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Abstract
Background: Natural killer (NK) cells may be impaired in patients with persistent hepatitis C virus (HCV) infection, but studies to date have yielded inconsistent findings due to patient and virus heterogeneity and difficulties obtaining appropriate controls.
Aims: To overcome these variables, we have examined numbers, phenotypes, cytotoxic activities and cytokine profiles of circulating NK cells from Irish women who acquired infection through administration of HCV genotype 1b-contaminated anti-D immunoglobulin from a single source and matched controls.
Results: Comparing 29 women who developed persistent infection with 21 who spontaneously resolved infection and 26 controls, we found that NK cell numbers were consistently lower in the persistently infected group (p = 0.02 and 0.002). This decrease was due to depletions of NK cells expressing low levels of CD56 (CD56dim NK cells; p = 0.004 and 0.0001), whilst CD56bright NK cells were expanded (p = 0.004 and 0.0001). Compared to HCV resolvers, CD56dim NK cells from persistently infected patients less frequently expressed CD16 and more frequently expressed NKG2A/C/E. These phenotypic changes did not significantly affect natural or interleukin-2-induced cytotoxicity by peripheral blood mononuclear cells against K562 and Daudi targets. Greater frequencies of CD56bright NK cells from chronic HCV patients produced interferon-γ compared with HCV responders (p = 0.05) and controls (p = 0.0001) after phorbol ester stimulation in vitro.
Conclusions: Alterations in NK subset distributions in chronic HCV infection may explain why previous reports of impaired NK cell functions were difficult to confirm. Altered NK cell functions may contribute to impaired cellular immune responses and chronicity of disease following HCV infection.
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Hepatitis C virus (HCV) establishes persistent infection in about 80% of infected individuals despite the generation of virus-specific antibody and T lymphocyte responses.1 2 Natural killer (NK) cells are key players in immune responses to viruses. They can kill virus-infected cells via perforin release and induction of apoptosis and can provide early bursts of cytokines, such as interferon-γ (IFN-γ), that have direct antiviral effects and can activate and polarise cytotoxic and helper T (Th) lymphocyte responses.3 4 NK cells are activated and regulated by signals through a variety of stimulatory, co-stimulatory and inhibitory receptors (NKRs), which bind components of pathogens, host cells or cytokines.5 6 Some T cells also express NKRs and mediate functions of both T cells and NK cells.7 8 NK cells and NKR+ T cells make up ∼13% of the lymphocyte pool in human blood and 50% in liver.7 The phenotypes and/or functional activities of various populations of these cells have been reported to be impaired in patients with chronic HCV infection.9–16 Furthermore, HCV has evolved mechanisms by which it can inhibit the responses of NK cells and possibly NKR+ T cells.17–20 The rapid antiviral activities of NK cells and NKR+ T cells place them as candidate therapeutic targets for HCV disease, and phase I trials using autologous NKR+ cells expanded ex vivo are under way for treatment of other diseases that require boosted Th1 and cytotoxic T cell responses.21 22 However, because of logistical difficulties in obtaining samples from patients with acute and resolved HCV infection, the heterogeneity of HCV subtype, viral load and route of transmission, and the presence of concurrent infections in different patients, the above studies have yielded conflicting results and the roles of NK cells and NKR+ T cells in immunity against HCV remain poorly understood.
In this study, we have examined the numbers, phenotypes, cytotoxic activities and cytokine release profiles of circulating NK cells and NKR+ T cells from patients recruited from a homogeneous cohort of Irish women, all of whom acquired HCV from a single source. Comparing 29 women who developed persistent HCV infection with 21 who spontaneously resolved HCV infection and 26 uninfected control subjects, we provide evidence that chronicity is associated with altered distributions of NK cell subpopulations with functional consequences.
METHODS
Study population
We studied a well-defined cohort of Irish women, who were infected with HCV genotype 1b after receiving contaminated anti-D immunoglobulin from a single source during 1977–1978.23 24 All patients attended the hepatitis C clinic at St Vincent’s University Hospital, Dublin. Fifty consecutive patients, who were negative for hepatitis B virus and human immunodeficiency virus infection, had no other risk factors for chronic liver disease, and consumed <14 U alcohol per week, were enrolled in the study. Informed written consent was obtained from each patient. All 50 patients tested positive for antibodies to HCV using an enzyme immunoassay (Abbott Diagnostics, Wiesbaden, Germany) and an immunoblot assay (RIBA-3; Chiron, Emeryville, CA, USA). None of the patients had received antiviral therapy at the time of entry to the study. Twenty-nine patients (median age 55, range 44–66 years) remain chronically infected with the virus as determined by testing consistently positive for HCV RNA by reverse transcriptase–polymerase chain reaction (RT-PCR), (Amplicor; Roche Diagnostic Systems, Nutley, USA), over an 8 year follow-up period. Of these, 18 had viral loads of >2×106 copies/ml and 11 had <2×106 copies/ml at the time of study. Eight had high alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels while these were normal in the remaining 13. According to the Ishak scoring system for fibrosis, two of the chronically infected patients scored 3, four scored 2, four scored 1, and 19 scored 0. Twenty-one patients (median age 51, range 42–61 years) achieved spontaneous (untreated) viral clearance and tested negative for HCV RNA by RT-PCR, on at least six occasions, over a 4 year follow-up period. All who resolved HCV infection had normal ALT and AST levels and fibrosis scores of 0. Blood samples from 26 healthy, HCV-uninfected female donors were studied as controls.
Sample preparation
Venous blood samples were collected in heparinised tubes and peripheral blood mononuclear cells (PBMCs) were prepared by standard Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation.
Antibodies and flow cytometry
Fluorochrome-labelled monoclonal antibodies specific for human CD3, CD16, CD45, CD56, CD94, CD158a, CD158b, CD158e, CD161, NKG2A/C/E, IFN-γ and tumour necrosis factor-α (TNF-α) and isotype-matched controls were obtained from BD-Pharmingen (Oxford, UK). The expression of cell surface or intracellular antigens by fresh or cultured PBMC or purified PBMC subsets was detected by monoclonal antibody staining and four-colour flow cytometry (FACSCalibur, Becton Dickinson, Oxford, UK) using CellQuest software (Becton Dickinson). Lymphocytes were defined by forward and side scatter parameters and by expression of CD45. Frequencies of lymphocyte subpopulations were determined as percentages of CD45+ lymphocytes.
Cytotoxicity assays
Natural and IL-2-induced cytotoxicity by PBMCs against the target cell lines K562 and Daudi were assayed in standard 4 h 51Cr-release assays. All PBMCs were cryopreserved and upon recovery were cultured for 48 h in RPMI 1640 containing 25 mmol/l HEPES, 2 mmol/l l-glutamine, 50 μg/ml streptomycin, 50 U/ml penicillin, and 10% fetal calf serum in the absence or presence of 50 U/ml recombinant human IL2 (National Cancer Institute at Frederick, Frederick USA), to measure natural and IL-2-induced cytotoxicity, respectively. PBMCs were then incubated with target cells labelled with sodium [51Cr]chromate (MP Biochemicals, Mechelen, Belgium), at PBMC/target ratios of 5, 25 and 50 in triplicate wells of 96-well plates and chromium release was measured after 4 h by scintillation counting. Specific lysis was calculated as described previously.7
Detection of intracellular cytokines
PBMCs (106 cells/ml of complete RPMI medium) were stimulated with 10 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich, Poole, UK) plus 1 μg/ml ionomycin (Sigma-Aldrich) in the presence of brefeldin A (10 μg/ml, Sigma) for 4 h in 24-well plates. As controls, unstimulated cells were treated similarly. Cells were then stained with monoclonal antibodies specific for surface CD3 and CD56 and intracellular IFN-γ or TNF-α, and detected by three-colour flow cytometry as described previously.7 Results are expressed as percentages of electronically gated lymphocyte subsets that express cytokine.
Statistical analyses
The non-parametric Mann–Whitney U test was used to compare phenotype frequencies and cytotoxicity between patient groups and p-values of <0.05 were considered significant.
RESULTS
Circulating NK cells but not CD56+ T cells are depleted in patients with chronic HCV infection
Flow cytometric analysis of CD3 and CD56 expression by PBMC was used to define NK cells (CD3-CD56+) and CD56+ T cells (CD3+CD56+) (fig 1A). Total CD56+ cell frequencies were decreased in HCV-infected patients who failed to clear the virus (median 12%, range 2–17% of lymphocytes) compared with those who spontaneously cleared the virus (15%; range 6–27%; p = 0.03) and healthy controls (18%; range 7–37%; fig 1B). This decrease was not due to decreased CD56+ T cell frequencies but to decreased CD56+CD3− NK cell frequencies (9%; range 2–13%) compared to 11% of HCV resolvers (range 4–21%; p = 0.02) and 13% of controls (range 4–33%; p = 0.002; fig 1B). Lymphocyte subset frequencies did not correlate with viral load or degree of fibrosis (data not shown).
NK cell and CD56+ T cell phenotypes in patients with chronic and resolved HCV infection
The expression of three killer immunoglobulin-like receptor (KIR) families, CD158a, CD158b and CD158e, the human NKR-P1 molecule CD161, the lectin inhibitory receptor dimer components CD94 and NKG2A/C/E, and the Fc receptor for immunoglobulin G (IgG) CD16, by electronically gated NK cells and CD56+ T cells from patients and controls was examined by flow cytometry. Frequencies of CD158a, CD158b, CD158e, CD161 and CD94 expression by NK cells were similar in the three subject groups (fig 2A). Expression of CD16 by NK cells was significantly lower in the chronically infected patients (median 72%; range 26–94%) compared with the HCV resolvers (84%; 66–98%; p = 0.02) and controls (95%; 92–98%; p = 0.0001). Expression of NKG2A/C/E was increased in chronic HCV patients (55%; 31–80%) compared with the spontaneous resolver group only (48%; 5–71%; p = 0.05; fig 2A). The frequencies of CD158a, CD158b, CD158e, CD161, CD94, CD16 and NKG2A/C/E expression by CD56+ T cells were not significantly different in patients with chronic and resolved HCV infection and controls (fig 2B).
CD56bright and CD56dim subsets of NK cells in patients with chronic and resolved HCV infection
Two functional subsets of NK cells, defined by differing intensities of CD56 expression, are found in human blood25–27 (fig 1A). In our patient groups, CD56bright cells were found slightly but consistently more frequently in chronically infected patients (11% of NK cells; range 2–38%) compared with HCV resolvers (7%; 2–15%, p = 0.004) and controls (4%; 1–12%; p = 0.0001; fig 3A). In contrast, CD56dim NK cells were found consistently less frequently (89%; 62–98%) compared with HCV resolvers (93%; 85–98%;p = 0.004) and controls (96%; 87–98%; p = 0.0001; fig 3B). The majority of CD56bright NK cells were negative for KIR and CD16 expression and positive for CD94 and NKG2A/C/E (fig 3C) with similar percentages of CD56bright cells expressing these phenotypes in the chronically infected, HCV resolver and control groups. In contrast, within the CD56dim NK cell population, CD16 was expressed less frequently in chronic HCV patients (76%; 26–94%) compared with HCV resolvers (84%; 66–98%; p = 0.03) but not controls (73%), while NKG2A/C/E expression was more frequent in chronic HCV (56%; 31–79%) compared to resolvers (48%; 5–71%; p = 0.05) and controls (17%; 7–24%; p = 0.0001; fig 3D). The frequencies of CD158a, CD158b, CD158e, CD161 and CD94 expression by CD56dim cells were similar in all subject groups.
NK cytotoxicity is not significantly impaired in patients with chronic HCV infection
Natural and IL-2-induced cytotoxicity by PBMCs from 10 patients with resolved HCV infection, 11 with chronic HCV infection and six age-matched female control subjects, against K562 and Daudi target cells was measured in chromium-release assays. Figure 4 shows that PBMCs from all subject groups displayed moderate natural cytotoxicity against K562, but not Daudi cells, and IL2-induced cytotoxicity against both targets. For each type of cytotoxicity, the percentages of target cells that were specifically lysed were similar in all groups at all PBMC/target ratios tested (fig 4).
Cytokine expression by NK cell subsets in patients with chronic and resolved HCV infection
PBMCs were stimulated in vitro with PMA and ionomycin and intracellular expression of IFN-γ and TNF-α by individual lymphocyte subsets from 18 healthy control subjects, 10 HCV resolvers and eight chronically infected patients was examined by flow cytometry (fig 5A). The frequencies of IFN-γ expression by electronically gated T cells and CD56+ T cells were similar in all subject groups (fig 5B). NK cells from HCV resolvers and persistently infected patients produced IFN-γ at higher frequencies (14% and 18%) compared with controls (3%; p = 0.0004 and 0.0001). Medians of 43% (17–60%) of CD56bright NK cells from chronic HCV-infected patients expressed IFN-γ compared to 22% (range 6–45%; p = 0.05) from HCV resolvers and 10% of controls (5–22%; p = 0.0001). Analysis of CD56dim cells revealed no differences in the frequencies of IFN-γ-positive cells in the two patient groups (12% in each; fig 5B) but these frequencies were higher than those in controls (3%; p = 0.0008). The proportions of all lymphocyte subsets that expressed TNF-α upon stimulation were similar in all subject groups (data not shown).
DISCUSSION
An impairment of NK cell function in patients with chronic HCV infection is suggested by several observations. Circulating NK cells numbers and/or cytotoxic activities have been reported in several studies to be lower in chronically infected patients compared to healthy controls,9 10 12 14 although this has been refuted in other studies.16 28 Other workers have reported phenotypic and functional changes in particular subsets of NK cells in HCV-infected individuals.13 14 16 Khakoo and co-workers29 have provided evidence that inheritance of particular KIR genes, which control NK cell activity, may predispose HCV-infected individuals to chronic infection, while other studies suggest that HCV can modulate NK cell activity, either directly by binding of the HCV envelope-2 protein to CD8117 18 or indirectly by inducing the expression of inhibitory ligands for NK cells.19 20 HCV may also inhibit NK cell activation by altering the functional activities of myeloid and/or plasmacytoid dendritic cells.30–33 T cells that exhibit NK phenotypes and functions, including CD56+ T cells11 15 34 and natural killer T (NKT) cells expressing invariant Vα24Vβ11 T cell receptors,11 35 have also been reported to be depleted in blood and/or livers of patients with chronic HCV infection.
The diversity of observations in the above-mentioned studies is likely to be due, in part, to heterogeneity of patients, diversity of HCV subtypes and viral loads, variations in the duration of HCV infection and the presence of other microbial infections. We have attempted to overcome these variables by examining the numbers, phenotypes, cytotoxic activities and cytokine profiles of circulating NK cells and NKR+ T cells from patients recruited from a homogeneous cohort of Irish women who acquired HCV genotype 1b infection through contaminated anti-D immunoglobulin from a single source.23 24 We selected 50 untreated women from this cohort 24 years later, who had no other risk factors for the development of chronic liver disease. Twenty-one of these women spontaneously resolved HCV infection while 29 women remained persistently infected.
Our results indicate that NK cells, but not CD56+ T cells, were significantly depleted in our series of patients with chronic HCV infection compared to the HCV resolvers and controls. As previously reported,14 16 we found that this reduction was due to the CD56dim subset of NK cells. CD56dim NK cells normally account for >90% of peripheral NK cells. They usually express CD16, KIRs and homing markers for inflamed peripheral sites.26 They carry perforin, and are the main mediators of NK cytotoxicity.4 25 27 CD56bright NK cells were expanded in the patients with chronic HCV infection compared to resolvers and controls. These cells account for <10% of peripheral NK cells. They express homing markers for secondary lymphoid tissues where they accumulate.26 36 They do not express KIRs, contain low levels of perforin, and are only weakly cytotoxic. However, CD56bright NK cells are important secretors of cytokines, including IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor, interleukin 10 (IL10) and IL13.4 25 27
The depletions of CD56dim NK cells found in patients with persistent HCV infection were not mirrored by findings of decreased natural and IL2-induced cytotoxicity by PBMCs against K562 and Daudi target cells. Thus, our data agree with other studies,16 28 37 which reported no significant reductions in NK cytolytic activity in patients with chronic HCV infection, but disagree with several other studies.9 10 12 15 The low levels of cytotoxicity measured in the present study might have been improved if enriched NK cell populations were used as effectors; however, Morishima and co-workers,16 using PBMCs enriched for NK cells, also found no decrease in cytotoxicity in HCV patients. However, these authors reported an association between low NK cytotoxic activity and the development of fibrosis, suggesting that NK activities may influence the outcome of HCV infection.
NK cells are important early secretors of IFN-γ.4 25 27 We found that, as well as being expanded, CD56bright NK cells from patients with persistent HCV infection produced IFN-γ more frequently than those from HCV resolvers or controls in response to stimulation in vitro. CD56dim NK cells from persistent and resolved HCV patients also produced IFN-γ at greater frequencies compared to CD56dim cells from controls. These observations are consistent with a previous report14 that overall numbers of IFN-γ-expressing NK cells are increased in chronic HCV patients. We do not know if these increased frequencies of IFN-γ-producing NK cells, induced by non-specific stimulation in vitro, translate into increased IFN-γ levels in vivo, but it is likely that early IFN-γ release by NK cells could have a major impact on the hepatic immune system. IFN-γ production by NK cells is associated with both the induction of virus-specific T cell responses and liver injury in murine models of viral infection.38–40 We have reported11 that hepatic IFN-γ-producing CD4+ T cells are expanded in the livers of chronic HCV patients. IFN-γ-secreting NK cells may therefore contribute both to the polarisation of T cell responses and liver pathology.
The functional activities of NK cells are controlled by a variety of stimulatory, co-stimulatory and inhibitory receptors.5 6 To determine whether NKR expression is altered in chronic HCV infection we quantified positivity for CD16, CD161, CD94, NKG2A/C/E and the KIR isotype families, CD158a, CD158b and CD158e in the subject groups. Similar to the findings of De Maria and co-workers,37 we found that the proportions of CD56dim NK cells expressing CD16 were reduced in patients with chronic HCV infection. CD16, the Fc receptor for IgG, mediates antibody-dependent cellular cytotoxicity41 and decreased CD16-mediated NK cytotoxicity has been observed after ligation of CD81 by the HCV E2 protein.18 Future studies using anti-CD16 antibody-redirected killing assays may confirm or refute a role for CD16 downregulation in HCV persistence. In the present study, the proportions of CD56dim NK cells expressing NKG2A/C/E were higher in patients with chronic HCV infection compared to resolvers and controls. This is consistent with previous reports of expansions of NKG2A+ NK cells in HCV-infected patients,37 42 but the antibody used in our study failed to distinguish the inhibitory NKG2A isotype from the non-inhibitory NKG2C and E isotypes, all of which can dimerise with CD94 to form a receptor for HLA-E.43 44 Redirected cytotoxicity assays using antibodies to NKG2A/C/E may ascertain whether the increased expression of this receptor family by NK cells from chronic HCV patients leads to augmented or diminished cytotoxicity.
The results of the present study provide evidence that circulating cytotoxic CD56dim NK cells are depleted and IFN-γ-producing CD56bright NK cells are expanded in patients with chronic HCV infection compared with HCV resolvers and controls in a homogenous subject population. These altered NK cell subset distributions are unlikely to be due to selective sequestration in the liver because NK cells are not expanded in HCV-infected livers11 34 and their numbers in blood positively correlate with those in liver.45 The changes could reflect alterations in the rates of production of the two NK cell subsets. Consistent with this idea, defects in the production of IL15, a key cytokine involved in NK cell development and homeostasis, have been reported in patients with chronic HCV infection.14 32 Alternatively, the altered NK cell subset distributions in persistently infected patients could be the result of decreased rates of differentiation of CD56bright cells into CD56dim NK cells.36 46 47 Future studies are required to ascertain how these phenotypic and functional changes to NK cells arise.
Our subject population provides the advantage of being able to study a homogenous patient cohort with regard to age, sex and ethnicity, all infected for the same length of time with a similar dose of a single HCV isolate from a single source. However, although this stringent patient selection procedure minimises the number of variables that need to be controlled, the results may not be typical of HCV infection. These patients exhibited an unusually high rate of spontaneous viral clearance and most had mild disease with only slightly elevated liver enzymes and normal liver histology.23 24 It is possible that they exhibit less-pronounced changes to NK cells. Therefore, similar studies on other patient cohorts, including males, patients with cirrhosis and end-stage liver disease, patients infected with other HCV genotypes, via other modes of transmission and with different viral loads are needed to demonstrate the universality of the findings.
Acknowledgments
We are very grateful to all who provided blood samples for this study.
REFERENCES
Footnotes
Funding: This work was supported by the Irish Health Research Board and Science Foundation, Ireland.
Competing interests: None.
Ethics approval: This study received ethics approval from the Research and Ethics Committee at St Vincent’s University Hospital, 11 December 2002, and conforms to the guidelines of the 1975 Declaration of Helsinki.
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