Immune Netw.  2010 Aug;10(4):126-134. 10.4110/in.2010.10.4.126.

Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity

• Lee MJ
• Jin YH
• Kim K
• Choi Y
• Kim HC
• Park S

• KMID: 2150671
• DOI: http://doi.org/10.4110/in.2010.10.4.126

Abstract

BACKGROUND
CD8+ T cells contribute to the clearance of Hepatitis B virus (HBV) infection and an insufficient CD8+ T cell response may be one of the major factors leading to chronic HBV infection. Since the HBx antigen of HBV can up-regulate cellular expression of several immunomodulatory molecules, we hypothesized that HBx expression in hepatocytes might affect CD8+ T cell activity.
METHODS
We analyzed the activation and apoptosis of CD8+ T cells co-cultured with primary hepatocytes rendered capable of expressing HBx by recombinant baculovirus infection.
RESULTS
Expression of HBx in hepatocytes induced low production of interferon-gamma and apoptosis of CD8+ T cells, with no effect on CD8 T cell proliferation. However, transcriptional levels of H-2K, ICAM-1 and PD-1 ligand did not correlate with HBx expression in hepatocytes.
CONCLUSION
Our results suggest that HBx may inhibit CD8+ T cell response by regulation of interferon-gamma production and apoptosis.

Keyword

Hepadnaviridae; T-lymphocytes; Cytotoxic; Viral proteins; Apoptosis; Interferon-gamma

MeSH Terms

Apoptosis
Baculoviridae
Cell Proliferation
Hepadnaviridae
Hepatitis
Hepatitis B
Hepatitis B virus
Hepatocytes
Intercellular Adhesion Molecule-1
Interferon-gamma
T-Lymphocytes
Trans-Activators
Viral Proteins
Intercellular Adhesion Molecule-1
Interferon-gamma
Trans-Activators
Viral Proteins

Figure

• 

  Figure 1 Baculovirus-mediated HBx expression in hepatocytes. (A) HEK293 cells were transfected with a baculoviral transfer vector containing the HBx gene (pAcSG2-CMV- HBx) or with an empty control vector (pAcSG2-CMV). Western blotting was performed 48 h later on cell lysates using rabbit sera specific to HBx (upper panel) and anti-tubulin antibody (lower panel). CON denotes untransfected cells. (B) Huh7 cells were infected with recombinant baculoviruses containing HBx gene (HBx) or with control baculoviruses (CMV). Purified baculoviruses at a multiplicity of infection (M.O.I) of 1000 were added into Huh7 cell culture and 1 h later the culture medium was replaced. Cell lysates obtained 48 h later were subjected to Western blotting to detect HBx. (C) Primary murine hepatocytes were isolated and infected with recombinant baculoviruses at a M.O.I. of 1000 for 1 h. At the indicated time points after infection, total RNA was prepared from cells and DNase I treated RNA was reverse transcribed. PCR for the amplification of HBx transcript (upper and middle panels) was performed using cDNA or RNA as template. RT-PCR for beta-actin was performed (lower panel). HBx denotes hepatocytes infected with baculoviruses containing HBx gene, CMV denotes hepatocytes infected with control baculoviruses and CON denotes uninfected hepatocytes.

• 

  Figure 2 HBx expression in hepatocytes enhances apoptosis of antigen-stimulated CD8+ T cells. Primary hepatocytes isolated from C57BL/6 male mice were infected with baculoviruses at a M.O.I. of 1000 for 1 h and then the culture medium was replaced. Twenty-four hours later, purified Matahari CD8 T cells were added to cultures of primary hepatocytes. After 48 h of co-culture, CD8 T cells were harvested and the frequency of apoptotic CD8+ T cells was analyzed by TUNEL and flow cytometry. The TUNEL histograms that are displayed are gated on CD8+ cells. Culture of uninfected hepatocytes (CON), culture of hepatocytes infected with baculoviruses containing HBx gene (HBx) or with control baculoviruses (CMV) Upper and lower panels depict separate independent experiments.

• 

  Figure 3 HBx expression in hepatocytes does not modulate proliferation of antigen-stimulated CD8+ T cells. Primary hepatocytes isolated from C57BL/6 male mice were infected with baculoviruses at a M.O.I. of 1000 for 1 h and then the culture medium was replaced. Twenty-four hours later purified Matahari CD8 T cells labeled with CFSE were added without IL-2 (A) or with IL-2 (B) to cultures of primary hepatocytes. After 48 h of co-culture, CD8 T cells were harvested and analyzed by flow cytometry. CFSE histograms are gated on CD8+ cells. Culture of hepatocytes infected with baculoviruses containing HBx gene (HBx) or with control baculoviruses (CMV)

• 

  Figure 4 HBx expression in hepatocytes reduces IFN-γ production in co-culture with antigen-stimulated CD8+ T cells. Primary hepatocytes isolated from C57BL/6 male mice were infected with baculoviruses at a M.O.I. of 1000 for 1 h and then the culture medium was replaced. Twenty-four hours later purified Matahari CD8 T cells were added and 48 h later the supernatant was obtained from the co-culture, culture of hepatocytes (hepatocyte only) and culture of purified Matahari CD8+ T cells (Lympho). The IFN-γ concentration was determined using ELISA. The data is mean of two independent experiments. HBx denotes hepatocytes infected with baculoviruses containing HBx gene, CMV denotes hepatocytes infected with control baculoviruses and CON denotes uninfected hepatocytes.

• 

  Figure 5 Baculoviral infection rather than HBx expression increases the mRNA levels of H-2K and ICAM-1 in hepatocytes. Primary hepatocytes isolated from C57BL/6 male mice were cultured for the indicated time as uninfected (CON) or infected either with baculoviruses containing HBx gene (HBx) or with control baculoviruses (CMV) at a M.O.I. of 1000 for 1 h and then the culture medium was replaced. After culture for the indicated time relative mRNA levels of H-2K (A) and ICAM-1 (B) were analyzed using semi-quantitative RT-PCR. Band intensity of PCR product is normalized to that of β-actin in each sample.

• 

  Figure 6 Baculoviral infection rather than HBx expression increases the PD-L1 mRNA levels in hepatocytes. Primary hepatocytes were isolated from C57BL/6 male mice (A and normal in B) and from HBx transgenic mice (transgenic in B). Hepatocytes were cultured for the indicated time. Relative mRNA levels of PD-L1 were analyzed using semi-quantitative RT-PCR. Band intensity of PCR product is normalized to that of β-actin in each sample. Data represents the mean±standard deviation of three independent experiments. In A HBx denotes hepatocytes infected with baculoviruses containing HBx gene, CMV denotes hepatocytes infected with control baculoviruses and CON denotes uninfected hepatocytes.

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