Article

Genetically Associated CD16(+)56(-) Natural Killer Cell Interferon (IFN)-alpha R Expression Regulates Signaling and Is Implicated in IFN-alpha-Induced Hepatitis C Virus Decline

Department of Medicine, Division of Infectious Diseases, University Hospital Case Medical Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
The Journal of Infectious Diseases (Impact Factor: 6). 04/2012; 205(7):1131-41. DOI: 10.1093/infdis/jis027
Source: PubMed

ABSTRACT

Natural killer (NK) cells likely contribute to outcome of acute hepatitis C virus (HCV) infection and interferon (IFN)-induced control of chronic HCV infection. We previously observed IFN-αR and NKp30 expression associated with IFN-α-dependent NK cell activity.
Here, we examined CD16(+)56(-), CD16(+)56(+), and CD16(-)56(+) NK cell subset IFN-αR and NKp30 expression in relation to magnitude of HCV genotype 1 decrease during pegylated IFN-α plus ribavirin therapy.
We observed greater baseline IFN-αR and NKp30 expression on CD16(+)56(+) and CD16(-)56(+) NK subsets in HCV-infected patients than in healthy control subjects. Baseline CD16(+)56(-) NK IFN-αR expression was associated with IFN-α-induced pSTAT1, and both were associated with magnitude of HCV decrease during pegylated IFN-α plus ribavirin therapy. Baseline CD16(+)56(-) NK IFN-αR expression was associated with race and interleukin 28B genotype, negatively associated with aspartate aminotransferase-to platelet ratio index, and positively associated with increase in NKp30 expression after in vivo IFN-α exposure. Finally, in vitro IFN-α2a-activated NK cytolysis of HCV-infected target cells was in part dependent on NKp30, and CD16(+)56(-) NK cell IFN-αR expression correlated with cytolytic activity.
IFN-αR expression on CD16(+)56(-) NK cells during chronic HCV infection may in part be genetically determined, and level of expression regulates IFN-α signaling, which in turn may contribute to control of HCV infection.

Full-text

Available from: Donald D. Anthony
MAJOR ARTICLE
Genetically Associated CD16
1
56
2
Natural Killer
Cell Interferon (IFN)–aR Expression Regulates
Signaling and Is Implicated in IFN-a–Induced
Hepatitis C Virus Decline
Sara J. Conry,
1,
a
Qinglai Meng,
1,
a
Gareth Hardy,
2
Nicole L. Yonkers,
2
Julia M. Sugalski,
1
Amy Hirsch,
3
Perica Davitkov,
4
Anita Compan,
3
Yngve Falck-Ytter,
5
Ronald E. Blanton,
6
Benigno Rodriguez,
7
Clifford V. Harding,
2
and
Donald D. Anthony
1,3,8
1
Department of Medicine, Divisions of Infectious and Rheumatic Diseases, University Hospitals Case Medical Center,
2
Department of Pathology,
3
Cleveland Veterans Administration Medical Center,
4
Department of Medicine, University Hospitals Case Medical Center,
5
Department of Medicine,
Division of Gastrointestinal Disease, University Hospitals Case Medical Center,
6
Center For Global Health and Diseases,
7
Department of Medicine,
Division of Infectious Disease, and
8
Case Center for AIDS Research, Case Western Reserve University, Cleveland, Ohio
Background. Natural killer (NK) cells likely contribute to outcome of acute hepatitis C virus (HCV) infection
and interferon (IFN)–induced control of chronic HCV infection. We previously observed IFN-aR and NKp30
expression associated with IFN-a–dependent NK cell activity.
Methods. Here, we examined CD16
1
56
2
, CD16
1
56
1
, and CD16
2
56
1
NK cell subset IFN-aR and NKp30
expression in relation to magnitude of HCV genotype 1 decrease during pegylated IFN-a plus ribavirin therapy.
Results. We observed greater baseline IFN-aR and NKp30 expression on CD16
1
56
1
and CD16
2
56
1
NK
subsets in HCV-infected patients than in healthy control subjects. Baseline CD16
1
56
2
NK IFN-aR expression
was associated with IFN-a–induced pSTAT1, and both were associated with magnitude of HCV decrease during
pegylated IFN-a plus ribavirin therapy. Baseline CD16
1
56
2
NK IFN-aR expression was associated with race and
interleukin 28B genotype, negatively associated with aspartate aminotransferase-to platelet ratio index, and positively
associated with increase in NKp30 expression after in vivo IFN-a exposure. Finally, in vitro IFN-a2a–activated NK
cytolysis of HCV-infected target cells was in part dependent on NKp30, and CD16
1
56
2
NK cell IFN-aR expression
correlated with cytolytic activity.
Conclusions. IFN-aRexpressiononCD16
1
56
2
NK cells during chronic HCV infection may in part be
genetically determined, and level of expression regulates IFN-a signaling, which in turn may contribute to control
of HCV infection.
Acute hepatitis C virus (HCV) infection becomes per-
sistent in a majority of cases [1], and the long-term risk
of cirrhosis, liver failure, cancer, and mortality among
those with chronic infection highlight the importance
of effective therapy [1, 2]. HCV magnitude decrease at
4 and 12 weeks of pegylated interferon (IFN)–a plus
ribavirin therapy is predictive of sustained virologic
response [3]. Although mechanisms underlying IFN-a
responsiveness remain unclear, factors associated with
response to IFN-a therapy include HCV genotype,
age, race, human immunodeficiency virus (HIV) co-
infection, baseline HCV level, and polymorphism near
the interleukin 28B (IL-28B) (IFN-k3) gene locus [39].
Despite introduction of protease inhibitors, the need
to combine these agents with IFN means that response
to newer regimens continues to depend on factors
regulating IFN-a responsiveness.
Received 20 July 2011; accepted 7 November 2011; electronically published
20 February 2012.
a
S. J. C. and Q. M. contributed equally to this work.
Presented in part: 2011 HCV International Meeting, Seattle, Washington.
September 2011. Poster 2.14.
Correspondence: Donald D. Anthony MD, PhD, Biomedical Research Building
1028, Case Western Reserve University, 2109 Adelbert Rd., Cleveland, OH 44106
(dda3@case.edu).
The Journal of Infectious Diseases 2012;205:1131–41
Ó The Author 2012. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com
DOI: 10.1093/infdis/jis027
NK IFN-aR and NKp30 in IFN-a HCV Therapy
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Natural killer (NK) cells provide essential host defense
during mouse hepatic viral [10, 11] and human herpesvirus
infection [1215]. They are innate lymphocytes with cytokine-
producing, chemokine-producing, and cytotoxic activities
regulated by activating and inhibitory receptors [16, 17].
Evidence for NK cells contributing to control of HCV derives
from observations that genetically determined NK-KIR (Killer
Immunoglobulin like Receptor)/ligand pairing correlates with
the course of acute HCV infection [18] and that, during
chronic infection, NK KIR2DL3, NKG2C, and NKp30 ex-
pression are associated with response to IFN-a–based therapy
[1921]. In addition, TRAIL expression is upregulated on NK
cells during IFN-a–based therapy, and this correlates with in
vitro cytolysis of HCV JFH-1–infected Huh 7.5 cells [22].
We observed NK IFN-aR and NKp30 expression to asso-
ciate with IFN-a–dependent killer activity during HIV in-
fection [23]. NK cells of individuals with preserved activity
appeared to have enhanced IFN-aR expression. We hypoth-
esized that IFN-aR expression is upregulated during chronic
viral infection, in turn determining IFN-a–dependent func-
tion. Here, we evaluated NK cell subset IFN-aRandNKp30
expression in HCV genotype 1–infected patients at baseline,
longitudinally over the course of IFN-a–based therapy, and in
relation to IFN-a signaling capacity and viral decrease.
MATERIALS AND METHODS
Participants
Study participants signed Cleveland Veterans Affairs Medical
Center or University Hospitals Case Medical Center in-
stitutional review board informed consent. HCV-infected pa-
tients (n 5 21) were chronically infected (antibody positive for
$6 months; HCV RNA positive) with HCV genotype 1, naive to
HCV therapy, and scheduled to begin pegylated IFN-a2a
(180 lg/week) plus weight-based ribavirin (1000–1200 mg/day)
therapy. Healthy control subjects (n 5 10) were recruited from
a comparable age range. Clinical characteristics for study par-
ticipants are shown in Table 1. HCV-infected and healthy
control groups differed by sex and age; thus, analyses comparing
groups required consideration of these factors. Three partic-
ipants were treated with a half-dose of pegylated IFN-a2a
because of baseline thrombocytopenia or neutropenia. Anal-
ysis was performed with all participant sample data and in
the absence of these 3 participant samples. All 21 participants
began therapy; 20 continued to receive full-dose therapy at
4 weeks (1 hepatic decompensation–related discontinuation),
19 continued to receive therapy at 8 weeks (1 mental health–
related discontinuation), and 15 continued to receive therapy
at 12 weeks (4 additional therapy-related discontinuations).
Table 1. Clinical Characteristics
Longitudinal Therapy Study Cross Sectional NK Cytolysis Study
Variable HCV Healthy Control HCV Healthy Control
No. 21 10 15 11
Age, y 57 (50–65)
a
44 (37–50) 55 (50–64)
a
31 (26–53)
Sex Male 95%
a
Male 50% Male 93%
a
Male 64%
Female 5% Female 50% Female 7% Female 36%
Race or ethnic group Black 48% Black 30% Black 60%
a
Black 9%
White 52% White 70% White 33% White 55%
Hispanic 7% Hispanic 18%
Asian 18%
Genotype 1 (100%) 1 (87%)
2 (13%)
HCV level (IU/mL) 996 438 1 373 190
(171 031–5 384 560) (36 893–18 800 880)
AST level (U/mL) 58 (23–230) 43 (21–162)
ALT level (U/mL) 47 (14–175) 48 (18–185)
APRI 0.72 (0.20–5.10) 0.44 (0.15–2.59)
<0.4, 33%; 0.4–1.5, 29%;
>1.5, 38%
<0.4, 47%; 0.4–1.5, 40%;
>1.5, 13%
PLT (10
9
platelets/mL) 201 (88–262) 212 (139–370)
Albumin level (g/dL) 3.7 (1.8–4.4) 4.0 (3.6–4.4)
Values are expressed as median (range) for HCV level (by branched chain method or branched DNA), albumin level, PLT count, AST level, ALT level, age, and APRI;
calculated as described [24]. Proportions of subjects within each category are given for HCV genotype, sex, APRI, and race.
Abbreviations: ALT, alanine aminotran sferase; APRI, AST-to-PLT ratio index; AST, aspartate aminotransferase; HCV, hepatitis C virus; NK, natural killer; PLT,
platelet.
a
P # .05 compared with healthy controls.
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Ribavirin dose reduction was required at week 8 (n 5 1) and
week 12 (n 5 2).
For in vitro NK cytolytic function assays, a separate non-
overlapping cohort of chronic HCV-infected patients naive to
therapy was recruited (n 5 15), along with age range–matched
healthy control subjects (n 5 11) (Table 1).
Clinical Laboratories
HCV branched-chain polymerase chain reaction (PCR; sensitivity,
615 IU/mL) and HCV transcription-mediated amplification PCR
(sensitivity, 15 IU/mL) were performed, and aspartate amino-
transferase (AST), alanine aminotransferase (ALT), platelet (PLT),
total bilirubin, and albumin levels were measured in a single
clinical laboratory. AST-to-PLT ratio index (APRI) was calculated
as described elsewhere [24]. IL-28B rs12979860 single-nucleotide
polymorphism (SNP) genotype was determined using a strand-
specific PCR method (Monogram Biosciences).
NK Cell Subset Frequency, IFN-aR, NKp30, TRAIL, and CD161
Expression
Freshly prepared peripheral blood mononuclear cells (PBMCs)
were stained in real time with anti–CD3-PerCP (clone SK7),
anti–CD16-APC-Cy7 (clone 3G8), anti–CD56-PE-Cy7 (clone
NCAM16.2), anti–CD161-APC (clone DX12), anti–NKp30-PE
(clone P30-15; BD Biosciences), and anti–IFN-aR1-FITC (clone
85228; R&D Systems) or isotype controls. IFN-aR1-PE (clone
85228; R&D Systems) was used for bulk NK cytolytic assays.
Flow cytometric analysis was performed on a BD LSRII flow
cytometer (BD Biosciences) with FACSDiva Software (BD
Biosciences).
For NK cell TRAIL expression, cryopreserved PBMCs were
stained with anti-CD3PerCP (clone SK7), anti-CD56APC (clone
NCAM16.2), anti-CD16FITC (clone 3G8; BD Biosciences), and
anti-TRAIL-PE (clone RIK-2) or PE-labeled mouse immuno-
globulin G (IgG) 1 (BD Biosciences). Analysis was performed
on a BD FACSCalibur flow cytometer (BD Biosciences) with
CELLQuest software (BD Biosciences).
IFN-a–Induced pSTAT1
Freshly prepared PBMCs (1 3 10
6
) were analyzed in real time
for IFN-a2a–induced pSTAT1 by preincubating cells at 4°Cfor
30 minutes with anti–CD16-FITC (clone 3G8), anti–CD56-PE
(clone NCAM16.2), and anti–CD3-APC (clone SK7; BD Bio-
sciences); washing; resuspending in RPMI 10% fetal calf serum
(Hyclone); and culturing for 15 minutes at 37°C with 0, 1000,
3000, or 10 000 U/mL of IFN-a2a (PBL Biomedical Labs). Cells
were washed, fixed with BD Cytofix, permeabilized with BD
Phosflow Perm Buffer III (BD Biosciences), and stained for
30 minutes at room temperature with anti-human pSTAT1
PerCPCy5.5 (clone 4a) or isotype control (BD Biosciences).
Flow cytometric analysis was performed on a BD LSRII flow
cytometer with FACSDiva software (Supplementary Figure 1),
and specific expression of pSTAT1 was calculated as the mean
fluorescence intensity (MFI) above isotype control.
IFN-a–Induced NK Subset IFN-g by Intracellular Flow
Cytometry and IFN-a–Induced PBMC IFN-g by Enzyme-Linked
Immunosorbent Spot Assay
Cryopreserved PBMCs were thawed, plated at 10
6
cells per well,
incubated for 20 hours at 37°C (Brefeldin A [Sigma-Aldrich] was
added after 2 hours) in the presence or absence of IFN-a2a (1000
U/mL; PBL Biomedical Labs), fixed, permeabilized, and stained
using intracellular cytokine staining protocol with anti–CD3-APC
(clone SK7), anti–CD14-PerCP (clone MuP9), anti–CD16 APC-
Cy7 (clone 3G8), anti–CD56-PE-Cy7 (clone NCAM16.2), and
anti–IFN-c2FITC (clone 25723.11; BD Biosciences). Thawed
cells were also plated at 300 000 and 600 000 cells per well in
precoated IFN-c enzyme-linked immunosorbent spot assay
plates (Millipore) and cultured for 20 hours at 37°Cinthepres-
ence or absence of IFN-a2a (1000 IU/mL), and IFN-c–secreting
cell frequency was determined as previously described [23].
NK Cytolysis of JFH-1–Infected Huh 7.5 Cell Cultures
Huh 7.5 cells were provided by Dr C. M. Rice (Apath LLC). The
pJFH1 plasmid was provided by Dr T. Wakita (National In-
stitute of Infectious Diseases, Tokyo, Japan). Infectious JFH1
virus was prepared as previously described [25]; 2000 Huh 7.5
cells were infected by 1 multiplicity of infection of JFH-1 for
2 days in 384-well plates prior to NK cell coculture.
Bulk NK cells were prepared by negative bead selection
(Stemcell Technologies) depleting CD3/4/14/19/20/36/66b/123/
HLA-DR/glycophorin-A–bearing cells (purity, .95%). Bulk NK
cells or flow-sorted NK subsets (BD FACSAria; BD Biosciences)
were stimulated with 500 U/mL of IFN-a-2a or media alone for
16 hours, washed, added to JFH-1–infected or –uninfected Huh
7.5 target cells at indicated effector: target ratios, and cocul-
tured for 5 hours. A total of 50 lL of supernatant was analyzed
for cytolytic activity, measuring glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) release with use of aCella-Tox (Cell
Technology) as described elsewhere [26, 27] by luminometer
(VICTOR3V; PerkinElmer). Mean serum concentration of IFN-
a2a during pegylated IFN-a2a (180 lg/week) therapy is 8 ng/mL
[28]. IFN-a2a used here is 3–5 pg/U. Therefore, 500–3000 U/mL
culture concentration is equivalent to 1.5–15 ng/mL, a range
similar to serum IFN-a2a concentrations during therapy.
Statistical Analysis
Statistical analyses were performed using SPSS for Windows,
version 19.0 (SPSS). We used the Mann–Whitney U test for
2-way comparisons of continuous variables across and within
groups. Associations between continuous variables were evalu-
ated using Spearman rank correlation coefficient. Intragroup
variable comparison over time in the same participants was
analyzed using Wilcoxon signed-rank test. Linear regression
analysis was used to evaluate the joint effects of IFN-aRraceand
APRI on viral decrease. Jonckheere-Terpstra test for ordered
alternatives was used to analyze ascending trends across the 3
NK IFN-aR and NKp30 in IFN-a HCV Therapy
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IL-28B genotypes. All tests of statistical significance were 2-sided,
and P values #.05 were considered statistically significant.
RESULTS
IFN-aR Expression Is Higher in Chronic HCV–Infected Patient
CD16
1
56
1
and CD16
2
56
1
NK Cells Than in Controls, and
HCV-Infected Patient IFN-aR Expression Differs by Race on
CD16
1
56
2
and CD16
1
56
1
NK Cells
We first evaluated NK subset frequency and IFN-aRand
NKp30 expression. Peripheral blood frequencies of CD16
1
56
2
,
CD16
1
56
1
, and CD16
2
56
1
NK cells were quantified as shown
(Figure 1A). CD16
1
56
2
NK cell frequencies were higher in
chronic HCV infection (Figure 1B), as previously described [29].
IFN-aR and NKp30 expression were greater in CD16
1
56
1
and
CD16
2
56
1
NK subsets in chronic HCV–infected patients than
in controls (P 5 .04 and P 5 .02, respectively, for IFN-aR and
P 5 .005 and P 5 .02, respectively, for NKp30), whereas ex-
pression of IFN-aR on CD16
1
56
2
NK cells did not differ
significantly (Figure 1C and 1D). Because sex differed between
groups, we also analyzed data for male patients only. Differ-
ences between groups were preserved when analysis was re-
stricted to male participants (P 5 .002 and P 5 .008). NK
TRAIL expression is associated with in vitro killing in HCV-
infected Huh 7.5 cells [22]. TRAIL expression was found to be
greater in CD16
1
56
2
and CD16
1
56
1
NK cells in chronic
HCV–infected patients than in those in controls. Because age
differed between HCV-infected and control groups, we evalu-
ated associations between receptor expression (IFN-aR,
NKp30, CD161, and TRAIL) and clinical variables (AST,
ALT, PLT, total bilirubin, age, race, APRI, and HCV level) in
the HCV-infected group. No correlation between IFN-aR
or NKp30 and age was observed. We did, however, observe
Figure 1. Natural killer (NK) subset interferon (IFN)–aR expression is increased during hepatitis C virus (HCV) infection, and expression differs by race.
Peripheral blood mononuclear cells were stained with CD3, CD16, CD56, IFN-aR, and NKp30 or isotype control, and flow cytometric analysis was
performed on a BD LSRII. A, Gating strategy used to define NK cell subsets in both healthy control and HCV-infected subjects, followed by example of
IFN-aR and NKp30 expression on NK cells of 1 healthy control subject. Isotype shown in nonshaded area, and antibody shown in shaded area. Specific
NKp30 and IFN-aR expression were determined as mean fluorescence intensity above isotype control. NK subset frequency (B), IFN-aR(C), NKp30 (D),
and TRAIL (E) expression comparison between healthy control subjects (ctrl; n 5 10) and HCV-infected patients (n 5 21). IFN-aR(F) and NKp30 (G) are
shown as a function of race in the HCV-infected group (W 5 white, B 5 black). P values #.05 are shown.
1134
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IFN-aR expression, but not NKp30, TRAIL, or CD161ex-
pression, to be higher on CD16
1
56
2
and CD16
1
56
1
NK cells
of white, compared with black, HCV-infected patients. APRI
also negatively correlated with IFN-aR expression on CD16
1
56
2
NK cells only (r 520.43; P 5 .05).Ideally,immunepa-
rameters would also be compared as a function of sustained
virologic response vs nonresponse to therapy. However, al-
though 20 of the initial 21 participants continued therapy
at 4 weeks, only 15 continued therapy at 12 weeks. Four were
nonresponders, 3 were partial responders, 3 were responder-
relapsers, 2 were sustained virologic responders, and the results
for 3 are pending. Because of the number of adverse effect–
related dropouts after 4 weeks of therapy, week 4 data were
viewed as the most appropriate to focus on here.
Magnitude of HCV Decrease at 4 Weeks of Pegylated IFN-a Plus
Ribavirin Therapy Is Associated With Baseline CD16
1
56
2
NK
Cell IFN-aR Expression and IL-28B Genotype
We observed an expected degree of variability in magnitude
of viral decrease during the early phase of pegylated IFN-a plus
ribavirin therapy, in part associated with race (week 4, 2.2 vs
0.52 log
10
decrease in white and black participants, respectively;
P 5 .01) and APRI (r 5 -0.70; P 5 .001), as expected. Race and
APRI were not associated (P 5 .2). IL-28B rs12979860 genotype
was available for 18 participants (3 [TT], 11 [CT], 4 [CC]). In
these genotype groups, 66%, 45%, and 0% of participants, re-
spectively, were black. As expected, IL-28B genotype was also
associated with 1-month viral decrease (median, 0.45, 1.39, and
2.14 log decrease in the TT, CT, and CC groups, respectively;
P 5 .04, Figure 2).
When evaluating whether NK IFN-aR, NKp30, CD161, or
TRAIL expression was predictive of HCV level decrease,
baseline NK IFN-aR expression correlated with magnitude
of HCV level decrease at 4 weeks, although only in the
CD16
1
56
2
NK-cell subset (Figure 2). When 3 participants
receiving submaximal IFN-a doses were removed from the
analysis, the relationship was preserved (r 5 0.59; P 5 .01).
In addition, this relation held for HCV level decrease at week
12, again selectively in the CD16
1
56
2
NK-cell subset
(r 5 0.73; P 5 .02). Linear regression analysis indicated that
the relationship between CD16
1
56
2
NK IFN-aRexpression
and magnitude of viral decrease was not significantly mod-
ified by race or APRI (P 5 .3 and P 5 .5 for interaction of
race or APRI with IFN-aR expression). Furthermore, anal-
ysis of this relation in APRI subgroups (above and below
median APRI) indicated that the same correlation tended
to hold (r 5 0.6, P 5 .09; r 5 0.58, P 5 .06). Baseline NKp30
expression did not significantly correlate with magnitude of
viral decrease, nor did baseline CD161 or TRAIL expression.
IL-28B genotype was associated with CD16
1
56
2
NK IFN-aR
Figure 2. Magnitude of hepatitis C virus (HCV) level decrease during pegylated interferon (IFN)–a plus ribavirin therapy is associated with baseline
CD16
1
56
2
natural killer (NK) cell interferon (IFN)–aR expression. A, Correlation between log viral decrease at 4 weeks of treatment and baseline IFN-aR
in HCV-infected patients (n 5 20). B, Interleukin 28B (IL-28B) genotype vs week 4 log HCV decrease (n 5 18); C, IL-28B genotype vs baseline CD3
16
1
56
NK IFN-aR mean fluorescence intensity (n 5 18).
NK IFN-aR and NKp30 in IFN-a HCV Therapy
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Page 5
expression (median, 1815, 2165, and 3117 MFI in the TT,
CT, and CC groups, respectively; P 5 .02) (Figure 2).
Baseline CD16
1
56
2
IFN-aR Expression Is Associated With
IFN-a2a-Induced pSTAT1 and With In Vivo IFN-a Plus
Ribavirin–Induced NKp30, Both Associated With HCV Level
Decrease
To determine whether IFN-aR expression affects IFN-a sig-
naling, we measured IFN-a2a–induced NK subset pSTAT1 by
flow cytometry when possible. As shown in Figure 3A,level
of IFN-aRexpressioncorrelatedwithIFN-a2a–induced
CD16
1
56
2
NK cell pSTAT1 in chronic HCV–infected patient
samples (Figure 3B ). This relationship also held for 1000 and
10 000 U/mL IFN-a2a conditions in the CD16
1
56
2
NK subset
(r 5 0.72, P 5 .005; r 5 0.55, P 5 .05). In addition, pSTAT1
level correlated with IFN-aR expression on media-treated
CD16
1
56
2
NK cells (r 5 0.80; P 5 .001), suggesting reflection
of in vivo IFN-a signaling. The relationship was not observed
for the other NK subsets (Figure 3B), although for the
CD16
2
56
1
subset, an association was observed between IFN-aR
and the delta pSTAT1 (change from unstimulated condition)
induced by 1 concentration of IFN-a-2a (3000 U/mL; r 5 0.57;
P 5 .03). When evaluating IFN-a-2a–induced pSTAT1 by race,
CD16
1
56
2
and CD16
2
56
1
NK cells from white participants
Figure 3. Baseline CD16
1
56
2
interferon (IFN)–aR expression is associated with IFN-a–induced CD16
1
56
2
pSTAT1 and magnitude of hepatitis C virus
(HCV) level decrease. A, Representative example of IFN-a2a–induced pSTAT1 expression on each of the 3 natural killer (NK) cell subsets over IFN-a2a
concentration range. B, Correlation between baseline IFN-a2a (3000 U/mL) induced pSTAT1 and baseline IFN-aR mean fluorescence intensity (MFI) on
each of the 3 NK subsets (12 hepatitis C virus [HCV]–infected patients). C, Correlation between baseline spontaneous (0 U/mL IFN-a) induced pSTAT1 and
baseline IFN-aR MFI for CD3
16
1
56
NK cells of HCV-infected patients (n 5 8). D, Racial comparison of IFN-a2a–induced pSTAT1 on each of the 3 NK
subsets at 3000 U/mL IFN-a2a. E, Correlation between log of viral decrease at 4 weeks of treatment and baseline IFN-a2a–induced pSTAT1 on each of
the 3 NK subsets at 3000 U/mL IFN-a2a (11 HCV-infected patients).
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were observed to have more IFN-a-2a–induced pSTAT1 than
cells from black participants (Figure 3D). When evaluating
IFN-a-2a–induced pSTAT1 in relation to therapy-induced
viral decrease, IFN-a-2a–induced CD16
1
56
2
NK cell pSTAT1
at baseline correlated with IFN-a therapy–induced HCV level
decrease at week 4 (Figure 3E). This relationship also held
for 0, 1000, and 10 000 U/mL IFN-a2a culture conditions
(r 5 0.76, P 5 .004; r 5 0.73, P 5 .007; r 5 0.59, P 5 .04). This
relationship also existed for the CD16
2
56
1
NK subset at 1 IFN-
a2a concentration (Figure 3E).
We next focused on downstream consequences of IFN-a
signaling. Baseline IFN-c–producing PBMC frequency and NK
subset IFN-c in response to IFN-a2a were not correlated with
the magnitude of HCV level decrease. IFN-a has been shown
to induce NK cell NKp30 expression [30]. We therefore
evaluated IFN-aR and NKp30 expression longitudinally over
IFN-a therapy. Expression of NKp30 was greater in CD16
1
56
2
NK cells at week 4 of therapy (Figure 4A), and there was a trend
toward week 4 CD16
1
56
2
NK cell subset NKp30 expression
to correlate with week 4 viral decrease, although only when
considering participants who received full-dose pegylated IFN-
a2a (r 5 0.44; P 5 .07). In addition, baseline CD16
1
56
2
NK cell
subset IFN-aR expression correlated with week 4 NKp30 ex-
pression, consistent with IFN-aR–mediated regulation of NKp30
expression (Figure 4B). CD16
2
56
1
NKp30 modestly increased
at week 4 (Figure 4A), although this was not correlated with
baseline IFN-aR expression. At the same time, CD16
1
56
2
NK
subset frequency decreased in the peripheral blood at week 4
(week 0 vs 4: median, 2.73% [interquartile range {IQR},
1.59%–3.20%] vs 0.81% [IQR, 0.44–1.70]; P , .01) as pre-
viously described [29]. IFN-aR expression was somewhat in-
creased at week 4, compared with baseline, in CD16
1
56
2
NK
cells (week 0 vs 4: median MFI, 2020 [IQR, 1596–2802] vs 2979
[IQR, 2479–5129]; P , .05).
NKp30 Contributes to Cytolysis of HCV JFH1-Infected Huh
7.5 Cells, CD16
1
56
2
NK Cells Contribute to Activity, and
CD16
1
56
NK IFN-aR Expression Is Associated With
Cytolytic Activity
To analyze the role of NKp30 in HCV-infected target cell
cytolysis, we performed 5-hour cocultures of negatively se-
lected bulk NK cells and HCV JFH1–infected Huh 7.5 cells.
NK-dependent cytolysis of Huh 7.5 targets was enhanced by
the presence of HCV infection and IFN-a2a pretreatment, as
previously described [22]. Activity was partially dependent on
TRAIL (Figure 5), as previously described [22], mainly in the
presence of IFN-a2a pretreatment (Figure 5C). Blockade of
NKp30 inhibited killing efficiency in both the absence and
presence (Figure 5C) of IFN-a2a pretreatment, although the
effect was more robust in the absence of IFN-a2a. These data
indicate a role for NKp30 in HCV-infected target killing.
We next evaluated the role of NK subsets in HCV-infected
target killer function. CD16
1
56
2
cells of healthy controls were
found to be capable of cytolytic activity (Figure 5D), although at a
somewhat reduced efficiency in comparison with CD16
2
CD56
1
NK cells. Cytolytic activity was enhanced by IFN-a2a treatment
Figure 4. Baseline CD16
1
56
2
interferon (IFN)–aR expression is associated with pegylated IFN-a plus ribavirin–induced CD16
1
56
2
natural killer (NK)
p30 expression. A, NKp30 expression over course of treatment on each of the 3 NK subsets (20 hepatitis C virus [HCV]–infected patients). B, Correlation
between baseline IFN-aR expression and week 4 NKp30 expression on each of the 3 NK subsets.
NK IFN-aR and NKp30 in IFN-a HCV Therapy
d
JID 2012:205 (1 April)
d
1137
Page 7
of all 3 NK subsets, particularly for CD16
2
CD56
1
NK cells.
These data indicate that all 3 NK subsets are capable of contrib-
uting with varying degrees to HCV-directed cytolytic activity.
We next evaluated NK subset IFN-aR expression in relation
to cytolytic function in groups of persons not overlapping with
the therapy cohort (Table 1). Healthy control CD16
1
56
2
NK
IFN-aR expression at baseline was correlated with IFN-
a–activated bulk NK cytolysis of HCV-infected Huh 7.5 target
cells, whereas IFN-aR expression on CD16
1
56
1
or CD16
2
56
1
subsets was not (Figure 6A). This relation also held at E:T 1.25:1
(r 5 0.8; P 5 .005). No relationship was observed between
IFN-aRexpressioninanyNKsubsetandcytolysisofun-
infected Huh 7.5 target cells for either healthy control subject
or HCV-infected participant NK samples or media-treated
healthy control NK cell cytolysis of HCV-infected Huh 7.5
targets. In contrast, HCV-infected patient CD16
1
56
2
NK cell
IFN-aR expression was associated with NK cytolysis of HCV-
infected targets when NK cells were treated with either media or
IFN-a2a (Figure 6B). This relationship held for all E:T in the
presence of IFN-a2a stimulation (E:T 1.25:1, r 5 0.56, P 5 .03;
E:T 0.6:1, r 5 0.53, P 5 .04).
DISCUSSION
Data here highlight racially associated NK cell IFN-aR expres-
sion during chronic HCV infection coinciding with known ra-
cial differences in IFN-a therapy efficacy. CD16
1
56
2
NK cell
IFN-aR expression at baseline correlated positively with IFN-a
signaling capacity and magnitude of viral decrease in HCV ge-
notype 1–infected patients treated with pegylated IFN-a plus
ribavirin. One consequence of IFN-a signaling is enhanced
NKp30 expression, and CD16
1
56
2
NK cell IFN-aR expression
at baseline correlated both with increased CD16
1
56
2
NK cell
NKp30 expression at week 4 of therapy and magnitude of
viral decrease. NKp30 and CD16
1
56
2
NK cells contributed to
HCV-infected target killing efficiency in vitro. Furthermore,
CD16
1
56
NK cell IFN-aR expression correlated with HCV-
infected cell targeting, suggesting that IFN-a–dependent activity
of this subset may be more dependent on IFN-aR expression
than that of other NK subsets (in which other factors are likely
to be rate limiting). These data suggest that CD16
1
56
2
NK cell
IFN-aR expression level may contribute to control of HCV
infection during IFN-a plus ribavirin therapy and suggest
a potential mechanism by which racial differences in IFN-a
therapy response are mediated. Because the IL-28B promoter
polymorphism is also associated with CD16
1
56
2
NK cell
IFN-aR expression, one potential mechanism underlying the
IL-28B genetic association may be directly or indirectly
through IFN-aRexpression.
Peripheral blood NK subset skewing exists during chronic
HCV infection, with increased CD16
1
56
2
and decreased
CD16
1
56
1
NK subset frequencies [29, 31]. CD16
1
56
2
NK
cells have lower TNF-a–andIFN-c–secreting activity but
Figure 5. Interferon (IFN)–a–activated natural killer (NK) cytolysis of hepatitis C virus (HCV)–infected targets is partially dependent on NKp30 and
partially mediated by CD16
1
56
2
NK cells. A, Negatively selected NK cells from 1 healthy control subject were cultured for 16 h with media or 500 U/mL
IFN-a2a, washed, and cocultured with HCV JFH-1–infected target cells for 5 h, and cytolysis was measured (y-axis) for differing E:T ratios (x-axis).
Background target cell cytolysis in the absence of added NK cells was ,10% in each experiment, and this background was subtracted from the data
shown. Ten microliters of lytic reagent was added to target cells as a positive control. Percentage cytotoxicity was calculated as: [(experimental
glyceraldehyde 3-phosphate dehydrogenase [GAPDH] release-spontaneous GAPDH release from effector cells alone spontaneous GAPDH release from
target cells alone)/(maximum induced GAPDH release from target cells-spontaneous GAPDH release from target cells)] 3 100. B, Negatively selected
NK cells from 3 healthy control subjects were cultured for 16 h with media then cocultured with HCV JFH1–infected target cells at 2.5:1 E:T 5 h as in (A).
For antibody-blocking experiments, NK cells were treated with 10 lg/mL of goat polyclonal anti-NKp30 IgG and vs isotype control (goat IgG) (R&D
Systems); or mouse anti-TRAIL monoclonal antibodies (clone, 2E5) vs isotype control (mouse IgG1) (Enzo Life Sciences) at 37°C for 1 h, then added to
target cell cultures. Means and standard deviations are shown. C, Same as (B), except NK cells were treated with IFN-a2a, then washed before coculture
with HCV-infected target cells. D, Bulk NK cells from 3 healthy controls were cultured in the presence vs absence of 500 U/mL IFN-a2a for 16 h, washed,
and cocultured with HCV JFH-1–infected target cells at E/T 1.2:1, or stained with antibodies to CD3, CD16, and CD56 followed by flow cytometric–based
cell sorting of NK subsets that were utilized in killer assays at E:T 1.2:1.
1138
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Conry et al
Page 8
similar CD107a and chemokine production in response
to K562 targets [29]. Further investigation of IFN-aR–
dependent activity of this subset is warranted.
Race is known to associate with response to IFN-a–based
therapy [3]. We found that NK subset IFN-aRexpressionand
IFN-a2a–induced pSTAT1 associate with race, providing
1 plausible mechanistic link between race and IFN-a response.
Of note, the black population in America is admixed 10%–20%
with the population of European ancestry [32]. This means that
the differences observed here are minimum estimates. IL-28B
SNPs (within the IFN-k gene region) are thought to account for
a substantial portion of racially based variability in pegylated
IFN-a plus ribavirin therapy response [8, 9, 3335]. IL-28B
SNPs have been associated with variable levels of IFN-k
messenger RNA expression [34, 35], although the mecha-
nism accounting for the IL-28B SNP link to IFN-a therapy
response is not known [36]. We identify an association be-
tween IL-28B genotype and CD16
1
56
2
NK subset IFN-aR
expression, providing 1 possible mechanistic link. Such an
association does not necessarily indicate a direct effect of
IL-28B on NK cells, although NK cell IL-28B receptor expres-
sion has been described without identified function [37].
Change in NK subset frequency and/or phenotype observed
in the peripheral blood over the course of therapy may reflect
anatomic compartment redistribution, cell differentiation, cell
expansion, cell death, or change in receptor expression in
the same cells. Peripheral blood CD16
1
56
2
NK cells have been
previously observed to decrease in frequency over the first
4 weeks of therapy [38]. Results here are in agreement. Whether
these cells redistribute, differentiate, or die is unknown.
IFN-aR expression was observed to negatively correlate with
APRI, indicating that disease stage may affect NK subset IFN-aR
expression. Of note, the relationship between viral decrease and
IFN-aR expression tended to exist in both high and low APRI
subgroups, indicating that the relationship between IFN-aR
expression and viral decrease is likely to be independent of APRI.
This was also supported by linear regression analysis. However,
linear regression analysis also revealed that the relationship be-
tween IFN-a-2a–induced pSTAT1 and viral decrease tended to
be attenuated in persons with higher APRI (P 5 .07). On the
surface, these relationships appear to be complex, especially
because peripheral blood NK IFN-aR expression may or may
not reflect that in the liver. One possibility is that hepatic pa-
renchymal sufficiency may be required to facilitate NK IFN-aR
expression. In addition, STAT1 levels have been associated with
IFN-a2a–induced NK pSTAT1 activity [39], and NK STAT1
levels may differ as a function of liver disease.
Week 4 NKp30 expression correlates with baseline CD16
1
56
2
NK cell IFN-aR expression. In addition, IFN-aR and NKp30
expression level were associated with each other on the same
cells at week 4 (data not shown), indicating that NKp30 up-
regulation likely occurred on the same cell expressing higher
levels of IFN-aR. The latter is supported by prior data in-
dicating that IFN-a stimulation results in upregulation of NK
NKp30 expression [30]. It is plausible that 1 mechanism un-
derlying variability in IFN-a–mediated control of HCV in vivo
Figure 6. CD16
1
56
2
natural killer (NK) interferon (IFN)–aR expression is correlated with in vitro hepatitis C virus (HCV)–targeted NK cytolytic function.
Negatively selected NK cells from healthy control subjects or HCV-infected patients were cultured for 16 h with media or IFN-a2a, washed, then
cocultured with HCV JFH1–infected target cells at 2.5:1 E:T 5 h as in Figure 5 (E:T 0.6:1 and 1.25:1 also performed, not shown). Background target cell
cytolysis in the absence of added NK cells was ,10% in each experiment, and this background is subtracted from the data shown. A, Healthy control
(n 5 11) subject NK subset IFN-aR expression vs IFN-a2a–treated NK cytolytic activity. B, HCV-infected subject (n 5 15) CD16
1
56
2
NK subset IFN-aR
expression vs NK cytolytic activity in absence or presence of IFN-a2a pretreatment.
NK IFN-aR and NKp30 in IFN-a HCV Therapy
d
JID 2012:205 (1 April)
d
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Page 9
is through the level of NK subset IFN-aR expression that de-
termines IFN-a–induced signaling magnitude, in turn leading
to enhanced NKp30 (and other modulators of effector function),
which in turn contribute to control of HCV infection. Certainly,
NKp30 expression during IFN-a therapy has recently been
shown to associate with favorable response to therapy [20, 21].
Although this is a small data set, clearly, further investigation
of this NK subset is warranted, with specific emphasis on in-
vestigation of genetically encoded and environmental factors
contributing to enhanced IFN-aR expression and mechanisms
underlying downstream associations with magnitude of viral
decrease.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://www.oxfordjournals.org/our_journals/jid/). Supplementary
materials consist of data provided by the author that are published to benefit
the reader. The posted materials are not copyedited. The contents of all
supplementary data are the sole responsibility of the authors. Questions or
messages regarding errors should be addressed to the author.
Notes
Acknowledgments. We thank the study participants, and Laura Napo-
litano, Yolanda Lie, and Eoin Coakley at Monogram Biosciences for helping
to make IL-28B genotyping possible.
Financial support. This work was supported by the National Institutes
of Health (grant numbers R01 DK068361 and R01 AI069195).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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    [Show abstract] [Hide abstract] ABSTRACT: In the last few years major progress has been made in better understanding the role of natural killer (NK) cells in hepatitis C virus (HCV) infection. This includes multiple pathways by which HCV impairs or limits NK cells activation. Based on current genetic and functional data, a picture is emerging where only a rapid and strong NK cell response early on during infection which results in strong T cell responses and possible subsequent clearance, whereas chronic HCV infection is associated with dysfunctional or biased NK cells phenotypes. The hallmark of this NK cell dysfunction is persistent activation promoting ongoing hepatitis and hepatocyte damage, while being unable to clear HCV due to impaired IFN-γ responses. Furthermore, some data suggests certain chronically activated subsets that are NKp46(high) may be particularly active against hepatic stellate cells, a key player in hepatic fibrogenesis. Finally, the role of NK cells during HCV therapy, HCV recurrence after liver transplant and hepatocellular carcinoma are discussed.
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