Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0604372 on August 31, 2004

Published online before print August 31, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0604372v1
76/6/1171    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Y.
Right arrow Articles by Ho, W.-Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.
Right arrow Articles by Ho, W.-Z.
(Journal of Leukocyte Biology. 2004;76:1171-1179.)
© 2004 by Society for Leukocyte Biology

Natural killer cells inhibit hepatitis C virus expression

Yuan Li, Ting Zhang, Chun Ho, Jordan S. Orange, Steven D. Douglas and Wen-Zhe Ho1

Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children’s Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine

1 Correspondence: Division of Allergy & Immunology, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, 34th Street & Civic Center Blvd., Philadelphia, PA 19104. E-mail: ho{at}email.chop.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Natural killer (NK) cells are critical in host innate defense against certain viruses. The role of NK cells in controlling hepatitis C virus (HCV) remains obscure. We examined whether NK cells are capable of inhibiting HCV expression in human hepatic cells. When NK cells are cultured with the HCV replicon-containing hepatic cells, they have no direct cytolytic effect but release soluble factor(s) suppressing HCV RNA expression. Media conditioned by NK cell lines (NK-92 and YTS) or primary NK cells isolated from healthy donors contain interferon {gamma} (IFN-{gamma}) and potently inhibit HCV RNA expression. Ligation of CD81 on NK cells inhibits IFN-{gamma} production and results in decreased anti-HCV activity. In addition, the antibodies to IFN-{gamma} or IFN-{gamma} receptors abolish the anti-HCV activity of NK cell-conditioned media. The role of IFN-{gamma} in NK cell-mediated, anti-HCV activity is supported by the observation that NK cell-conditioned media enhanced expression of signal transducer and activator of transcription-1, a nuclear factor that is essential in IFN-{gamma}-mediated antiviral pathways. NK cell-conditioned media have the ability to stimulate intracellular IFN-{alpha} expression in the hepatic cells, suggesting a mechanism responsible for NK cell-mediated, anti-HCV activity. Thus, NK cells hold the potential to play a vital role in controlling HCV replication in hepatic cells using an IFN-{gamma}-dependent mechanism.

Key Words: interferon • CD81 • STAT


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV) is a positive-strand RNA virus of the flavivirus family, which was molecularly cloned in 1989 [1 ]. HCV infects over 170 million people worldwide, and ~70% of infected people develop a chronic infection, which can lead to serious liver disease, including cirrhosis and hepatocellular carcinoma [2 3 4 5 ]. HCV uses complex and unique mechanisms to prevent, evade, or subvert innate and adaptive immune responses and to establish persistent infection and chronic hepatitis [6 ]. Treatment of HCV infection with interferon {alpha} (IFN-{alpha}) and ribavirin is associated with a sustained clinical response rate of less than 50% [7 8 9 ]. The limited therapeutic efficacy of available treatment and the absence of an effective HCV vaccine to prevent HCV infection constitute a strong rationale for investigation of human host defense mechanisms against HCV with the goal of development of immune-based therapies.

The interaction between HCV and the innate immune system plays a critical role in the immunopathogenesis of HCV disease. The liver as the primary site of HCV replication is significantly enriched for cells of the innate immune response. The normal liver contains a high percentage of CD3–CD56+ natural killer (NK) cells, which are one of the three main intrahepatic lymphocytes along with T cell receptor {gamma}/{delta}+ and V{alpha}24+ T cells [10 ]. A hallmark of HCV-associated liver pathology is massive lymphocyte infiltration in the infected organ [10 , 11 ]. NK cells specifically recognize infected cells without undergoing rearrangement of their germ-line DNA and may be critical for controlling the spread of intracellular pathogens. NK cells can provide a critical first line of defense mechanism against viral infections through their rapid cytotoxic activity and production of cytokines, such as IFN-{gamma} [12 ]. NK cells participate in the immune response against a number of virus infections including herpesviruses, influenza virus, and human immunodeficiency virus (HIV) [12 ]. The role of NK cells in controlling HCV replication remains obscure, but their importance is highlighted by the observation that a HCV protein (E2) inhibits signaling in NK cells, suggesting an attempt by HCV to specifically evade these defenses [13 , 14 ].

One of the major obstacles for the investigation of innate host defense against HCV infection is the lack of cell and small animal models for HCV replication. Recently established HCV subgenomic replicon systems [15 16 17 ] have greatly facilitated the analysis of the dynamics of HCV replication and virus-host interaction. This system has allowed the examination of antiviral properties of immune cells under physiologic conditions of viral replication [15 16 17 ]. In this study, we investigated the anti-HCV activity of NK cells in the HCV replicon system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human (rh)IFN-{alpha} and IFN-{gamma} and the monoclonal antibodies (mAb) against IFN-{alpha}, IFN-{gamma}, and IFN-{gamma} receptors (IFN-{gamma}R1 and IFN-{gamma}R2) were obtained from R&D Systems (Minneapolis, MN). Purified mouse mAb against human CD81 (clone JS-1) was purchased from BD PharMingen (San Diego, CA) and horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) Ab was purchased from Jackson ImmunoResearch Labs (West Grove, PA). The mAb against HCV NS5 was a gift of Dr. Bill Sun (Thomas Jefferson University, Philadelphia, PA).

Cells
The human NK cell line (NK-92) [18 ] was derived from an individual with lymphoma and was supplemented with 100 U/ml rh-interleukin (IL)-2 (Hoffmann-LaRoche, Nutley, NJ) for continuing in vitro growth. The human YTS NK cell line (YTS) is a subclone of the YT lymphoid cell line derived from a patient with NK cell leukemia and is IL-2-independent [19 ]. IM-9 cells (human B lymphoblasts) were obtained from American Type Culture Collection (Manassas, VA). Primary NK cells were isolated from peripheral blood of four adult healthy donors lacking antibodies to HIV and HCV. The Institutional Review Board of the Children’s Hospital of Philadelphia (PA) approved the studies. NK cells were enriched by immunomagnetic-negative selection (Miltenyi Biotec, Auburn, CA). The purity (% of CD56+CD3–) of NK cells measured by fluorescence-activated cell sorting analysis was greater than 95%. Enriched NK cells were maintained in 24-well plates at a density of 106 cells/well in 1 ml RPMI plus 10% (v/v) fetal bovine serum (HyClone, Logan, UT), supplemented with IL-2 (100 U/ml). Media conditioned by NK-92 and YTS cells (conditioned media) were collected every 48 h during cell passages and from primary NK cell cultures 72 h after IL-2 stimulation. All conditioned media were filtered through 0.22-µm pore-size filters and were stored at –70°C in aliquots. Huh.8 and Huh7 cells were obtained from Dr. Charles Rice (Washington University School of Medicine and Apath, L.L.C., St. Louis, MO). FCA-1 cells were obtained from Dr. Christoph Seeger (Fox Chase Cancer Center, Philadelphia, PA). Huh.2 cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, MD). Huh7, the parental cell line of Huh.8 and FCA-1, is derived from a human hepatoma [20 ]. Huh.8 contains a G418-selectable HCV RNA replicon with wild-type HCV nonstructural protein NS5A sequence [16 ]. Using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay, which we have developed recently [21 ], we were able to detect 2500–5000 copies of HCV mRNA per Huh.8 cell. Huh.2 is a cell clone containing a G418-electable HCV replicon with HCV NS5A mutation (S1172P) [16 ]. FCA-1 cells contain a subgenomic replicon from a known, infectious HCV clone [22 ] with several consensus mutations in NS3 as well as in NS5A (NS3: E177G; NS5A: D1229E, I1299V) [22 ]. FCA-1, Huh.2, Huh.8, and Huh7 cells were maintained as described [23 ]. The HCV replicon-containing cells (Huh.8, Huh.2, and FCA-1) grown in 24-well plates were incubated with or without NK cell-conditioned media for up to 96 h. No cytotoxic effect (trypan blue-dye staining) of NK cell-conditioned media on FCA-1, Huh.2, or Huh.8 cells was observed with the concentrations of NK cell-conditioned media used in the experiments (data not shown). The cell viability was measured by cell proliferation assay. In all cases, the limulus amebocyte lysate assay demonstrated that the media and reagents are endotoxin-free.

Biological assays
NK cells (primary NK cells, NK-92, and YTS) were cocultured with 51Cr-labeled hepatic cells (FCA-1 and Huh7) at specified effector-to-target (E:T) cell ratios, and lytic activity was calculated from the 51Cr released into supernatant after 4 h incubation as described [24 ]. For coculture experiments, 0.4 µm pore transwell tissue-culture plates (Costar, Cambridge, MA) were used. HCV replicon cells (105 cells) were incubated in the lower compartment with different numbers of NK cells in the upper compartment. The hepatic cells in lower compartments were collected 48 h after coculture for RNA extraction and HCV RNA real-time RT-PCR. CD81 ligation on NK cells was performed by incubation, 1 x 105 NK cells, in a microtiter plate (enzyme immunoassay/radioimmunoassay plate, Corning Inc., Acton, MA) well, which had been coated with anti-CD81 mAb (5 µg/ml) using sodium bicarbonate buffer (pH 8.2). NK cell-conditioned media were collected and pooled 72 h postincubation as described above.

HCV RNA quantification
Total RNA (1 µg) was extracted from FCA-1, Huh.2, Huh.8, and Huh7 cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as described [21 , 23 ]. HCV real-time RT-PCR assay, which we recently developed, was used for the quantification of HCV RNA [21 ].

Immunoassays
For immunofluorescent evaluation, FCA-1, Huh.2, and Huh.8 cells were cultured in the presence or absence of NK cell-conditioned media (10%, v/v) on glass coverslips (12 mm) in a 24-well plate. The cells were fixed with 75% ice-cold acetone 48 h post-treatment and then pretreated with a blocking solution for 10 min. The coverslips were then incubated with a mouse monoclonal anti-HCV NS5 Ab (1:100) in blocking solution at room temperature for 60 min and subsequently incubated with HRP-conjugated goat anti-mouse IgG Ab (1:100) for 30 min and followed by Fluorophore Tyramide (NEN Life Science Products Inc., Boston, MA) working solution for 15 min (in dark). The coverslips were washed three times with 1x phosphate-buffered saline (PBS), mounted in vectorshield (Vector Labs, Burlingame, CA), and viewed with a fluorescence microscope (Zeiss, Germany). For immunoblot, total cell lysates were prepared using lysis buffer (Promega, Madison, WI) from FCA-1, Huh.2, and Huh.8 cells (105 cells/well in a 24-well plate) treated with or without NK cell-conditioned media (10%, v/v). Protein concentrations were determined by a DC protein assay kit (Bio-Rad, Hercules, CA). HCV NS5 protein was detected using a nitrocellulose (NC) membrane in a Bio-Dot SF apparatus (Bio-Rad, Hercules, CA) loaded with 0.5 µg total protein extracted from FCA-1, Huh.2, and Huh.8 cells, treated with or without NK cell-conditioned media (10%, v/v). After blocking with PBS containing 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated with the mAb to HCV NS5 protein at 4°C overnight. After washing three times with PBS, the NC membrane was incubated with a HRP-conjugated goat anti-mouse IgG Ab for 1 h. The bound Ab was visualized by developing the membrane in a SuperSignal West Pico chemiluminescent substrate kit (Pierce, Rockford, IL). Enzyme-linked immunosorbent assay (ELISA) kits for IFN-{gamma} were purchased from Endogen, Inc. (Cambridge, MA), and assays were performed as instructed by the manufacturer. Western blot assay was performed as described [23 , 25 ].

Transfection and luciferase assays
Two plasmids, pIFNA4-Luc and wild-type IFN regulatory factor 7 (IRF-7) [26 ], were gifts from Dr. Rong-Tuan Lin at McGill University (Montreal, Quebec). Transfection of plasmid DNA was carried out with FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as recommended by the manufacturer. Luciferase activities in cell lysates were quantified using a luciferase assay system (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) [23 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NK cells have no specific cytolytic activity against HCV replicon cells
We first examined whether NK cells have a direct cytolytic effect on HCV replicon-containing cells. NK cell lines (NK-92 and YTS) and primary NK cells were cocultured with FCA-1 cells or Huh7 cells at different E:T ratios in 24-well culture plates. In the 51Cr release assay, NK-92 cells at the highest E:T ratio of 4.8:1 had little cytolytic effect (~20% specific lysis) on FCA-1 cells. However, this effect was also observed in Huh7 cells. There was no cytotoxicity observed at lower E:T ratios (Fig. 1 ), which were used in our subsequent experiments. In addition, cytolytic activity by YTS cells or primary NK cells was not observed against FCA-1 or Huh7 cells, even at the highest E:T ratio (4.8:1; Fig. 1 ).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. NK cells have no cytolytic activity on hepatic cells. Primary NK cells or NK cell lines (NK-92 and YTS) were cocultured with hepatic cells (FCA-1 and Huh7) labeled with 51Cr (incubated with 150 µCi 51Cr for 1 h) at desired E:T ratios (4.8:1, 2.4:1, 1.2:1, and 0.6:1). Cytolytic activity was measured by 51Cr release into supernatant after 4 h incubation. The percentage of specific lysis was calculated by the following: (experimental release–spontaneous release)/(maximum release–spontaneous release) x 100. The results shown are mean ± SD of triplicate cultures, representative of three separate experiments.

 
NK cells produce anti-HCV soluble factors
To determine whether NK cells release soluble factor(s) that suppress HCV replicon expression, a transwell coculture system was used. NK cells in the top compartment were cocultured with HCV replicon-containing cells (FCA-1, Huh.2, and Huh.8) plated in the bottom compartment. HCV replicon expression was markedly inhibited (up to 90% of suppression) in FCA-1, Huh.2, and Huh.8 cells cocultured with NK cells (Fig. 2A ). The degree of inhibition positively correlated with the number of NK cells added to HCV replicon cell cultures (Fig. 2B) .



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. NK cells release soluble factor(s) to suppress HCV RNA expression in HCV replicon-containing hepatic cells. (A and B) Coculture of NK cells with HCV replicon cells (FCA-1, Huh.2, and Huh.8). FCA-1, Huh.2, and Huh.8 cells (105 cells/well) were cultured in the lower compartment of a 24-well transwell coculture system. The numbers of NK cells and IM-9 cells added in the top compartment are indicated. After 48 h coculture, total cellular RNA extracted from the hepatic cell cultures was subjected to real-time RT-PCR for HCV and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as HCV RNA levels relative (% of Control) to the control (no NK cells on the top compartment, which is defined as 100%). (C) NK cell-conditioned media (CM) inhibit HCV RNA expression in HCV replicon-containing hepatic cells. FCA-1, Huh.2, and Huh.8 cells (105 cells/well) were cultured in the presence or absence of conditioned media from primary NK cells or NK cell lines (NK-92 and YTS) or IM-9 cells for 48 h. Total cellular RNA extracted from the cell cultures was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative (% of Control) to the control (without treatment with NK cell-conditioned media, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments.

 
NK cell-conditioned media inhibit HCV replicon expression
As NK cells released soluble factor(s) that inhibit HCV RNA expression, we examined whether NK cell-conditioned media have the same activity. NK cell-conditioned media efficiently inhibited HCV RNA expression (Fig. 2C) , and the observed activity was NK cell-specific, as the control cell line (IM-9) had no effect on HCV replicon expression (Fig. 2A and 2C) . The inhibitory effect of NK cell-conditioned media was not limited to a particular clone of HCV replicon cells, as NK cell-conditioned media suppress HCV RNA expression in all three replicon cell lines (FCA-1, Huh.2, and Huh.8; Fig. 2C ). The anti-HCV capacity of NK cell-conditioned media was concentration-dependent (Fig. 3A ), and the maximal activity was observed 48 h after the introduction of NK cell-conditioned media (Fig. 3B) . As HCV NS5 protein plays a critical role in HCV replication, we also examined NS5 protein expression in HCV replicon-containing cells treated with or without NK cell-conditioned media. HCV NS5 protein expression was inhibited in the cells treated with NK cell-conditioned media as determined by immunofluorescent staining (Fig. 4A ) and immunoblot assay (Fig. 4B and 4C) .



View larger version (31K):
[in this window]
[in a new window]
  		Figure 3. Dose-dependence (A) and time-course (B) of inhibition of HCV replicon expression by NK cell-conditioned media. Huh.8, Huh.2, and FCA-1 cells (105 cells/well) were cultured in the presence or absence of NK cell-conditioned media (CM) at indicated concentrations. At different time-points, total cellular RNA extracted from the cell cultures was subjected to real-time RT-PCR for HCV RNA quantification. The data are expressed as HCV RNA levels relative (% of Control) to control (without treatment with NK CM, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments.

 


View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. Effect of NK cell-conditioned media on HCV NS5 protein expression in HCV replicon-containing hepatic cells as determined by immunofluorescence staining (A) and by immunoblot assay (B and C). FCA-1, Huh.2, and Huh.8 cells (105 cells/well) plated on the coverslips or wells of a 24-well plate were cultured in the presence or absence of NK cell-conditioned media (CM). After 48 h incubation, cells on the coverslips were stained with the Ab against HCV NS5 antigen. For immunoblot assay, equal amounts (10 µg) of protein extracted from NK CM-treated and untreated (Control) HCV replicon cells were applied onto a NC membrane. The signal intensities of protein bands in the blot shown (B) were quantified as densitometry scanning units (DSU) by a densitometer and were shown (C). One representative of three separate experiments is shown.

 
IFN-{gamma} is the NK cell-produced anti-HCV factor
As NK cells play a significant role in antiviral defense, in part, through their ability to secrete cytokines such as IFN-{gamma} [27 ], we examined whether IFN-{gamma} is responsible for the observed NK cell-mediated, anti-HCV activity. The NK cell lines and primary NK cells isolated from health donors produced IFN-{gamma} (Fig. 5A ). Neutralization of IFN-{gamma} with blocking Ab completely reversed the anti-HCV effect of NK cell-conditioned media derived from NK-92 and primary NK cell cultures (Fig. 5B) . To further confirm that IFN-{gamma} is directly responsible for the anti-HCV ability of NK cell-released factor(s), we used antibodies to IFN-{gamma}R1 and IFN-{gamma}R2 to block IFN-{gamma} binding to the human hepatic cells before the addition of NK cell-conditioned media. Pretreatment of the cells with these antibodies blocked the anti-HCV activity of NK cell-conditioned media (Fig. 5C) . In contrast, Ab to IFN-{alpha} had no effect on the anti-HIV activity of NK cell-conditioned media (data not shown). In addition, other factors [tumor necrosis factor {alpha}, macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, and regulated on activation, normal T expressed and secreted], added to HCV replicon cell cultures, had no effect on HCV replicon expression (data not shown), further suggesting the specificity of IFN-{gamma}.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. IFN-{gamma} is responsible for the anti-HIV activity of NK cell-conditioned media. (A) IFN-{gamma} production by primary NK and NK cell lines. NK cell-conditioned media were prepared as stated in Materials and Methods. IFN-{gamma} levels in media from primary NK cells and NK cell lines (NK-92 and YTS) were determined by ELISA. (B) Anti-IFN-{gamma} Ab blocks NK cell-conditioned, medium-mediated, anti-HCV activity. FCA-1 cells (105 cells/well) plated in 24-well plates were cultured in the presence or absence of NK cell-conditioned media (CM) with or without Ab against IFN-{gamma} (10 µg/ml). For the cultures in the presence of NK cell-conditioned media and the Ab against IFN-{gamma}, the NK cell-conditioned media were preincubated with the Ab against IFN-{gamma} for 30 min before being added to FCA-1 cell cultures. IFN-{gamma} (1000 U/ml) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN-{gamma}. Mouse IgG2A was used to determine the specificity of the Ab to IFN-{gamma}. (C) Antibodies to IFN-{gamma}R abrogates anti-HCV activity of NK cell-conditioned media. FCA-1 cells (105 cells/well) plated in 24-well plates were incubated with or without the antibodies to IFN-{gamma}R1 (10 µg/ml) and/or IFN-{gamma}R2 (10 µg/ml) for 1 h prior to the addition of the NK-92-conditioned media. Goat IgG was used as a control Ab to determine the specificity of the antibodies to IFN-{gamma}R. Total cellular RNA extracted from FCA-1 cells was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification 48 h post-exposure to NK cell-conditioned media. The data are expressed as HCV RNA levels relative (% of Control) to control (no Ab treatment and no NK cell-conditioned media added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. +, In the presence; –, in the absence.

 
Cross-linking NK cells with the Ab to CD81 results in loss of anti-HCV activity
CD81 belongs to a family of molecules designated tetraspanins, which are characterized by four transmembrane domains forming two extracellular loops. CD81 is found on the surface of almost all nucleated cells, where it is a component of multiple and distinct receptor complexes depending on the cell type [28 ]. CD81 cross-linking via immobilized mAb specific for CD81 can inhibit IFN-{gamma} production by NK cells after exposure to IL-2 [14 ]. Thus, we examined whether CD81 cross-linking reduces anti-HCV ability of NK cells. We observed that CD81 cross-linking led to decreased IFN-{gamma} expression in primary NK cells and NK cell lines at the mRNA and protein levels (Fig. 6A and 6B ). This reduction of IFN-{gamma} by NK cells following CD81 cross-linking was confirmed by diminished anti-HCV activity of the NK cell-conditioned media (Fig. 6B) .



View larger version (49K):
[in this window]
[in a new window]
  		Figure 6. CD81 cross-linking-mediated reduction in IFN-{gamma} expression by NK cells corresponds to the diminished anti-HCV activity. (A and B) Inhibition of IFN-{gamma} expression in primary NK and NK cell lines mediated by CD81 cross-linking. IFN-{gamma} mRNA (A) in NK cells was determined by RT-PCR (inset, upper panel, IFN-{gamma}; lower panel, ß-actin) and quantified by real-time RT-PCR (SYBR Green). IFN-{gamma} protein (B) released from NK cells was measured by ELISA. The results shown are mean ± SD of triplicate cultures. (C) CD81 cross-linking results in diminished inhibition of HCV replicon expression. FCA-1 cells were cultured for 48 h in conditioned media (10%, v/v) from NK cells (NK-92, YTS, and primary NK cells) in the presence or absence of anti-CD81 Ab. Total cellular RNA extracted from the cell cultures was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative (% of Control) to the control (without treatment with NK CM, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments.

 
NK cell-conditioned media enhance expression of signal transducer and activator of transcription (STAT)1 and IFN-{alpha}
STAT1 plays a significant role in signaling for IFN-mediated, biologic effects [29 ]. We hypothesized that NK cell-conditioned media that contain IFN-{gamma} enhance STAT1 expression in the hepatic cells. As expected, NK cell-conditioned media up-regulated STAT1 protein expression in a HCV replicon-containing cell line (FAC-1) and its parental cell line (Huh7; Fig. 7 ). As intracellular IFN-{alpha} produced by the hepatic cells has a central role in inhibiting HCV replication (our unpublished data), we also examined whether NK cell-conditioned media induce intracellular IFN-{alpha} expression in the hepatic cells. We showed that Huh7 and FCA-1 cells treated with NK cell-conditioned media express higher levels of endogenous IFN-{alpha} than untreated control cells (Fig. 8A ). To investigate whether the induction of intracellular IFN-{alpha} is a result of the activation of the IFN-{alpha} promoter by NK cell-conditioned media, we transfected the hepatic cells with the plasmid carrying the IFN-{alpha} promoter. We demonstrated that NK cell-conditioned media activated IFN-{alpha} promoter in Huh7 cells (Fig. 8B) .



View larger version (45K):
[in this window]
[in a new window]
  		Figure 7. Effect of NK cell-conditioned media on STAT1 protein expression in the hepatic cells. FCA-1 and Huh7 cells were cultured in NK (NK-92 and YTS) cell-conditioned media (10%, v/v) for 48 h. Equal amounts (10 µg) of total proteins extracted from NK CM-treated and untreated (control) FCA-1 and Huh7 cells were applied onto a NC membrane for Western blot assay using the Ab specific to STAT1 and actin. The arrows indicate the position of STAT-1 (91 kD) or actin (42 kD). The insets below the panels show the signal intensities of protein bands of the representative blot expressed as DSU. The results were recorded on the film (2 min exposure). One representative of three separate experiments is shown.

 


View larger version (28K):
[in this window]
[in a new window]
  		Figure 8. NK cell-conditioned media induce IFN-{alpha} in the hepatic cells. (A) NK cell-conditioned media induce endogenous IFN-{alpha}. FCA-1 and Huh7 cells were cultured in NK (NK-92 and YTS) cell-conditioned media (10%, v/v) for 48 h. Total cellular RNA extracted from the cell cultures was subjected to real-time RT-PCR for IFN-{alpha} RNA quantification. The data are expressed as IFN-{alpha} RNA levels relative (fold) to the control (without NK cell-conditioned medium treatment, which is defined as 1). (B) NK cell-conditioned media enhance IRF-7-induced IFN-{alpha} promoter activation. Huh7 cells cultured in the presence or absence of NK (NK-92 and YTS) cell-conditioned media were cotransfected with the plasmid containing the IRF-7 gene and the plasmid containing IFN4A, an IFN-{alpha} promoter, linked with a luciferase gene. Luciferase activities were determined in the cell extracts. The data are expressed as lucifease activity relative (fold) to control (without transfection or treatment with NK cell-conditioned media). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although direct NK-mediated cytotoxicity is vital in limiting certain viral infections, the antiviral cytokines such as IFN-{gamma}, produced by NK cells, also have significant roles in the control of viral infections [30 ]. In this study, we took advantage of the newly developed HCV replicon system to examine the anti-HCV activity of NK cells, which release soluble factors that potently inhibit HCV replicon replication, which was determined by the transwell coculture and NK cell-conditioned medium experiments (Figs. 2 and 3) . The NK cell-mediated, anti-HCV activity is innate, as the primary NK cells used in the experiments were isolated from HCV seronegative subjects. We subsequently identified that IFN-{gamma} is vital in NK cell-mediated, anti-HCV activity. IFN-{gamma} is a powerful inhibitor of HCV replicon replication [31 , 32 ]. Antibodies to IFN-{gamma} and/or IFN-{gamma}R completely abolished NK cell-mediated, anti-HCV replicon expression in the hepatic cells (Fig. 5B and 5C) , indicating IFN-{gamma} in NK cell-conditioned media has the major role in controlling HCV replicon expression. We also examined whether NK cell-conditioned media activate STAT1, an important member of the STAT family, which is essential for the innate immune response to viral infection [33 , 34 ]. As STAT1 is required for the induction of IFN-dependent responses and plays a dedicated role in signaling for IFN-mediated biologic effects [29 ], our data, showing that NK cell-conditioned media enhanced STAT1 protein expression in HCV replicon cells and the parental hepatic cell (Huh7; Fig. 7 ), provide additional evidence that IFN-{gamma} is indeed responsible for NK cell action on HCV replicon expression. In addition, the magnitude of the anti-HCV ability of NK cells from these donors is comparable with the NK cells isolated from HCV-infected subjects (data not shown). We did not observe a direct NK cell-mediated cytolytic effect on HCV replicon cells (Fig. 1) . A possible explanation is that HCV replicon cells do not produce infectious HCV particles and do not contain the HCV antigens necessary for NK cell recognition.

The intrahepatic immune system is likely to play a key role in determining the outcome of HCV infection. This system is characterized by a unique repertoire of lymphocytes [35 , 36 ]. In addition to conventional CD4+ and CD8+ T cells and B cells, the liver contains large numbers of NK cells. An important question is why intrahepatic NK cells fail to stop spreading of HCV infection and eliminate the virus in the liver in the majority of HCV-infected subjects. One of the possibilities is that HCV has the ability to disrupt the host immune defense against virus. HCV-encoded proteins, such as envelope protein E2, the nonstructural protein 5A, and core proteins, have been shown to have immunosuppressive potentials [37 38 39 ]. As NK cells are a critical component of host innate immune response against viral infection [40 ], they may be important targets for HCV. Inhibition of the NK cell-mediated innate immune response against HCV could provide HCV with a growth advantage. There have been a number of reports on abnormalities of NK cell function in HCV-infected subjects. Corado et al. [41 ] reported that NK cell-mediated cytotoxicity is significantly diminished in HCV-infected subjects. HCV inhibits NK cell function by engaging CD81 through its E2 envelope protein [13 , 14 ]. Unlike ligation of CD81 in T cells, cross-linking CD81 on NK cells with immobilized HCV E2 protein or with anti-CD81 Ab blocks NK cell-mediated cytolytic activity and IFN-{gamma} production [13 , 14 ]. We also showed that cross-linking of NK cells with the Ab to CD81 resulted in reduction of IFN-{gamma} production by NK cells, which was correlated with the diminished anti-HCV ability (Fig. 6A and 6B) . Thus, the mechanism of HCV E2-mediated effects on NK cells is most probably a CD81-mediated inhibition of IFN-{gamma} production.

Patients with chronic HCV infection are currently treated with IFN-{alpha} alone or in combination with ribavirin. Sustained response rates, however, are limited to 10–20% in cases of IFN-{alpha} monotherapy and 30–40% with IFN-{alpha} plus ribavirin combination therapy [42 ]. Recently, IFN-{alpha} monotherapy has been improved by using pegylated IFN-{alpha}, which has an extended half-life, reduced immunogenicity, and enhanced biological activity. However, even with pegylated IFN-{alpha} treatment, there is still no cure for a large proportion of patients with chronic HCV infection [43 , 44 ]. Our in vitro data, in conjunction with the previous findings showing that IFN-{gamma} has the ability to suppress expression of subgenomic and genomic HCV RNA [31 , 32 ], indicate that IFN-{gamma} may be an alternative treatment for chronic HCV infection. It has been reported that priming with the IFN-{gamma} prior to the initiation of IFN-{alpha} treatment in patients with refractory, chronic HCV infection leads to a more favorable host immune response and might contribute to viral clearance [45 ]. IFN-{gamma} brings an additive antiviral environment when combined with IFN-{alpha} treatment in patients with chronic HCV infection [46 ]. Our data, showing that NK cell-conditioned media enhance IFN-{alpha} promoter activation and induce intracellular IFN-{alpha} expression in the hepatic cells (Fig. 8) , provide a mechanism of IFN-{gamma}-mediated, anti-HCV activity in the replicon cells and further support the notion of using IFN-{gamma} as an additional therapeutic agent for HCV infection. Thus, the combination therapy with IFN-{alpha} and IFN-{gamma} should be explored in spite of the fact that treatment of HCV with IFN-{gamma} alone is not effective in a randomized and controlled trial [47 ].

Taken together, our data showing that NK cells are capable of secreting IFN-{gamma} to powerfully suppress HCV RNA expression indicate that NK cells are indeed involved in the host innate immune defense against HCV infection. NK cells may play a vital role in protection against HCV infection and progression of HCV disease. The importance of our study is highlighted by the observations that HCV compromises functions of NK cells through CD81 ligation and inhibits NK cell-induced intracellular IFN-{alpha} expression in the hepatic cells. Further study will determine whether the balance can be moved toward NK cells in innate immune defense against HCV infection and disease.


   ACKNOWLEDGEMENTS
 
This work was supported by the National Institutes of Health Grants DA12815 and DA16022 (to W-Z. H.), MH49981 and AA13547 (to S. D. D.), and AI55602 (to J. S. O.). We thank Dr. Charles Rice for generously providing us with the Huh.8 and Huh7 cell lines. We also are grateful to Dr. Christoph Seeger for providing the FCA-1 cells for this study.

Received June 29, 2004; revised August 6, 2004; accepted August 10, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., Houghton, M. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome Science 244,359-362[Abstract/Free Full Text]
  2. Alter, M. J., Margolis, H. S., Krawczynski, K., Judson, F. N., Mares, A., Alexander, W. J., Hu, P. Y., Miller, J. K., Gerber, M. A., Sampliner, R. E. (1992) The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic Non-A, Non-B Hepatitis Study Team N. Engl. J. Med. 327,1899-1905[Abstract]
  3. Alter, M. J., Kruszon-Moran, D., Nainan, O. V., McQuillan, G. M., Gao, F., Moyer, L. A., Kaslow, R. A., Margolis, H. S. (1999) The prevalence of hepatitis C virus infection in the United States, 1988 through 1994 N. Engl. J. Med. 341,556-562[Abstract/Free Full Text]
  4. Tong, M. J., el-Farra, N. S., Reikes, A. R., Co, R. L. (1995) Clinical outcomes after transfusion-associated hepatitis C N. Engl. J. Med. 332,1463-1466[Abstract/Free Full Text]
  5. Kiyosawa, K., Sodeyama, T., Tanaka, E., Gibo, Y., Yoshizawa, K., Nakano, Y., Furuta, S., Akahane, Y., Nishioka, K., Purcell, R. H. (1990) Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus Hepatology 12,671-675[Medline]
  6. Racanelli, V., Rehermann, B. (2003) Hepatitis C virus infection: when silence is deception Trends Immunol. 24,456-464[CrossRef][Medline]
  7. Alter, H. J., Seeff, L. B. (2000) Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome Semin. Liver Dis. 20,17-35[CrossRef][Medline]
  8. Davis, G. L., Esteban-Mur, R., Rustgi, V., Hoefs, J., Gordon, S. C., Trepo, C., Shiffman, M. L., Zeuzem, S., Craxi, A., Ling, M. H., Albrecht, J. (1998) Interferon {alpha}-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. International Hepatitis Interventional Therapy Group N. Engl. J. Med. 339,1493-1499[Abstract/Free Full Text]
  9. McHutchison, J. G., Gordon, S. C., Schiff, E. R., Shiffman, M. L., Lee, W. M., Rustgi, V. K., Goodman, Z. D., Ling, M. H., Cort, S., Albrecht, J. K. (1998) Interferon {alpha}-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group N. Engl. J. Med. 339,1485-1492[Abstract/Free Full Text]
  10. Nuti, S., Rosa, D., Valiante, N. M., Saletti, G., Caratozzolo, M., Dellabona, P., Barnaba, V., Abrignani, S. (1998) Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for V{alpha}24+ T cells and rapid elimination of effector cells by apoptosis Eur. J. Immunol. 28,3448-3455[CrossRef][Medline]
  11. Bianchi, L. (1983) Liver biopsy interpretation in hepatitis. Part II: histopathology and classification of acute and chronic viral hepatitis/differential diagnosis Pathol. Res. Pract. 178,180-213[Medline]
  12. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P., Salazar-Mather, T. P. (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines Annu. Rev. Immunol. 17,189-220[CrossRef][Medline]
  13. Crotta, S., Stilla, A., Wack, A., D’Andrea, A., Nuti, S., D’Oro, U., Mosca, M., Filliponi, F., Brunetto, R. M., Bonino, F., Abrignani, S., Valiante, N. M. (2002) Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein J. Exp. Med. 195,35-41
  14. Tseng, C. T., Klimpel, G. R. (2002) Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions J. Exp. Med. 195,43-49
  15. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L., Bartenschlager, R. (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line Science 285,110-113[Abstract/Free Full Text]
  16. Blight, K. J., Kolykhalov, A. A., Rice, C. M. (2000) Efficient initiation of HCV RNA replication in cell culture Science 290,1972-1974[Abstract/Free Full Text]
  17. Rosenberg, S. (2001) Recent advances in the molecular biology of hepatitis C virus J. Mol. Biol. 313,451-464[CrossRef][Medline]
  18. Gong, J. H., Maki, G., Klingemann, H. G. (1994) Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells Leukemia 8,652-658[Medline]
  19. Cohen, G. B., Gandhi, R. T., Davis, D. M., Mandelboim, O., Chen, B. K., Strominger, J. L., Baltimore, D. (1999) The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells Immunity 10,661-671[CrossRef][Medline]
  20. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., Sato, J. (1982) Growth of human hepatoma cell lines with differentiated functions in chemically defined medium Cancer Res. 42,3858-3863[Abstract/Free Full Text]
  21. Yang, J. H., Lai, J. P., Douglas, S. D., Metzger, D., Zhu, X. H., Ho, W. Z. (2002) Real-time RT-PCR for quantitation of hepatitis C virus RNA J. Virol. Methods 102,119-128[CrossRef][Medline]
  22. Guo, J. T., Bichko, V. V., Seeger, C. (2001) Effect of {alpha} interferon on the hepatitis C virus replicon J. Virol. 75,8516-8523[Abstract/Free Full Text]
  23. Li, Y., Zhang, T., Douglas, S. D., Lai, J-P., Xiao, W-D., Pleasure, D. E., Ho, W-Z. (2003) Morphine enhances hepatitis C virus (HCV) replicon expression Am. J. Pathol. 163,1167-1175[Abstract/Free Full Text]
  24. Douglas, S. D., Durako, S. J., Tustin, N. B., Houser, J., Muenz, L., Starr, S. E., Wilson, C., . Adolescent Medicine, H. I. V. A. R. N. (2001) Natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents AIDS Res. Hum. Retroviruses 17,543-552[CrossRef][Medline]
  25. Zhang, T., Li, Y., Lai, J. P., Douglas, S. D., Metzger, D. S., O’Brien, C. P., Ho, W. Z. (2003) Alcohol potentiates hepatitis C virus replicon expression Hepatology 38,57-65[CrossRef][Medline]
  26. Sharma, S., tenOever, B. R., Grandvaux, N., Zhou, G. P., Lin, R., Hiscott, J. (2003) Triggering the interferon antiviral response through an IKK-related pathway Science 300,1148-1151[Abstract/Free Full Text]
  27. Biron, C. A. (1997) Activation and function of natural killer cell responses during viral infections Curr. Opin. Immunol. 9,24-34[CrossRef][Medline]
  28. Levy, S., Todd, S. C., Maecker, H. T. (1998) CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system Annu. Rev. Immunol. 16,89-109[CrossRef][Medline]
  29. Bach, E. A., Aguet, M., Schreiber, R. D. (1997) The IFN {gamma} receptor: a paradigm for cytokine receptor signaling Annu. Rev. Immunol. 15,563-591[CrossRef][Medline]
  30. Orange, J. S., Wang, B., Terhorst, C., Biron, C. A. (1995) Requirement for natural killer cell-produced interferon {gamma} in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration J. Exp. Med. 182,1045-1056[Abstract/Free Full Text]
  31. Frese, M., Schwarzle, V., Barth, K., Krieger, N., Lohmann, V., Mihm, S., Haller, O., Bartenschlager, R. (2002) Interferon-{gamma} inhibits replication of subgenomic and genomic hepatitis C virus RNAs Hepatology 35,694-703[CrossRef][Medline]
  32. Liu, C., Zhu, H., Tu, Z., Xu, Y. L., Nelson, D. R. (2003) CD8+ T-cell interaction with HCV replicon cells: evidence for both cytokine- and cell-mediated antiviral activity Hepatology 37,1335-1342[CrossRef][Medline]
  33. Meraz, M. A., White, J. M., Sheehan, K. C., Bach, E. A., Rodig, S. J., Dighe, A. S., Kaplan, D. H., Riley, J. K., Greenlund, A. C., Campbell, D., Carver-Moore, K., DuBois, R. N., Clark, R., Aguet, M., Schreiber, R. D. (1996) Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway Cell 84,431-442[CrossRef][Medline]
  34. Durbin, J. E., Hackenmiller, R., Simon, M. C., Levy, D. E. (1996) Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease Cell 84,443-450[CrossRef][Medline]
  35. Doherty, D. G., O’Farrelly, C. (2000) Innate and adaptive lymphoid cells in the human liver Immunol. Rev. 174,5-20[CrossRef][Medline]
  36. Seki, S., Habu, Y., Kawamura, T., Takeda, K., Dobashi, H., Ohkawa, T., Hiraide, H. (2000) The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses Immunol. Rev. 174,35-46[CrossRef][Medline]
  37. Gale, M. J., Jr, Korth, M. J., Tang, N. M., Tan, S. L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R., Katze, M. G. (1997) Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein Virology 230,217-227[CrossRef][Medline]
  38. Taylor, D. R., Shi, S. T., Romano, P. R., Barber, G. N., Lai, M. M. (1999) Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein Science 285,107-110[Abstract/Free Full Text]
  39. Large, M. K., Kittlesen, D. J., Hahn, Y. S. (1999) Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence J. Immunol. 162,931-938[Abstract/Free Full Text]
  40. Lanier, L. L., Phillips, J. H. (1996) Inhibitory MHC class I receptors on NK cells and T cells Immunol. Today 17,86-91[CrossRef][Medline]
  41. Corado, J., Toro, F., Rivera, H., Bianco, N. E., Deibis, L., De Sanctis, J. B. (1997) Impairment of natural killer (NK) cytotoxic activity in hepatitis C virus (HCV) infection Clin. Exp. Immunol. 109,451-457[CrossRef][Medline]
  42. Moradpour, D., Blum, H. E. (1999) Current and evolving therapies for hepatitis C Eur. J. Gastroenterol. Hepatol. 11,1199-1202[Medline]
  43. Zeuzem, S., Feinman, S. V., Rasenack, J., Heathcote, E. J., Lai, M. Y., Gane, E., O’Grady, J., Reichen, J., Diago, M., Lin, A., Hoffman, J., Brunda, M. J. (2000) Peginterferon {alpha}-2a in patients with chronic hepatitis C N. Engl. J. Med. 343,1666-1672[Abstract/Free Full Text]
  44. Heathcote, E. J., Shiffman, M. L., Cooksley, W. G., Dusheiko, G. M., Lee, S. S., Balart, L., Reindollar, R., Reddy, R. K., Wright, T. L., Lin, A., Hoffman, J., De Pamphilis, J. (2000) Peginterferon {alpha}-2a in patients with chronic hepatitis C and cirrhosis N. Engl. J. Med. 343,1673-1680[Abstract/Free Full Text]
  45. Katayama, K., Kasahara, A., Sasaki, Y., Kashiwagi, T., Naito, M., Masuzawa, M., Katoh, M., Yoshihara, H., Kamada, T., Mukuda, T., Hijioka, T., Hori, M., Hayashi, N. (2001) Immunological response to interferon-{gamma} priming prior to interferon-{alpha} treatment in refractory chronic hepatitis C in relation to viral clearance J. Viral Hepat. 8,180-185[CrossRef][Medline]
  46. Kumashiro, R., Ide, T., Sasaki, M., Murashima, S., Suzuki, H., Hino, T., Morita, Y., Miyajima, I., Ogata, K., Tanaka, E., Yoshida, H., Tanikawa, K., Sata, M. (2002) Interferon-{gamma} brings additive anti-viral environment when combined with interferon-{alpha} in patients with chronic hepatitis C Hepatol. Res. 22,20-26[CrossRef][Medline]
  47. Saez-Royuela, F., Porres, J. C., Moreno, A., Castillo, I., Martinez, G., Galiana, F., Carreno, V. (1991) High doses of recombinant {alpha}-interferon or {gamma}-interferon for chronic hepatitis C: a randomized, controlled trial Hepatology 13,327-331[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Exp. Med.Home page
P. P. Banerjee, R. Pandey, R. Zheng, M. M. Suhoski, L. Monaco-Shawver, and J. S. Orange
Cdc42-interacting protein 4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse
J. Exp. Med., October 1, 2007; 204(10): 2305 - 2320.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
L. Golden-Mason, N. Castelblanco, C. O'Farrelly, and H. R. Rosen
Phenotypic and Functional Changes of Cytotoxic CD56pos Natural T Cells Determine Outcome of Acute Hepatitis C Virus Infection
J. Virol., September 1, 2007; 81(17): 9292 - 9298.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. P. Windisch, M. Frese, A. Kaul, M. Trippler, V. Lohmann, and R. Bartenschlager
Dissecting the Interferon-Induced Inhibition of Hepatitis C Virus Replication by Using a Novel Host Cell Line
J. Virol., November 1, 2005; 79(21): 13778 - 13793.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
A. P. Williams, A. R. Bateman, and S. I. Khakoo
HANGING IN THE BALANCE: KIR and Their Role in Disease
Mol. Interv., August 1, 2005; 5(4): 226 - 240.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0604372v1
76/6/1171    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Y.
Right arrow Articles by Ho, W.-Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.
Right arrow Articles by Ho, W.-Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS