Activation of natural killer cells by hepatitis C virus particles in vitro

Abstract

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection.

Fig. 1

Production and infectivity of hepatitis C virus pseudoparticles (HCVpp). (a) HEK293T cells were transfected transiently with expression vectors encoding the HCV E1 and E2 glycoproteins, retroviral core proteins and a packaging-competent green fluorescent protein (GFP)-containing retroviral transfer vector to produce HCVpp. Left: phase contrast microscopical picture of HEK293T cells 48 h after transfection. Right: fluorescence picture of HEK293T cells 48 h after transfection showing ∼40% GFP-positive cells confirming HCVpp production. (b) Infectivity of HCVpp produced by HEK293T cells. Huh-7 human hepatocellular carcinoma cells were infected with 500 µl HEK293T supernatant containing HCVpp, or vesicular stomatitis virus (VSV)-Gpp as a positive control, or left untransfected; 72 h after infection Huh-7 cells were analysed by flow cytometry for GFP expression.

Fig. 2

Flow cytometry analysis of primary natural killer (NK) cells cultured in the presence of hepatitis C virus (HCV). NK cells from normal donor 1 were cultured for 5 days in Iscove's modified Dulbecco's medium/interleukin-2 (IMDM/IL-2) medium alone, in IMDM supplemented with HCV pseudoparticles (HCVpp)-containing supernatant, or in IMDM supplemented with HCV1b-containing human sera. Cells were stained with AlexaFluor 647-conjugated anti-CD16 and either anti-NKp44-phycoerythrin (PE), anti-CD94-PE or anti-CD81-PE monoclonal antibodies, respectively.

Fig. 3

Increased expression of activating receptors and decreased expression of inhibitory receptors on natural killer (NK) cells after exposure to hepatitis C virus (HCV). NK cells from five healthy donors (HD, panels on left side) and three chronic HCV patients (Pat., panels on right side) were cultured for 5 days in either NK cell medium [Iscove's modified Dulbecco's medium (IMDM)] with interleukin-2/phytohaemagglutinin-P (IL-2/PHA-P)/feeders alone, or supplemented (1:1) with Dulbecco's modified Eagle's medium (DMEM) conditioned by untransfected HEK293T cells (cDMEM), HCV pseudoparticles (HCVpp)-containing supernatant from transfected HEK293T cells, normal human serum (NHS) or human serum containing HCV1b, as indicated. Cells were analysed by multi-colour flow cytometry. (a) Frequencies of cells stained for either CD56 or CD16, and for one of the activating receptors NKG2D, NKp46, NKp44 or NKp30 are indicated. (b) Frequencies of cells stained for CD56 or CD16, and for one of the inhibitory receptors CD94/KLRD1 or AIRM1/p75, the KIR/CD158 receptor family, or for the cellular HCV-E2 ligand CD81/TAPA1 are indicated. The mean values of NK cell frequencies are shown. The differences between the cDMEM- and HCVpp-, as well as between the NHS- and HCV1b serum-treated groups, were evaluated by a paired two-tailed Student's t-test; n.s.: not significant; *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 3

Increased expression of activating receptors and decreased expression of inhibitory receptors on natural killer (NK) cells after exposure to hepatitis C virus (HCV). NK cells from five healthy donors (HD, panels on left side) and three chronic HCV patients (Pat., panels on right side) were cultured for 5 days in either NK cell medium [Iscove's modified Dulbecco's medium (IMDM)] with interleukin-2/phytohaemagglutinin-P (IL-2/PHA-P)/feeders alone, or supplemented (1:1) with Dulbecco's modified Eagle's medium (DMEM) conditioned by untransfected HEK293T cells (cDMEM), HCV pseudoparticles (HCVpp)-containing supernatant from transfected HEK293T cells, normal human serum (NHS) or human serum containing HCV1b, as indicated. Cells were analysed by multi-colour flow cytometry. (a) Frequencies of cells stained for either CD56 or CD16, and for one of the activating receptors NKG2D, NKp46, NKp44 or NKp30 are indicated. (b) Frequencies of cells stained for CD56 or CD16, and for one of the inhibitory receptors CD94/KLRD1 or AIRM1/p75, the KIR/CD158 receptor family, or for the cellular HCV-E2 ligand CD81/TAPA1 are indicated. The mean values of NK cell frequencies are shown. The differences between the cDMEM- and HCVpp-, as well as between the NHS- and HCV1b serum-treated groups, were evaluated by a paired two-tailed Student's t-test; n.s.: not significant; *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 4

Anti-CD81 antibodies counteract the induction of activating natural killer (NK) receptors by hepatitis C virus (HCV). Purified NK cells were cultured as described in the legend to Fig. 3. The anti-CD81 monoclonal antibody I·3·3·22 was added to culture wells at 1 µg/well as indicated. Frequencies of NK cells labelled for either CD56 or CD16, and one of the activating receptors, NKp46, NKp44, NKp30 or NKG2D, are shown.

Fig. 5

Augmented cytokine secretion by natural killer (NK) cells after culture in the presence of hepatitis C virus (HCV). Secretion of interferon (IFN)-γ (a) and tumour necrosis factor (TNF)-α (b) by NK cells from healthy donors (HD) and chronic HCV patients (Pat.) cultured for 5 days in either Iscove's modified Dulbecco's medium (IMDM) with interleukin-2/phytohaemagglutinin-P (IL-2/PHA-P)/feeders alone, or IMDM supplemented (1:1) with conditioned Dulbecco's modified Eagle's medium (DMEM), normal human serum, HCV pseudoparticles (HCVpp) or HCV1b-infected human serum, as indicated. Anti-HCV serum was added to wells as indicated. Values shown are means ± standard error of the mean from triplicate samples; statistical significances were determined by Student's t-test. *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 6

Augmented cytotoxicity by natural killer (NK) cells after culture in the presence of hepatitis C virus (HCV). Cytotoxicity assay using NK cells from healthy donor 1 and chronic HCV patient 1 cultured in Iscove's modified Dulbecco's medium/interleukin-2/phytohaemagglutinin-P (IMDM/IL-2/PHA-P) medium for control, or in IMDM supplemented with HCV pseudoparticles (HCVpp)-containing supernatant or with HCV-infected serum. T2 cells (a) and BxPC3 cells (b) were labelled with Muromonab-CD3-fluorescein isothiocyanate (OKT3-FITC) and HEA125-FITC, respectively, and used as targets for NK cells at a effector to target ratio of 10:1. Lysis is presented as percentage of dead cells (propidium iodide-positive cells) from all FITC-labelled target cells. The percentage of propidium-iodide-positive target cells in the absence of effector cells has been subtracted. Values shown are means ± standard error of the mean from duplicate samples; statistical significances were determined by Student's t-test. *P < 0·05.