Yonsei Med J.  2005 Dec;46(6):827-834.

Expression of Hepatitis C Virus Core Protein in Hepatocytes Does Not Modulate Proliferation or Apoptosis of CD8+ T Cells

• Jin YH
• Crispe IN
• Park S

Abstract

Hepatocytes are the primary targets of the hepatitis C virus (HCV). While immunosuppressive roles of HCV core protein have been found in several studies, it remains uncertain whether core protein expressed in hepatocytes rather than in immune cells affects the CD8+ T cell response. In order to transduce genes selectively into hepatocytes, we developed a baculoviral vector system that enabled primary hepatocytes to express a target epitope for CD8+ T cells, derived from ovalbumin (OVA), with or without HCV core protein. Culture of OVA-specific CD8+ T cells with hepatocytes infected with these baculoviral vectors revealed that core protein has no effect on proliferation or apoptosis of CD8+ T cells. Our results suggest that HCV core protein does not exert its suppressive role on the CD8+ T cell immune response through expression in hepatocytes.

Keyword

Hepacivirus; T cells; cytotoxic; viral core proteins; baculoviridae; co-culture techniques

MeSH Terms

Viral Core Proteins/*metabolism
Ovalbumin/genetics/immunology
Mice
Hepatocytes/cytology/*virology
Green Fluorescent Proteins/genetics
Genetic Vectors
Cell Proliferation
CD8-Positive T-Lymphocytes/*immunology
Baculoviridae/genetics
Apoptosis
Animals

Figure

• 

  Fig. 1 Baculovirus-mediated gene expression in hepatocytes. (A) Schematic representation of a baculoviral vector expressing HCV core and EGFP-OVA. (B) Primary mouse hepatocytes were observed after 24 h of infection with baculovirus expressing EGFP. (C) Liver cells infected with baculovirus expressing EGFP were incubated with DiI AcLDL (red) for the final 16 hours. Images from a fluorescence tissue culture microscope were merged using Adobe PhotoShop. (D) Hepatocytes infected with baculovirus expressing EGFP alone or EGFP-OVA were stained with Ab specific to the H-2Kb-OVA peptide complex and PE-conjugated secondary Ab (lower panels). (E) PCR was performed using DNase-treated total RNA or cDNA prepared from hepatocytes infected with baculovirus expressing HCV core protein. (F) Extracts of baculovirus-infected hepatocytes were subjected to SDS-PAGE, and HCV core protein was detected by immunoblotting with a specific rabbit antiserum. HCV, hepatitis C virus; EGFP-OVA, enhanced green fluorescent protein-ovalbumin.

• 

  Fig. 2 HCV core protein does not affect proliferation of CD8+ T cells. (A) Comparable levels of infection were demonstrated after 24 h of hepatocyte infection with baculoviruses expressing EGFP alone, EGFP-OVA, and HCV core-EGFP-OVA. (B) CFSE-labeled OT-1 CD8+ T cells were co-cultured with hepatocytes infected with baculovirus or pulsed with OVA peptide. Two days later, T cells were collected and analyzed by FACS. CFSE histograms of OT-1 T cells are gated on CD45.1+ CD8+ cells. Data represent three separate experiments.

• 

  Fig. 3 HCV core protein does not affect apoptosis of CD8+ T cells. OT-1 CD8+ T cells were added to hepatocyte cultures expressing EGFP-OVA with or without HCV core protein. CD8+ T cells were collected, stained, and analyzed by FACS 60 h after co-culture. TUNEL histograms of OT-1 T cells are gated on CD45.1+ CD8+ cells. Data represent three separate experiments.

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