Errata: Erratum Volume 53, Issue 1, 377, Article first published online: 12 January 2011
Potential conflict of interest: Nothing to report.
CD56pos natural killer (NK)/natural T (NT) cells are important innate effectors providing the first line of defense against viral infection. Enhanced NK activity has been shown to protect from human immunodeficiency virus-1 infection. However, the role played by these innate effectors in protection against or development of hepatitis C virus (HCV) infection is unknown. We characterized CD56pos populations in 11 injection drug users (IDUs) who remained uninfected despite being repeatedly exposed to HCV. NK profiles in exposed but uninfected (EU) individuals were compared with preinfection samples (median 90 days prior to HCV seroconversion) collected from 14 IDUs who were exposed and subsequently became infected (EI) and unexposed normal control subjects (n = 8). Flow cytometric analysis of CD56pos populations demonstrated that EUs had a higher proportion of CD56low mature (P = 0.0011) NK cells compared with EI subjects. Bead-isolated NKs (>90% purity) from EUs had significantly higher interleukin-2 (IL-2)–induced cytolytic activity against the NK-sensitive cell line K562 at an effector-to-target ratio of 10:1 (P < 0.0001). NKp30, a natural cytotoxicity receptor involved in NK activation, is highest on NK/NT cells in EUs relative to infected subjects. Using the JFH-1 infection system, we demonstrated that NKp30high cells in the absence of exogenous stimulation significantly reduce infection of hepatocytes. Conclusion: CD56pos populations in EUs are enriched for effector NKs displaying enhanced IL-2–induced cytolytic activity and higher levels of the natural cytotoxicity receptor NKp30-activating receptor. In addition, NKp30high cells are more effective in preventing infection of Huh-7.5 cells than their NKp30low/neg counterparts. These data support the hypothesis that NK cells contribute to anti-HCV defense in vivo in the earliest stages of infection, providing innate protection from HCV acquisition. (HEPATOLOGY 2010)
Hepatitis C virus (HCV), a member of the Flaviviridae family, is known for its high propensity to establish persistent infection.1, 2 The host immune response early in HCV infection is thought to determine subsequent outcome,3 suggesting an important role for innate immunity in viral elimination either directly, preventing establishment of infection, or indirectly, through priming of antigen-specific adaptive immune mechanisms. The observation that some injection drug users (IDUs) remain healthy with no evidence of infection despite continued long-term exposure to HCV4 strongly suggests a role for innate immunity in natural protection from HCV infection.
Natural killer (NK) cells are key innate immune effectors that provide the first line of defense against viral infection, shaping subsequent adaptive immunity.5 NK activity is stringently controlled by inhibitory NK cell receptors (NKRs), which in steady state conditions override signals provided by engagement of activating receptors.6 NKRs include the predominantly inhibitory killer immunoglobulin-like receptors (KIR); C-type lectin-like receptors of the CD94/NKG2 family comprising inhibitory (NKG2A) and activatory (NKG2C/D) isoforms; and the natural cytotoxicity receptors (NCRs), such as NKp30, NKp44, and NKp46, orphan receptors that deliver activatory signals.6, 7
In humans, NKs can be identified by the expression of N-CAM (CD56), and relative expression of this antigen identifies functionally distinct immature/regulatory (CD56bright) and effector (CD56dim) NK subsets. CD56dim NKs carry perforin and are the main mediators of cytotoxicity.8 Expression of CD56 and various NKRs is shared by another innate-like effector population, natural T (NT) cells. The functional properties of NTs are similar to NKs; therefore, in addition to NKs, NTs are likely to be involved in the first line of defense against viral infection. It is noteworthy that the liver, the preferred site of HCV replication, is highly enriched for innate immune effectors, in particular NK and NT cells.9
The phenotypes and/or functional activities of various populations of innate effectors have been reported to be impaired in patients with chronic HCV.10-20 Evidence suggests that inheritance of particular killer immunoglobulin-like receptor genes involved in the control of NK activity may predispose to chronic infection.21, 22 Other studies have shown that HCV can modulate NK activity, either directly by binding of the HCV envelope-2 (E2) protein to CD8123-25 or indirectly by inducing expression of inhibitory ligands for NKs.14, 26, 27 Data on the role of NKs in the setting of acute HCV infection are limited. However, we have demonstrated that reduced interleukin-2 (IL-2)–activated killing early in infection was associated with the ultimate development of persistence, suggesting a role for innate NK/NT cells in clearance of HCV in the acute setting.28 A role for these populations in conferring innate protection from HCV acquisition has yet to be established, though it has been suggested by an in vitro model in which NK cells were key to suppressing HCV infection of human hepatocytes.29
Enhanced NK activity30 has been shown to contribute to protection from human immunodeficiency virus (HIV)-1 infection in exposed individuals. However, the role played by innate CD56pos effector populations in protection against or development of HCV infection is unknown. To address this question, we characterized CD56pos NK and NT cells in preinfection blood samples from a high-risk, long-term exposed IDU cohort in which some individuals remained uninfected despite repeated exposure to HCV.4 We demonstrate relatively increased effector NK cell level as well as enhanced NK cytolytic function, which was associated with an increase in NCR NKp30 expression, in subjects who remain resistant to infection in the face of repeated exposures. We also demonstrate that NKp30high NK cells in the context of the JFH-1 in vitro infection system are more effective in preventing infection of Huh-7.5 cells than their NKp30low/neg counterparts in the absence of exogenous stimulation. Our data offer new insight into the mechanisms underlying protection from HCV infection that may have implications for improving immunotherapeutic strategies.
EI, exposed and subsequently infected; EU, exposed but uninfected; FACS, fluorescence-activated cell sorting; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IDU, injection drug user; IFN-γ, interferon-γ; IL-2, interleukin-2; LAK, lymphokine-activated killing; NCR, natural cytotoxicity receptor; NK, natural killer cell; NKR, natural killer cell receptor; NT, natural T cell; PMA, phorbol myristate acetate; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
Patients and Methods
The study group comprised 25 IDUs, 11 of whom remained uninfected despite being repeatedly exposed to HCV (EUs), and 14 IDUs who subsequently became infected (EIs). The average age of exposed individuals was 25 years; 84% were Caucasian, and 60% were female. The age, race, and sex distribution did not differ between the EU and EI groups. For the cohort of exposed individuals who subsequently became infected, preinfection samples (median 90 days prior to HCV seroconversion) were analyzed. All exposed individuals tested negative for hepatitis B virus/HIV. Eight individuals with no risk factors who tested negative for HCV/HIV served as unexposed normal control subjects. The study protocol was approved by the Institutional Review Boards at the University of Colorado and Johns Hopkins Medical Institutions. Written and oral consent was obtained from the study participants before samples were collected.
Sample Preparation and Storage.
Peripheral blood mononuclear cells were isolated by way of Ficoll (Amersham Biosciences, Piscataway, NJ) density gradient centrifugation and cryopreserved for subsequent analyses.
Flow Cytometric Analysis.
Flow cytometric analysis was performed using a BD FACSCalibur instrument (BD Biosciences, San Jose, CA) compensated with single fluorochromes and analyzed using CellQuest software (BD Biosciences). Flurochrome-labeled (FITC/PE/PerCP/APC) monoclonal antibodies specific for CD3/CD56 were obtained from BD Biosciences. Anti–TRAIL-PE monoclonal antibody was supplied by R&D Systems (Minneapolis, MN). Anti–NKp30-PE and NKp44-PE were obtained from Immunotech (Beckman Coulter, Fullerton, CA). Peripheral blood mononuclear cells (2.5 × 105) were stained for cell surface antigen expression at 4°C in the dark for 30 minutes, washed twice in 2 mL phosphate-buffered saline containing 1% bovine serum albumin and 0.01% sodium azide (fluorescence-activated cell sorting [FACS] wash), and fixed in 200 μL of 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Isotype-matched fluorescently labeled control antibodies were used to determine background levels of staining. Lymphocytes were identified by characteristic forward and side scatter parameters, and populations of interest were gated on patterns of CD56/CD3 staining within the lymphocyte population. Results are expressed as the percent positive of the gated population. Intracellular perforin staining was performed after permeabilization with 0.2% saponin using a δ-G9 antibody obtained from BD Biosciences.
Thawed mononuclear cell suspensions were enriched for NKs using the NK Isolation Kit II from Miltenyi Biotec (Gladbach, Germany) according to the manufacturer's instructions. The median purity of NKs was >90% in all cases. After isolation, NKs were cultured with or without IL-2 (25 ng/mL, R&D Systems) for 48 hours at 37°C and 5% CO2. Following culture, carboxyfluorescein succinimidyl ester–labeled target cells (K562s) were added to NKs at effector-to-target concentrations of 0:1 (negative control) and 10:1 (test) and incubated at 37°C for 4 hours. After incubation, cytotoxicity was measured using the flow cytometry–based Total Cytotoxicity & Apoptosis Detection Kit from Immunochemistry (Bloomington, MN). Immediately before acquisition, 7-aminoactinomycin D was added to effector-to-target populations and incubated for 15 minutes on ice. Cells treated with 0.1% Triton-X served as positive controls.
Degranulation was determined by way of flow cytometric analysis of increased CD107a (Lamp, BD Bioscences) expression on bead-isolated NKs after 4-hour stimulation with phorbol myristate acetate (PMA) (10 ng/mL) and ionomycin (1 μg/mL) in the presence of brefeldin A (Sigma-Aldrich) and CD107a. NKs cultured under the same conditions without PMA and ionomycin served as unstimulated controls.
Antibodies for intracellular interferon-γ (IFN-γ) were supplied by BD Biosciences. Thawed mononuclear cells were stimulated with PMA (10 ng/mL) and ionomycin (1 μg/mL) for 4 hours at 37°C in the presence of brefeldin A. After stimulation, cells were stained for surface antigens (as above), fixed for 30 minutes at 4°C in 100 μL Fix and Perm Medium A (Caltag, Burlingame, CA), permeabilized using 100 μL Fix and Perm Medium B (Caltag), and incubated with anti-cytokine monoclonal antibody for 1 hour at 4°C in the dark. Cell suspensions were then washed in FACS wash and fixed in 200 μL 2% paraformaldehyde and acquired after 1 hour. Cells cultured under the same conditions in the absence of PMA and ionomycin served as controls.
Hepatocyte Cytotoxicity Assay.
NKs were enriched using magnetic beads and were surface-stained for CD3, CD56, and NKp30 as described above. NKs (CD3−/CD56+) underwent FACS sorting on expression of NKp30 using a FACS Aria instrument (BD Biosciences). NKp30high and NKp30low/neg fractions were incubated for 48 hours with or without IL-2 (25 ng/mL) at 1 × 106/mL in 96-well round bottom plates. Huh-7.5 cells (Apath LLC, St. Louis, MO) were seeded at 1.25 × 105 cells/well in 24-well plates. After 24 hours, NKs were added at an NK/Huh-7.5 cell ratio of 5:1. Cells were infected simultaneously with JFH-1 (National Institute of Infectious Diseases, Tokyo, Japan) at a multiplicity of infection of 0.003. Five days after infection, cells were harvested for RNA extraction (RNeasy Mini Kit, Qiagen). RNA was transcribed to complementary DNA using the QuantiTect Reverse Transcription Kit (Qiagen), and HCV transcripts were detected using a 7300 Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA). A standard curve was created using JFH-1 plasmid stock (range, 1 × 107 to 1 × 101). Taqman Master Mix, primers, and probes were purchased from Applied Biosystems. HCV primer and probe sequences were as follows: forward, GCA CAC TCC GCC ATC AAT CAC T; reverse, CAC TCG CAA GCG CCC TAT CA; probe, 6FAM AGG CCT TTC GCA ACC CAA CGC TAC T TAMRA. NKs cultured as above were assessed for the expression of NKp30.
Results are expressed as the median (range). A nonparametric Mann-Whitney U test was used to compare differences between patient groups. Significance was set at P < 0.05. The JMP 6.0 statistical software package (SAS Institute, Inc., Cary, NC) was used.
CD56pos Cell Levels.
Flow cytometric analysis of CD56pos populations in preinfection blood samples demonstrated that the percentage of total CD56pos lymphocytes did not differ significantly between unexposed normal controls or exposed individuals, irrespective of subsequent outcome. However, as shown in Fig. 1, the lymphocyte subset distribution within the overall CD56pos population was altered in EIs, at a time prior to acquisition of HCV. This subgroup of exposed individuals had decreased levels of CD56low effector NKs (median, 51.48% [range, 26.12%-81.55%], percentage of total CD56pos lymphocytes) compared with the EU group (median, 75.20% [range, 58.60%-80.70%], P = 0.0011), which had similar levels to normal controls (median, 67.76% [range, 43.61%-80.5%]). A higher proportion of NT cells (CD3+/CD56+) contributed to the levels of total CD56pos lymphocytes in the EI group, which demonstrated lower levels of CD56low NKs (data not shown). These data suggest that decreased effector NK levels predispose to HCV acquisition in exposed individuals.
Impaired NK Cytolytic Activity but Intact IFN-γ Production Predates Acquisition of HCV Infection.
Because killing of virally infected cells represents the primary effector function of CD56low NKs, we next tested the cytolytic potential of isolated NKs in our cohorts. This flow-based cytotoxicity assay measures the cytolytic potential of NKs on a per-cell basis.28 As shown in Fig. 2A, NKs (>90% purity) from HCV-exposed EIs had reduced IL-2–induced cytolytic activity against the NK-sensitive cell line K562 at an effector-to-target ratio of 10:1 compared with EUs (P < 0.0001) and normal controls (P = 0.0227). Natural cytotoxicity, lysis in the absence of cytokine stimulation, was similar in all groups (data not shown). These data suggest that lower numbers of effector NKs, coupled with an impaired ability to exert cytolytic effector function in response to IL-2, predisposes to HCV acquisition in high-risk exposed individuals.
In addition to their cytolytic activity, NKs are characterized functionally by their ability to quickly produce IFN-γ, and in vitro studies suggest that it may be this aspect of their functionality that is important for control of virus replication.31, 32 Therefore, we tested the ability of NKs from our cohorts to produce IFN-γ using an intracellular cytokine flow-based assay. As shown in Fig. 2B, the ability to produce IFN-γ is intact for NKs in EIs. These data suggest that IFN-γ production by innate CD56pos NKs does not provide protection from HCV acquisition.
Phenotype of CD56pos Lymphocytes.
Activation of NKs largely depends on the NCR family of molecules and monoclonal antibodies to NCR block NK-mediated lysis of target cells.7 NCRs include NKp46 involved in natural cytotoxicity,33 as well as NKp30 and NKp44, which are expressed on activated NKs.34 Recent studies have highlighted the important role played by NCRs in immunosurveillance of viral infection. Impaired NK function in HIV-1–infected patients has been associated with decreased NCR expression.35 Susceptibility to NK cell lysis of herpes simplex virus–infected cells is dependent on NCR and independent of down-regulation of MHC class I molecules or induction of activating NKG2D ligands.36 Envelope proteins from the Dengue virus and West Nile virus (two other Flaviviruses) bind NKp44.37 Human cytomegalovirus pp65 protein binds NKp30, thereby inhibiting NK activation and promoting virus survival.38 The role played by NCR in chronic HCV infection remains controversial, with both increases and decreases in expression being reported.16, 39 Because we had demonstrated a significant decrease in lymphokine-activated killing (LAK) activity in the patient group that subsequently became infected, we characterized the expression of activating NCRs (p30 and p44), which has been shown to play a role in determining the cytolytic activity of activated NKs.6, 7 We included tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)—another NK/NT cell receptor involved in cell lysis—in our analysis because HCV core protein has been shown to sensitize hepatocytes to TRAIL-induced apoptosis.40
NCR NKp30 expression was significantly up-regulated on both total NKs and NTs in the EU cohort (Fig. 3A). Both CD56high and CD56low NK cell subsets express NKp30 at similar levels. There is a trend for increased NKp30 on both subsets (CD56high, P = 0.0666; CD56low, P = 0.0627). No significant difference in the expression of NCRp44 was demonstrated, although a trend toward reduced NCRp44 on NTs in the EI patient cohort was noted (Fig. 3B). TRAIL was unchanged on NKs and significantly down-regulated on NTs in the EI group (Fig. 3C). NKp30 was the only cytotoxicity receptor tested to be altered on NKs, suggesting that the increase in this receptor may play a role in the enhanced LAK activity in the EU group. This hypothesis is supported by the correlation shown between LAK activity and NKp30 expression on NKs in the entire exposed cohort (Fig. 3D). No correlation was seen for expression of NCR NKp44 (Fig. 3D) or TRAIL (data not shown) either on NKs or NTs. These data suggest that up-regulation of NKp30 may contribute to innate protection against HCV and that this receptor may represent a novel target for immune manipulation.
NKp30high NK Cells Protect Against HCV Infection In Vitro.
As NKp30 expression was significantly up-regulated on NKs and correlated with LAK activity in the patient cohort that remained uninfected despite repeated exposure, we tested the functional significance of NKp30 expression in a relevant replicon model. We used the Huh-7.5 JFH-1 in vitro HCV infection system to compare the ability of FACS sorted NKp30low/neg and NKp30high subsets of NKs to attenuate infection of hepatocytes by HCV. For each of the four normal subjects tested, unstimulated NKs expressing high levels of NKp30 were more effective in preventing infection of Huh-7.5 cells than their NKp30low/neg counterparts (P = 0.0361 for combined data). IL-2 stimulation of NKs overcomes the lack of NKp30 (Fig. 4). In a standard degranulation assay, NKp30high NKs demonstrated more efficient degranulation in response to short-term stimulation compared with their NKp30low counterparts (Fig. 5A). In addition NKp30high NKs express more perforin than NKp30low NKs in the resting state (Fig. 5B,C). IL-2 is likely to overcome the relatively impaired cytotoxicity of the NKp30low population through up-regulation of this receptor on NKs (Fig. 5D). These data provide further evidence that up-regulation of NKp30 in response to HCV exposure may provide protection from infection.
HCV infection represents a considerable public health burden. Efforts to develop a vaccine have been unsuccessful, and treatment of chronic HCV infection remains suboptimal.41 Understanding the immune correlates that contribute to innate protection from HCV acquisition will aid in the development of novel immune-based treatment strategies. The observation that some IDUs remain healthy with no evidence of infection despite continued long-term exposure to HCV4 strongly suggests a role for innate immunity in natural protection from HCV infection. However, because of logistical difficulties in obtaining samples from high-risk individuals prior to HCV infection, the hypothesis that innate immune effector populations contribute to natural resistance to HCV infection had not been tested.
Support for a role for innate effector populations in protection from viral infection in vivo is provided by studies that have demonstrated that enhanced activity of NK30 and NT42 cells contribute to protection from HIV-1 infection in high-risk exposed individuals. In vitro studies provide strong evidence that NK cells play a key role in suppressing HCV infection of human hepatocytes.29 Our unique cohort of prospectively collected peripheral blood samples from high-risk IDUs allows us to address the possible role of these cells in conferring protection from acquisition of HCV infection.
In the present study, we demonstrate that in patients who remain protected from HCV infection, total CD56pos populations are enriched for CD56low effector NKs displaying enhanced IL-2–induced cytolytic activity and higher levels of NKp30-activating NKRs. For the first time, these data support the hypothesis that NKs contribute to anti-HCV defense in the earliest stages of infection, providing protection from HCV acquisition. Of note, IFN-γ production by NKs was comparable with normal controls, suggesting that the cytolytic activity of NKs is more important than cytokine production in mediating protection. This may appear to be contradictory to in vitro studies, suggesting that IFN-γ is key for control of viral replication and HCV infection of human hepatocyte cell lines.29, 31, 32 The contribution of IFN-γ to viral control may vary at different stages of infection. Moreover, there is an association with viral clearance and higher LAK activity in the setting of acute HCV.28 It should be noted that we cannot in the functional assays distinguish the individual contribution of the CD56high/low NK subsets. However, our preinfection data suggest that cytotoxicity is important in protection and control early in infection, but that once chronic infection is established, IFN-γ production by these populations may become more critical for the control of virus.
Our phenotyping panel is not exhaustive, and further studies are required to determine the relative contribution of various NKRs to natural protection. These assays are beyond the scope of this study, because larger numbers of cells than are available to us would be required. However, the observed up-regulation of NKp30 and its correlation with LAK activity suggests a role in innate protection from HCV infection, although we cannot exclude the involvement of other receptors. Our study demonstrated a significant role for at least one NKR (NKp30) in providing innate protection from HCV infection; a larger cohort of patients may identify other important NKRs. The observation that NKp30high NKs significantly reduce infection in the JFH-1 in vitro infection system offers further support for a protective role for NKp30. Of note, this protection was provided without the need for exogenous stimulation by IL-2. This may be of particular importance before induction of adaptive immunity or in the setting of insufficient T cell priming and lack of CD4+ T cell help known to occur in HCV infection.43
In conclusion, our study provides new insight into the mechanisms underlying protection from HCV infection that may have implications for improving immunotherapeutic strategies.
The authors thank Dr. Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan) for kindly providing the JFH-1 plasmid. We also thank the Colorado Center for AIDS Research Laboratory Core for access to FACS.