ in the presence of expressed HBx was higher than upon culture with uninfected hepatocytes. Thus, in our co-culture system, CD8 T cells seem not to have been the exhausted phenotype. However, surface expression of PD-1, CD127, CTLA4 or CD57 on T cells needs to be investigated to confirm this suggestion.
PD-L1 and PD-1 interaction suppresses IFN-γ production by CD8 T cells (32
). Furthermore, a human hepatoma cell line upregulates apoptosis of co-cultured Jurkat T cells when its PD-L1 transcription is induced by IFN-α or -γ treatment (33
). PD-L1 transcription is enhanced in primary human hepatocytes infected with adenoviruses and in HepG2.2.15, a hepatocellular carcinoma cell line in which HBV replication occurs (33
). Thus, it has been speculated that HBV-induced intrahepatic PD-L1 expression may lead to apoptosis of HBV-specific CD8 T cells expressing high levels of PD-1. However, HBx expression alone did not enhance PD-L1 transcription in HBx transgenic murine hepatocytes and, regardless of HBx expression, upregulation of PD-L1 transcription by baculovirus-infection of hepatocytes was observed. Thus, induction of PD-L1 expression seems not involved in HBx-mediated suppression of CD8 T cell activity.
Contrary to previous reports, baculovirus mediated HBx expression in hepatocytes did not presently transactivate H-2K and ICAM-1. Previous reports showed that HBx expression induces transcription of MHC and ICAM-1 in human hepatoma cell lines compared to transfection of empty vector (19
). The discrepancy may be from the method for HBx gene introduction into cells; baculovirus infection may mask the effect of HBx on induction of these genes in our system. Additionally, primary murine hepatocytes may differ from human hepatoma cell lines in response to HBx.
In conclusion, the present study reveals that HBx downregulates CD8 T cell activity through the modulation of IFN-γ production and apoptosis, and indicates the irrelevance of PD-L1, ICAM-1 and H-2K to HBx-mediated CD8 T cell suppression. Exploration of molecules involved in HBx-induced CD8 T cell apoptosis and evaluation of the suppressive role of HBx in CD8 T cell immune response in vivo should be undertaken.