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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN-γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)-induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN-α-based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN-α-based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor–apoptosis-inducing ligand [TRAIL] expression and degranulation) and decreased IFN-γ production. NK cells from patients with a greater than 2 log10 first-phase HCV RNA decline to IFN-α-based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log10 first-phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN-α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN-α during the second-phase virological response. Conclusion: These data show that IFN-α-induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN-γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN-α-based therapy. (Hepatology 2012)

Natural killer (NK) cells are innate immune cells best known for their immediate effector functions against virus-infected cells and tumor cells.1 These effector functions include the destruction of target cells via perforin/granzyme-mediated lysis or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the production of cytokines, such as tumor necrosis factor alpha (TNF-α), macrophage-inflammatory protein 1 beta, and interferon gamma (IFN-γ).1 IFN-γ, in particular, has elicited great interest because it is abundantly produced, has direct antiviral activity, and provides a link between innate and adaptive immunity by contributing to the priming of cluster of differentiation (CD)4+ and CD8+ T cells and via the induction of chemokines to T-cell recruitment to the target organ.2

Different effector functions have traditionally been associated with specific NK cell subsets, which can be distinguished based on CD56 expression. Approximately 90% of NK cells in the peripheral blood express low levels of CD56 on their cell surface. These CD56dim NK cells respond quickly to viral infection, exert cytotoxicity, and produce chemokines and cytokines within hours.3-5 The remaining 10% of NK cells with high levels of CD56 expression (CD56bright) respond slower and produce large amounts of IFN-γ and TRAIL with little perforin/granzyme-mediated cytotoxicity.

We and others have recently shown that patients with chronic hepatitis C virus (HCV) infection display a polarized NK cell phenotype with increased cytotoxicity and TRAIL production and decreased IFN-γ production.6-8 Induction of cytotoxicity and production of IFN-γ require differential signal transducer and activator of transcription (STAT)1/4 signaling, as previously shown in a mouse model of lymphocytic choriomeningitis virus (LCMV)-induced hepatitis.9 In this model, virus-induced type I IFN results in the increased expression of STAT1, which competes with STAT4 in signaling events downstream of the IFN-α/β receptor.9 The result is preferential STAT1 over STAT4 phosphorylation, increased NK cell cytotoxicity, and decreased IFN-γ production.9, 10 Interestingly, Miyagi et al. demonstrated increased STAT1 levels in the NK cells of HCV-infected patients, as compared to healthy controls, and showed that in vitro stimulation with IFN-α resulted in preferential STAT1 over STAT4 phosphorylation.11 However, a demonstration that changes in IFN signaling correlate with changes in NK cell function in HCV-infected patients has not yet been provided. Furthermore, the kinetics of the in vivo responsiveness of NK cells to IFN in humans are not known and may be very important for the therapeutic use of IFN-α (e.g., for the therapy of chronic HCV infection).

To address these points, we performed a prospective analysis of STAT expression and phosphorylation in NK cells in chronic HCV infection and during the first 12 weeks of IFN-α-based therapy. This time period defines an early virological response (EVR), which is predictive of the ultimate treatment outcome.12 Changes in STAT signaling during this time period were correlated with changes in NK cell effector functions. In addition, the study included several time points during the first 48 hours of treatment, which allowed us to correlate changes in IFN-induced signaling in NK cells to the first-phase decline in HCV titer.13 The results provide novel insights into the mechanisms of IFN responsiveness and refractoriness of NK cells during viral infection and IFN-α-based therapy.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Study Cohort.

Peripheral blood NK cells were studied in 10 healthy subjects without HCV infection and 35 untreated patients with chronic HCV infection. Twenty-four patients with chronic HCV infection (Table 1) were prospectively studied during treatment with pegylated interferon (PegIFN) alpha-2a (180 μg/week subcutaneously) and weight-based ribavirin (RBV; 1,000 mg for <75 kg body weight and 1,200 mg for ≥75 kg body weight per oral [PO] daily for HCV genotypes 1 and 4 and 800 mg PO daily for HCV genotypes 2 and 3) 4 weeks and 0 hours before treatment and 6, 24, and 48 hours and 1, 2, 4, and 12 weeks after treatment initiation. The week 1, 2, 4, and 12 samples were drawn before the weekly PegIFN injection. Two patients consented to an additional blood draw 6 hours after the week 12 PegIFN injection. All subjects gave written informed consent under protocols approved by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Institutional Review Board, conforming to the ethical guidelines of the 1975 Declaration of Helsinki.

Table 1. Epidemiological and Clinical Data of HCV-Infected Patients
 Chronic HCV Patients, Not Treated With PegIFN/RBV (n = 35)Chronic HCV Patients, Studied During PegIFN/RBV Therapy (n = 24)
  • Abbreviations: HCV, hepatitis C virus; PegIFN, pegylated interferon; RBV, ribavirin; n.a., not applicable; IQR, interquartile range; SNP, single-nucleotide polymorphism; n.d., not done; SEM, standard error of the mean; ALT, alanine aminotransferase.

  • *

    One patient did not yet reach the week 12 time point and thus was not evaluated for early virological response.

Early virological response, n (%)n.a.23 (100)*
Gender (male/female)20/1516/8
Age at start of treatment, median (IQR) years49.0 (44-55)53.5 (50-55)
Ethnicity (Asian/African American/Caucasian/Hispanic)7/10/18/03/5/15/1
Body mass index, median (IQR)29.0 (23.7-34.1)28.9 (23.6-33.5)
IL-28B rs12979860 SNP (CC/CT/TT)n.d.13/6/3
HCV genotype (1/2/3/4/6)19/5/5/4/1/n.d.13/8/2/1
Serum HCV RNA titer at start of treatment, mean (± SEM) log10 IU/mL6.04 (± 0.11)6.30 (± 0.15)
ALT level at start of treatment, median (IQR) U/mL74 (44-88)80 (45-112)

NK Cell Analysis

IFN-α signaling

Expression of STAT1, phosphorylated STAT1 (pSTAT1), and pSTAT4 were assessed either directly in vivo or after in vitro stimulation of prewarmed heparinized blood without or with 600 ng/mL of consensus sequence IFN-α (InterMune Inc., Brisbane, CA) for 5 minutes at 37°C. Cells were fixed and erythrocytes were lysed by incubation with a 20-fold excess volume of Lyse/Fix buffer (BD Biosciences, San Jose, CA) for 10 minutes at 37°C. After centrifugation, cells were permeabilized with Perm Buffer (BD Biosciences) for 20 minutes on ice, washed twice, and resuspended in Staining Buffer (BD Biosciences).

All samples were stained with anti-CD56-PE (phycoerythrin) (Beckman Coulter, Brea, CA) and anti-CD20-PerCP/Cy5.5 to identify NK cells and B cells, respectively, and with anti-CD3/fluorescein isothiocyanate or anti-CD3-APC to exclude T cells. Cells were additionally stained with anti-STAT1-Alexa647, anti-pSTAT1-Alexa488 (which assesses tyrosine phosphorylation at Y701), or anti-pSTAT4-Alexa488 (assesses tyrosine phosphorylation at Y693) for 20 minutes at room temperature and analyzed on an LSRII with FacsDiva version 6.1.3 (BD Biosciences) and FlowJo version 8.8.2 (Tree Star, Ashland, OR) software.

Degranulation

Thawed peripheral blood mononuclear cells (PBMCs) were cultured overnight at 37°C in 5% CO2 in Roswell Park Memorial Institute 1640 medium with 10% fetal calf serum (Serum Source International, Charlotte, NC), 1% penicillin/streptomycin, 2 mM of L-glutamine, and 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Cellgro, Manassas, VA). The next day, PBMCs were counted and stimulated in the presence or absence of K562 cells (ATCC, Manassas, VA) to assess degranulation, as previously described,6 but in the absence of additional cytokines.

TRAIL expression

Thawed PBMCs were stained with ethidium monoazide, anti-CD19-PeCy5 (BD Biosciences), anti-CD14-PeCy5 (Serotec, Raleigh, NC), anti-CD56-PeCy7, anti-CD3-AlexaFluor700 (BD Biosciences), and anti-TRAIL-PE (BD Biosciences).

IFN-γ production

Thawed PBMCs were incubated with or without interleukin (IL)-12 (0.5 ng/mL; R&D Systems, Minneapolis, MN) and IL-15 (20 ng/mL; R&D Systems) for 14 hours, followed by the addition of brefeldin A for 4 hours and intracellular staining for IFN-γ, as previously described.6

Viral Kinetics.

HCV RNA levels were measured using Cobas TaqMan real-time polymerase chain reaction (Roche Diagnostics, Palo Alto, CA), with a lower limit of detection of 15 IU/mL. The first-phase virological response was defined as the logarithmic decline in HCV RNA titer during the first 48 hours of therapy.

Genotyping.

DNA samples were genotyped for the IL-28B rs12979860 polymorphism with a TaqMan genotyping assay (Applied Biosystems Inc., Foster City, CA).

Statistical Analysis.

GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA) and JMP (SAS Institute Inc., Cary, NC) software was used to perform the (1) Mann-Whitney nonparametric two-sample rank test to compare NK cells from healthy subjects and patients, and from patients with strong and weak first-phase responses, (2) repeated measures analysis of variance (ANOVA) to assess changes in STAT1 and pSTAT1 expression during treatment, (3) Wilcoxon signed rank test to determine changes in pSTAT1 and pSTAT4 levels and pSTAT1/pSTAT4 ratio from baseline to time points during treatment, and (4) Spearman correlation analysis to study changes in pSTAT1 signaling in relation to NK cell function. A two-sided P value of less than 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Increased STAT1 Expression in NK Cells During HCV Infection Is Further Enhanced During PegIFN/RBV Therapy.

We have previously described that NK cells are activated in HCV infection, but that activation does not result in equal stimulation of all effector functions.6 Specifically, NK cells of patients with chronic HCV infection display enhanced cytotoxicity, as evidenced by increased degranulation and TRAIL production and decreased IFN-γ production, as compared to uninfected controls.6 We demonstrated that this phenotype can be reproduced by in vitro stimulation of NK cells from healthy, uninfected controls with IFN-α.6

To evaluate how the IFN-α-based treatment of chronic HCV would modulate NK cell phenotype and function, we first studied the in vivo level of STAT1 in peripheral blood NK cells of chronically HCV-infected patients (Table 1) and healthy controls. The total NK cell population of HCV-infected patients, as well as their CD56bright and CD56dim subsets, showed increased levels of STAT1, when compared to healthy controls (Fig. 1A). This increase in STAT1 expression was not observed for T cells (Supporting Fig. 1A). We then prospectively followed a group of HCV-infected patients during the first 12 weeks of IFN-based therapy for HCV. All patients mounted an early virological response (i.e., serum HCV RNA levels at least 2 log10 lower at week 12 than before treatment). STAT1 expression increased significantly in the total NK cell population and the CD56bright and CD56dim subsets within 24 hours of therapy, and STAT1 levels increased further throughout the study period of 12 weeks (Fig. 1B-D; P < 0.0001 for all populations). The same increase in STAT1 expression was observed in CD3+CD56 T cells (Supporting Fig. 1B).

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Figure 1. Increased expression of STAT1 in NK cells during HCV infection is further enhanced by PegIFN/RBV therapy. (A) In vivo STAT1 expression in CD3CD56+ NK cells and their CD56bright and CD56dim subpopulations in HCV-infected patients and healthy, uninfected blood donors. (B-D) In vivo STAT1 expression levels of all CD3CD56+ NK cells (B) and their CD56bright (C) and CD56dim (D) subpopulations during therapy with PegIFN/RBV. Mean ± standard error of the mean (SEM) are shown for 14 patients undergoing PegIFN/RBV therapy. h, hour; wk, week. ***P < 0.001 by repeated measures ANOVA.

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PegIFN/RBV Therapy Induces Preferential Phosphorylation of STAT1 Over STAT4 in NK Cells.

To evaluate changes in signaling downstream of the IFN-α/β receptor, we next studied STAT1 and STAT4 phosphorylation at all treatment time points. Changes in pSTAT1 and pSTAT4 expression were greatest within the first 48 hours of therapy. In vivo pSTAT1 levels peaked in CD3CD56+ NK cells and in their CD56bright and CD56dim subsets within 6 hours of therapy (mean fluorescence intensity [MFI] 163 ± 16 at baseline and 205 ± 20 at maximum; P = 0.005, P = 0.018, and P = 0.003, respectively; Fig. 2A).

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Figure 2. PegIFN/RBV therapy results in preferential phosphorylation of STAT1 over STAT4 in NK cells. (A, B) Maximal changes in pSTAT1 (A) and pSTAT4 (B) expression levels in chronic HCV patients of total CD3CD56+ NK cells (left panel) and their CD56bright (middle panel) and CD56dim (right panel) subsets before and after PegIFN/RBV therapy initiation. (C) pSTAT1/pSTAT4 ratio (MFI) throughout the first 48 hours of therapy. *P < 0.05; **P < 0.01; ***P < 0.001 (comparing the indicated individual time points to the 0-hour time point).

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In contrast, pSTAT4 levels decreased in the overall CD3CD56+ NK cell population and in their CD56bright and CD56dim subsets in response to IFN-based therapy, reaching a minimum at the 48-hour time point (MFI 183 ± 10 at 0 hours and 149 ± 8 at 48 hours; P = 0.011, P = 0.023, and P = 0.028; respectively; Fig. 2B). Because STAT1 and STAT4 signaling molecules both compete for phosphorylation at the IFN-α/β receptor,9 these data suggest that an increase in the expression of STAT1 (Fig. 1) results in the preferential phosphorylation of STAT1 over STAT4 during IFN-based therapy (Fig. 2). Consistent with this interpretation, the pSTAT1/pSTAT4 ratio peaked 6 hours after initiation of therapy and remained increased up to 48 hours in the CD56dim NK cell subset (Fig. 2C).

PegIFN/RBV-Induced STAT1 Phosphorylation Correlates to Polarization of NK Cell Function.

In a detailed prospective analysis, we showed previously that NK cell effector functions are strongly induced in response to IFN-α.14 NK cell cytotoxicity, as determined by TRAIL expression (Fig. 3A, left panel) and degranulation (Fig. 3B, left panel), peaked as early as 6 and 24 hours, respectively. Conversely, the frequency of IFN-γ producing NK cells reached its minimum 6 hours after treatment initiation (Fig. 3C, left panel) and never increased above pretreatment levels at later time points.14 Importantly, the increase in cytotoxicity, as evidenced by TRAIL production, directly correlated with the increase in pSTAT1 levels (r = 0.586, P = 0.014; Fig. 3A, right panel), and the increase in NK cell degranulation followed the same trend (r = 0.453, P = 0.078; Fig. 3B, right panel). In contrast, the change in IFN-γ production correlated inversely with the increase in pSTAT1 levels (r = 0.549, P = 0.015; Fig. 3C, right panel). These results support the interpretation that the polarization of NK cell function in patients with chronic HCV is mediated by IFN-α, because IFN-based therapy further drives this functional dichotomy by the induction of pSTAT1.

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Figure 3. PegIFN/RBV-induced changes in pSTAT1 expression in NK cells correlate to changes in NK cell function. Changes in TRAIL production (A, left graph), degranulation (B, left graph), and IFN-γ production (C, left graph) in response to PegIFN/RBV therapy initiation, in correlation to changes in pSTAT1 expression level from 0 to 6 hours (A-C, right graphs). r, Spearman correlation coefficient.

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Prolonged PegIFN/RBV Therapy Results in Refractoriness of NK Cells to In Vitro IFN-α Stimulation.

To evaluate whether NK cells are maximally stimulated by IFN-based therapy in vivo we isolated PBMCs at numerous time points within the first weeks of treatment, subjected them to in vitro stimulation with IFN-α and determined their pSTAT1 levels. In vitro–induced pSTAT1 levels decreased after the initial 6 hours of PegIFN/RBV treatment, reached their minimum after the first week of PegIFN/RBV treatment, and remained low for the following 11 weeks of the study period (MFI at 0 hours: 407 ± 37; at 24 hours: 279 ±2 5; at week 12: 181 ± 24, P = 0.039; Fig. 4A). The same kinetics were observed when the in vitro inducibility of pSTAT1 was normalized either to in vivo pSTAT1 levels (Fig. 4B) or total STAT1 levels at each individual treatment time point (Supporting Fig. 2). These results demonstrate that maximal pSTAT1 induction was reached very early during PegIFN/RBV therapy (between 6 and 48 hours), and that NK cells remained refractory to further stimulation. To ensure that these observations were not a result of sampling at nadir time points (i.e., just before the weekly PegIFN injection), we studied 2 patients after the first injection and after the week 12 injection of PegIFN. In vivo pSTAT1 levels increased in NK cells within 6 hours after the first PegIFN injection (Fig. 4C). However, no increase was observed in the 6 hours following the week 12 PegIFN injection. Thus, prolonged exposure to IFN-α appears to render NK cell refractory over the course of treatment.

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Figure 4. Refractoriness of pSTAT 1 inducibility in NK cells during PegIFN/RBV therapy. (A) pSTAT1 levels in CD3CD56+ NK cells after in vitro stimulation with IFN-α (pSTAT1 inducibility). (B) In vitro pSTAT1 inducibility in CD3CD56+ NK cells at the indicated PegIFN/RBV time points relative to in vivo pSTAT1 expression at the same time points. Inducibility was calculated as the fold change in pSTAT1 MFI after in vitro treatment with IFN at each time point. Mean ± SEM are shown for 21 patients. *P ≤ 0.05, **P < 0.01 by repeated measures ANOVA. (C) In vivo pSTAT1 levels in NK cells before and 6 hours after PegIFN injection at start (left panel) and 12 weeks (right panel) of PegIFN/RBV therapy. h, hour; wk, week.

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Patients With a Weak First-Phase Virological Response Do Not Reach Maximal pSTAT1 Induction In Vivo.

To evaluate a potential association of NK cell responsiveness with treatment efficacy, we determined the decline in HCV RNA in the peripheral blood during the first 48 hours of treatment. This is defined as the first-phase virological response and predicts treatment outcome.13 Because chronic infection with HCV genotypes 1 and 4 requires a longer course of treatment than chronic infection with HCV genotypes 2 and 3,15 we limited this analysis to patients infected with HCV genotypes 1 and 4 (Table 2). Patients with a strong first-phase virological response (defined as greater than 2 log reduction in HCV RNA titer in the first 48 hours) displayed a significantly greater increase of in vivo pSTAT1 levels in NK cells during the first 6 (Fig. 5A,B) and 24 hours (Fig. 5C) of therapy than patients with a weak first-phase virological response (less than 2 log reduction). This was independent of the IL-28 genotype (another determinant of treatment outcome),16 because neither in vivo pSTAT1 levels nor in vitro pSTAT1 inducibility in NK cells correlated to the IL-28B single-nucleotide polymorphism (SNP) at rs12979860, an independent factor of treatment responsiveness.

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Figure 5. Correlation of pSTAT1 expression and first-phase virological response. (A) Fold change in in vivo pSTAT1 expression by CD3CD56+ NK cells during the first 6 hours of PegIFN/RBV therapy in individual patients with (solid lines) and without (broken lines) a greater than 2 log10 first-phase decline in HCV RNA titer. (B, C) Fold change in in vivo pSTAT1 expression by CD3CD56+ NK cells during the first 6 (B) and 24 hours (C) of PegIFN/RBV therapy in individual patients with and without a greater than 2 log10 first-phase decline in HCV RNA titer. (D) Fold change in pSTAT1 inducibility (pSTAT1 MFI after in vitro treatment with IFN normalized to in vivo levels before [0 hours] or 6 hours after initiation of PegIFN/RBV therapy) in individual patients with (solid lines) and without (broken lines) a greater than 2 log10 first-phase decline in HCV RNA titer. (E, F) Fold change in pSTAT1 inducibility in NK cells (pSTAT1 MFI after in vitro treatment with IFN normalized to in vivo levels before [0 hours] or 6 [E] or 24 hours [F] after initiation of PegIFN/RBV therapy) in individual patients with and without a greater than 2 log10 first-phase decline in HCV RNA titer.

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Table 2. Epidemiological and Clinical Data of Patients With and Without a Strong First-Phase Virological Response
 Patients With a Greater Than 2log10 48-h Decrease in HCV Titer (n = 4)Patients With a Less Than 2log10 48-h Decrease in HCV Titer (n = 10)
  • Abbreviations: HCV, hepatitis C virus; IQR, interquartile range; SNP, single-nucleotide polymorphism; SEM, standard error of the mean; ALT, alanine aminotransferase.

  • *

    One patient did not yet reach the week 12 time and thus was not evaluated for sustained virological response.

Decrease in HCV titer 0-48 hours, median log10 (IQR)2.59 (2.2-2.9)1.15 (0.7-1.6)
Early virological response, n (%)4 (100)9 (100)*
Gender (male/female)3/16/4
Age at start of treatment, median (IQR) years57.5 (54.3-60.8)53.0 (47.3-55.5)
Ethnicity (Asian/African American/Caucasian/Hispanic)1/1/2/01/3/5/1
Body mass index, median (IQR)30.8 (22.9-34.5)29 (25.6-35.9)
IL-28B rs12979860 SNP (CC/CT/TT)3/0/04/4/2
HCV genotype (1/4)4/07/1
Serum HCV RNA titer at start of treatment, mean (± SEM) log10 IU/mL6.6 (± 0.15)6.6 (± 0.31)
ALT level at start of treatment, median (IQR) U/mL61 (36-98)83 (62-179)

There are two possible explanations for the lower IFN responsiveness of NK cells in patients with a weak first-phase virological response. One possibility is that their level of NK cell responsiveness to IFN is genetically predetermined. The other possibility is that their NK cells are suboptimally stimulated in vivo. To differentiate between both possibilities, we subjected PBMCs of patients with and without a strong first-phase virological response to further in vitro stimulation with IFN-α. Interestingly, NK cells from patients with a <2 log10 first-phase HCV RNA decline exhibited greater in vitro inducibility of pSTAT1 than NK cells from patients with a greater first-phase response (Fig. 5D-F). These results suggest that NK cells of patients with a weak first-phase virological response are suboptimally stimulated in vivo.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study shows that IFN-α-induced modulation of STAT1 phosphorylation underlies the in vivo polarization of NK cells toward increased cytotoxicity and decreased IFN-γ production. This result is consistent with the observation that LCMV-induced IFN-α secretion in mice has been shown to increase STAT1 expression in NK cells, resulting in preferential STAT1 over STAT4 phosphorylation.6, 9, 11 It also extends the findings by Miyagi et al. on preferential STAT1 phosphorylation in HCV-infected patients,11 because we show that IFN-α exposure in vivo results in increased pSTAT1 levels, and that it correlates to increased TRAIL production and degranulation and decreased IFN-γ production (Fig. 3). The clinical relevance of IFN-α signaling in NK cells is suggested by our observation that NK cell responsiveness and refractoriness correlate with the first-phase virological response to IFN-α-based therapy (Fig. 5).

This analysis of NK cells is relevant for current research on biomarkers predicting IFN responsiveness and treatment outcome. Advantages of using NK cells as biomarkers of IFN responsiveness are that they are readily accessible from the peripheral blood, and that both in vivo and in vitro NK cell responsiveness can easily be assessed in a short, standardized flow-cytometry–based assay by checking pSTAT1 levels. How does our system compare to other biomarkers of IFN responsiveness? A well-established biomarker for IFN responsiveness is the intrahepatic expression of interferon-stimulated genes (ISGs). Typically, ISGs are most highly expressed pretreatment in HCV-infected patients who respond poorly to IFN-α-based therapy.17 As a potential explanation, it has been proposed that high baseline activation of the endogenous IFN system does not allow a further increase of ISGs during IFN-based therapy, possibly because the ISG response has already reached maximal levels and/or inhibitory autocrine feedback mechanisms have been induced.18 In contrast to these ISG data, we did not find any evidence that pretreatment pSTAT1 levels or in vitro inducibility differed among HCV-infected patients (data not shown). Thus, pSTAT1 induction is an independent measure for IFN responsiveness and may complement ISG analysis.

How does the NK cell response correlate to the treatment response? Because all patients in our study achieved an EVR to PegIFN/RBV therapy at week 12, we were not able to assess NK cell responses in the context of the ultimate treatment outcome. On the other hand, we believe that late time points of PegIFN/RBV therapy are less relevant for our study, because NK cells exhibited their greatest response within the first days of therapy in parallel to the first-phase virological response (Figs. 1-4). Our data clearly indicate that near-maximal NK cell activation can be reached within hours of the first injection of PegIFN, because the response to additional in vitro stimulation with IFN-α was significantly reduced at later treatment time points (Fig. 4). Here, we made the interesting observation that NK cells from patients with a weak first-phase decline in HCV titer, who displayed lower levels of in vivo pSTAT1 induction than patients with a strong first-phase decline in HCV titer, nevertheless retained responsiveness to in vitro stimulation with IFN-α. Thus, both patient groups differed in their in vivo responsiveness to IFN-based therapy, but not in their overall response to IFN-α (Fig. 5A-C). These results suggest that NK cell responsiveness depends, to a certain extent, on the environment. One explanation is that in vivo levels and pharmacokinetics of IFN differ among patients. Another possible explanation is that certain factors, such as suppressive cytokines, interfere with the responsiveness of NK cells to PegIFN therapy in vivo, and that these are overcome once NK cells are stimulated with high doses of IFN-α in vitro. However, removal of inhibitory factors can be excluded, because the in vitro NK cell stimulation was performed in whole blood. A third possibility is that genetic determinants, such as IL-28B SNP at rs1297986016 and killer cell immunoglobulin-like receptor/human leukocyte antigen compound genotype,19 cannot completely be ruled out because of the small size of the analyzed patient cohort (Tables 1 and 2). However, if rs12979860 SNPs play a role, it would be an indirect, rather than direct, effect on NK cells, because NK cells retain their responsiveness to in vitro stimulation with IFN-α (Fig. 5D-F) and because they do not respond directly to type III IFN, including IL-28B.20 Thus, our study opens the interesting possibility that in vivo responsiveness to IFN-α-based therapy may be improved.

Another relevant result of this study was the observed refractoriness of NK cells to in vitro IFN-α stimulation, which occurred in all patients within the first week of IFN-α-based therapy and was maintained for the entire study (Fig. 4A,B). NK cells were not only refractory to in vitro IFN-α stimulation, but exhibited refractoriness in vivo, as shown in the patients who consented to a blood draw before and 6 hours after the week 12 PegIFN injection and did not exhibit an increase in vivo pSTAT1 levels during this period (Fig. 4C). This refractoriness to STAT1 phosphorylation is striking, because STAT1 levels continued to increase, whereas pSTAT1 levels declined in NK cells. There are at least three possible explanations: First, the half-life time of STAT1 is longer than that of pSTAT1, because STAT1 has been shown to persist for many days in response to IFNs, whereas pSTAT1 levels decrease by Src homology region 2-domain phosphatase (SHP)1, SHP2, and suppressor of cytokine signaling 1–dependent negative regulation and tyrosine-phosphatase–mediated dephosphorylation. Second, the accumulated unphosphorylated STAT1 itself is able to induce the expression of a subset of ISGs, such as 2′-5′-oligoadenylate synthetase, myxovirus resistance 1, and STAT1, creating a pSTAT1-independent positive feedback loop.21 Third, STAT1 can also be induced independently from signaling via the IFN-alpha/beta receptor in NK cells, as suggested by the observation that NK cells from STAT1-deficient mice show a greater level of impairment in cytotoxicity and ability to reject transplanted tumors than NK cells from mice that lack IFN receptors.22

Collectively, the results suggest that continued exposure to high levels of IFN-α may reign in the NK cell response to prevent collateral damage. Similar mechanisms may be operative in acute HCV infection, which is known to induce high levels of type I IFN-induced genes without evidence of significant liver injury throughout the incubation phase of 1-2 months.23 Thus, IFN-α-induced NK cell refractoriness may contribute to the often observed, but in its mechanisms not yet understood, clinically asymptomatic nature of acute HCV infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Xiongce Zhao, NIDDK, for statistical analysis.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24628_sm_SuppFig1.tif442KSuppl. Fig. 1: STAT1 expression in T cells. (A) In vivo STAT1 expression in CD3+CD56- T cells in HCV-infected patients and healthy, uninfected blood donors. n.s., not significant. (B-D) In vivo STAT1 expression levels in T cells during therapy with PegIFN/RBV. Mean ± SEM are shown for 14 patients undergoing PegIFN/RBV therapy. h, hour; wk, week. *** P<0.001 by repeated measures ANOVA.
HEP_24628_sm_SuppFig2.tif317KSuppl. Fig. 2: In vitro pSTAT1 inducibility in CD3-CD56+ NK cells normalized to STAT1 levels. Mean ± SEM are shown for 14 patients undergoing PegIFN/RBV therapy. h, hour; wk, week. ***P<0.001 by repeated measures ANOVA.

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