Apoptosis of activated CD4+ and CD8+ T cells is enhanced by co-culture with hepatocytes expressing hepatitis C virus (HCV) structural proteins through FasL induction - PubMed (original) (raw)

Apoptosis of activated CD4+ and CD8+ T cells is enhanced by co-culture with hepatocytes expressing hepatitis C virus (HCV) structural proteins through FasL induction

Khadija Iken et al. Virology. 2006.

Abstract

A central unresolved issue in hepatitis C virus (HCV) infection is how the virus establishes chronic infection. Recent studies suggest that the liver microenvironment leads to apoptosis of activated T cells, which may be involved in the tolerance to liver allograft. Here, We report that murine hepatocytes expressing a transgene encoding the HCV structural proteins core, envelope 1 (E1) and envelope 2 (E2) enhance apoptosis of activated T cells. Unlike normal liver, which appears to selectively remove only activated CD8+ T cells, enhanced apoptosis was seen for both CD4+ and CD8+ T cells. Enhanced apoptosis of activated T lymphocytes was associated with upregulation of FasL by HCV transgenic hepatocytes and was specifically inhibited by anti-FasL blocking antibody. Increased apoptosis of activated T cells induced by HCV structural proteins could amplify the ability of the liver to down-modulate T cell responses, leading to attenuation of anti-viral responses and facilitating viral persistence.

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Figures

Fig. 1

FACS analysis of apoptosis in CD4+ and CD8+ T cells. In this representative experiment, FACS analysis was performed on activated T cells after staining with FITC-conjugated anti-CD4 or anti-CD8 monoclonal antibody, PE-conjugated annexin V and propidium iodide (PI), 15 h after co-culture with HCV transgenic (Tg+ HC) or non-transgenic (Tg− HC) hepatocytes. Shown is a representative FACS profiles after gating on CD4+ (upper panel) or CD8+ (lower panel) T cells. A representative experiment is shown.

Fig. 2

Increased apoptosis of activated CD4+ and CD8+ T cells after co-culture with transgenic hepatocytes expressing HCV core, E1 and E2 proteins. Apoptosis of activated T cells was studied by 3-color FACS analysis, 4 or 15 h after co-culture with HCV transgenic (Tg+, stippled) or non-transgenic (Tg−, open) hepatocytes. (A) T cells in the early phase of apoptosis (annexin V-positive, PI-negative) are shown. Increased apoptosis of activated T cells after co-culture with HCV transgenic (HCV Tg+) compared to non-transgenic (HCV Tg−) hepatocytes was seen at both 4 and 15 h and was seen for both CD4+ (circles) and CD8+ (squares) cells. Data of five to eight independent experiments were analyzed by Mann–Whitney U test. (B) The relative increase of early apoptotic cells increases after co-culture with Tg hepatocytes. In this example, data for Tg hepatocytes were normalized to the appropriate Tg− control. Results for both CD4+ and CD8+ remain statistically significant at 4 and 15 h. (C) All annexin V+ cells, showing increased apoptosis of both CD4+ and CD8+ T cells at 15 h, and an increase in CD4+ AV+ cells at 4 h. (D) Relative increase in all AV+ cells.

Fig. 3

Increased expression of Fas ligand in HCV transgenic hepatocytes expressing structural proteins. (A) Cell lysates (200 μg) were subjected to Western blot analysis using specific polyclonal rabbit anti-mouse Fas ligand antibodies. The level of cellular murine beta actin is shown as an internal control for comparison. One representative experiment is shown. (B) Induction of mRNA for FasL by HCV structural proteins. Whole-liver tissue from Tg− and Tg+ mice was harvested; RNA was extracted and analyzed by reverse transcription PCR for FasL transcripts. Results for GAPDH mRNA are shown as controls. Results are a representative experiment of 3 individual experiments. (C) Differences in surface expression of FasL analyzed by flow cytometry. Freshly isolated HC were stained with 0.5 μg/ml FasL. Tg+ HC (thick line) had increased expression of FasL compared to Tg− HC (dashed line), which had only minimal increase compared to the isotype control (thin dashed line). One representative experiment of three is shown. (D) Activated lymphocytes show no difference in surface FasL expression after co-culture with either Tg− or Tg+ hepatocytes.

Fig. 4

HCV induces apoptosis through Fas–FasL cascade. (A) Anti-Fas ligand antibodies inhibit enhanced apoptosis of activated CD4+ and CD8+ T cells induced by transgenic hepatocytes expressing HCV core, E1 and E2 proteins. Apoptosis of activated T cells was studied by 3-color FACS analysis 15 h after co-culture with HCV transgenic hepatocytes (Tg+ HC), in the presence of 10 μg/ml rabbit anti-mouse Fas ligand polyclonal antibodies (Ab) or purified rabbit IgG (IgG) as an isotype control or media (Med). Results are based on binding of annexin V after gating on viable CD4+ and CD8+ cells. Shown are means and standard deviations of 5 experiments.

Fig. 5

Increased apoptosis of activated T cells after adoptive transfer into HCV Tg mice. Cells were activated with conA and given via intraportal injection as in the text. The degree of apoptosis in CFSE+ cells by AV staining was determined 16 h following adoptive transfer. Median values and intraquartile ranges (IQR) of 3 independent experiments with 2–3 mice in each group are shown are shown. (B) HCV Tg+ or non-Tg mice were treated with 20 μg/mouse of anti-FasL Ab (Ab-1, Oncogene Research) or the isotype control. Data (median (IQR)) for 3 HCV Tg+ and 3 non-Tg mice (difference not significant) in each of two independent experiments. Statistical significance by Mann–Whitney.

Fig. 6

Caspase-3 activity of activated lymphocytes in HCV Tg+ and non-HCV Tg− mice. Caspase-3 activity was measured in lysates from cells by release of pNA from peptide substrates as described in Materials and methods. Results represent the average of three independent experiments.

References

1. Bain C, Fatmi A, Zoulim F, Zarski JP, Trepo C, Inchauspe G. Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gastroenterology. 2001;120:512–524. - PubMed
1. Baum LG, Pang M, Perillo NL, Wu T, Delegeane A, Uittenbogaart CH, Fukuda M, Seilhamer JJ. Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells. J Exp Med. 1995;181:877–887. - PMC - PubMed
1. Belz GT, Altman JD, Doherty PC. Characteristics of virus-specific CD8(+) T cells in the liver during the control and resolution phases of influenza pneumonia. Proc Natl Acad Sci USA. 1998;95:13812–13817. - PMC - PubMed
1. Bertolino P, Heath WR, Hardy CL, Morahan G, Miller JF. Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2 Kb in the liver. Eur J Immunol. 1995;25:1932–1942. - PubMed
1. Cooper S, Erickson AL, Adams EJ, Kansopon J, Weiner AJ, Chien DY, Houghton M, Parham P, Walker CM. Analysis of a successful immune response against hepatitis C virus. Immunity. 1999;10:439–449. - PubMed

Apoptosis of activated CD4+ and CD8+ T cells is enhanced by co-culture with hepatocytes expressing hepatitis C virus (HCV) structural proteins through FasL induction - PubMed (original) (raw)