Consistent Beneficial Effects of Killer Cell
Immunoglobulin-Like Receptor 2DL3 and Group 1
Human Leukocyte Antigen-C Following Exposure to
Hepatitis C Virus
Susanne Knapp,1Usama Warshow,2Doha Hegazy,2Louise Brackenbury,1I. Neil Guha,3Andrew Fowell,3
Ann-Margaret Little,4Graeme J. Alexander,5William M.C. Rosenberg,6Matthew E. Cramp,2and Salim I. Khakoo1
Natural killer cells are a key component in the immune control of viral infections. Their
functions are controlled by inhibitory receptors for major histocompatability complex
(MHC) class I, including the killer cell immunoglobulin-like receptors (KIR). KIR2DL3 in
combination with its cognate human leukocyte antigen (HLA)-C ligand has been shown to
be associated with spontaneous resolution of viremia following hepatitis C virus (HCV)
outcomes following HCV exposure, we studied individuals with apparent resistance to HCV
infection who remain seronegative and aviremic despite long-term injection drug use and
also individuals chronically infected with HCV who successfully clear HCV with treatment.
Homozygosity for KIR2DL3 in combination with group 1 HLA-C allotypes was more
frequent in exposed seronegative aviremic individuals as compared to those with chronic
HCV (25.0% versus 9.7%, P ? 0.003, odds ratio [OR] ? 3.1, 95% confidence interval [CI]
? 1.3-7.1) in a model similar to that found for those spontaneously resolving HCV. In
individuals undergoing treatment for HCV, those with KIR2DL3 and group 1 HLA-C were
1.1-4.5). KIR and HLA-C protection in both treatment response and spontaneously resolv-
ing HCV was validated at the allelic level, in which KIR2DL3-HLA-Cw*03 was associated
was associated with spontaneous resolution of HCV infection (P ? 0.01, OR ? 2.3, 95%
the outcome of HCV infection. This advantage extends to the allelic level for both gene
families. (HEPATOLOGY 2010;51:1168-1175.)
uals exposed to HCV fail to spontaneously clear the virus,
infection. The virus poses a significant challenge
to the immune system as the majority of individ-
and hepatocellular carcinoma. This failure to mount a suc-
cessful immune response is multifactorial in nature and in-
cludes abnormalities in T, B, and dendritic cell responses.
Abbreviations: HCV, hepatitis C virus; HLA, human leukocyte antigen; HLA-C1, group 1 HLA-C allotype; IFN, interferon; KIR, killer cell immunoglobulin-like
receptor; MHC, major histocompatability complex; PEG-IFN: pegylated-interferon; SVR, sustained virological response.
From the1Department of Hepatology, Division of Medicine, Imperial College London, UK;2Hepatology Research Group, Peninsula Medical School and Hepatology
Department Derriford Hospital, Plymouth, UK;3Department of Hepatology, University of Southampton, Southampton, UK;4Anthony Nolan Research Institute, Royal
Free Hospital, London, UK;5Department of Medicine, University of Cambridge, Cambridge, UK;6Institute of Liver Studies, University College, London, UK.
Received July 27, 2009; accepted November 22, 2009.
Supported by a Wellcome Trust Senior Clinical fellowship (to S.I.K.) and a grant from the DANA foundation.
London W2 1PG, UK. E-mail: email@example.com; fax: 44 207 724 9369.
Copyright © 2009 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
Natural killer (NK) cells are a subset of lymphocytes
activate dendritic cells and secrete Th1-type cytokines to
augment antiviral cytotoxic T-cell responses. Their re-
sponses are controlled by multiple activating and inhibi-
tory receptors and it is thought that the net inhibitory or
activating signal derived from these receptors determines
whether or not they become activated. NK cells are en-
riched in the liver in comparison to peripheral blood.1
However, their role in the outcome of HCV infection
low levels of perforin.4Conversely, more recent work has
suggested that NK cell cytotoxicity in chronic HCV may
not be impaired,5and may even be augmented, with an
impairment of Th1 type cytokine secretion, and aug-
mented interleukin 10 (IL-10) secretion.6-8Phenotypic
studies have also given conflicting results in terms of ex-
NK cell function have been related to the binding of the
HCV envelope proteins to CD81. Cross-linking of this
molecule can impair NK cell function in vitro.10,11How-
ever, this effect cannot be demonstrated for infectious
HCV particles and so is thought unlikely to occur in
vivo.12More recently, the changes in NK cell phenotype
and function have been associated with chronic exposure
to interferon-alpha (IFN-?).8
tion to the NK cell receptor repertoire are the killer-cell im-
munoglobulin-like receptors (KIR). The ligands for these
I, which are important for both NK cell development and
function.13,14The KIRs are expressed exclusively on the
CD56dimsubset of NK cells,22and this perforin and gran-
zyme-rich subset is reduced in individuals with chronic
HCV infection.15One feature of KIR, in comparison to
other NK cell receptors, is their substantial genetic diversi-
ty.16The KIR locus is rapidly evolving and exhibits signifi-
cant population diversity.17Thus, different individuals can
have different numbers of KIR genes. Furthermore, the li-
gand for the inhibitory KIR are subsets of the polymorphic
HLA class I molecules, HLA-A,-B, and -C, and this gener-
ates a further level of diversity.18
that may be determined by the host immune response.
Individuals may remain HCV Ab-negative and HCV
RNA-negative (seronegative, aviremic), HCV Ab-posi-
tive HCV RNA-negative (spontaneous resolver), and
HCV Ab-positive HCV RNA-positive (chronically in-
fected). This latter group may be further subdivided into
those who clear HCV RNA following therapy (sustained
virological responders) and those who do not. KIR2DL3
binds a subset of HLA-C allotypes that include HLA-
Cw*01, -Cw*03, -Cw*07, -Cw*08 and Cw*12, Cw*14
and Cw*16. Collectively, these are called the group 1
HLA-C (HLA-C1) allotypes. HLA-Cw*01 was shown to
be protective against HCV infection, and it was subse-
quently shown in two independent studies that individu-
als who spontaneously resolve infection are more likely to
have the gene KIR2DL3 in combination with its cognate
HLA-C1 ligands than those who remain chronically in-
fected.19,20This receptor:ligand combination is thought
KIR:HLA-C receptor:ligand pairings and thus permit a
more responsive NK cell phenotype.21,22Furthermore,
although KIR3DS1 is protective against HCV-associated
hepatocellular carcinoma, it is not known if the HLA-C-
specific KIR influences the course of the other stages of
Seronegative aviremic individuals with a long history
of likely HCV exposure through high-risk injection drug
use have detectable HCV-specific T-cell responses in up
markers of infection.24,25These individuals have been
previously termed “exposed uninfected”31with “unin-
fected” indicating the absence of demonstrable infection,
as defined by conventional antibody or RNA testing, at
the time of recruitment. The presence of T-cell responses
to nonstructural antigens may indicate that at some stage
they have had a replicative infection the magnitude of
which is not clear.25Nevertheless, they appear to have a
favorable immune response to HCV infection and, con-
sistent with an augmented T-cell response being impor-
It is noteworthy that IL-12 is also an NK cell activating
Another potent NK cell activating cytokine is interfer-
on-alpha (IFN-?). It increases both NK cell cytotoxicity
and IFN-? production. IFN-? in combination with riba-
virin forms the cornerstone of current anti-HCV thera-
pies. Treatment with IFN-? and ribavirin is effective in
over 50% of treated individuals, with the most important
determinant of outcome being viral genotype. However,
and host genetic factors also play a role.27-29Moreover, it
has also been shown that treatment-induced or enhanced
cellular immune responses are important in the outcome
of the treatment of HCV infection.30-32Successful treat-
ment with IFN-? has been associated with augmented
HEPATOLOGY, Vol. 51, No. 4, 2010KNAPP ET AL. 1169
up-regulated in chronic HCV infection.6-8However,
monocyte-derived dendritic cells from HCV-infected in-
dividuals.33Therefore, the balance of activating and in-
hibitory interactions may be important in determining
the outcome of IFN-? treatment in HCV infection, and
this may be related to effects on target cells, in addition to
the NK cells themselves. The role of NK cell receptors in
to study one aspect of this complexity, namely, to deter-
mine whether the gene combination of KIR2LD3 and
group 1 HLA-C is beneficial across the spectrum of po-
tential clinical outcomes following HCV exposure.
Patients and Methods
Seronegative Aviremic Cohort. Forty-eight HCV
exposed but seronegative aviremic cases were recruited
from Dartmoor prison (n ? 19) and from various needle
29), between 2001 to 2007, with Ethical Committee ap-
proval. Individuals with a substantial history of past or
present intravenous drug use, sharing of needles or other
drug injection equipment, and who tested negative for
both HCV antibody and HCV RNA were included. No
antibodies was determined by third-generation enzyme-
linked immunosorbent assay (ELISA; Abbott IMx, Ab-
bott Diagnostics, Maidenhead, UK), and HCV RNA by
commercially available qualitative polymerase chain reac-
tion (PCR; Amplicor, Roche, Basel, Switzerland). All
were HIV-and hepatitis B surface antigen (HBsAg)-neg-
ative. Detailed information about drug-injecting behav-
(Table 1). Mean duration of drug use was 8.4 years with
Intravenous Drug Use (IDU) Cohort. A total of 257
clinics at Southampton General Hospital from 2003 to
2007, with Ethical Committee approval. These individu-
als were selected on the basis of race and having intrave-
nous drug usage as their only parenteral risk factor for
acquiring HCV (Table 1). HBsAg-and HIV-positive in-
dividuals were excluded. All individuals were positive by
second-generation ELISA and confirmed viremic using
the HCV COBAS Amplicor system (Roche). The mean
Interferon-Treated Cohort. In all, 208 individuals
with chronic HCV underwent treatment for chronic
HCV infection. These individuals were selected on the
basis of having completed ?80% of a course of IFN-?-
based treatment, and of these 32, individuals had dosage
modifications to their treatment regimen. A total of 165
as a risk factor for HCV infection. Of these, 18 had re-
ceived blood products, 20 had an unknown route of in-
fection, and five had potential exposure by needlestick
injury or tattooing. Individuals coinfected with HBV or
from the hepatology clinics at Southampton General
Hospital, with Ethical Committee approval between
2003 and 2007. All had viremia confirmed by the CO-
BAS Amplicor system. HCV genotyping was performed
using quantitative PCR (iQur, Southampton, UK). De-
tails of this cohort are given in Table 2. Individuals were
had a negative HCV-RNA test at the end of treatment
of treatment using the same assays.
Retrospective Analysis. For the retrospective analysis
of resolved against chronically infected individuals the
Table 1. Demographics and Drug-Injecting Behavior of
Exposed Seronegative Aviremic and Chronically Infected IDU
Aviremic n (%)
Number of patients
Age at first injection drug use
Duration of injection drug use
Lifetime injection episodes
Frequency of injection drug
use ( n)
4-6 times a day
2-3 times a day
Every few days/weekly
Monthly or less
Currently using (n)
Sharing behavior (n)
Shared other drug injecting
Shared with someone
known to have HCV
28.8 ? 5.2
43.9 ? 9.8 ?0.001
19 ? 5—
8.4 ? 7.5 (0.5-24)—
1170KNAPP ET AL. HEPATOLOGY, April 2010
uals was used.19This group consisted of 341 individuals
(122 HCV Ab-positive, RNA-negative, and 219 HCV
Ab-positive, RNA-positive). The mean age was 41.9
years, 339 (99%) were Caucasian, and 227 (66.5%) were
male. There was no significance in these parameters be-
tween the Ab-positive, RNA-negative, and Ab-positive,
RNA-positive groups.19These individuals were recruited
between 1999 to 2003.
KIR and HLA Genotyping. Genomic DNA was ex-
tracted from peripheral blood lymphocytes convention-
ally using a salt precipitation method or the QIAamp
blood kit (Qiagen, Crawley, UK). KIR genotyping was
performed using the Lifematch kit (Tepnel, Stamford,
CT) and by PCR using sequence specific primers as de-
scribed.19HLA typing was performed by direct sequenc-
ing of PCR products.34HLA types that were not resolved
by sequencing or which gave unusual results were also
tested by sequence specific oligonucleotide probe typing
(PCR-SSOP), using commercial kits (Dynal, RELI SSO,
Statistical Analysis. Statistical analysis was per-
formed using SPSS v. 17. The data presented were ana-
lyzed by chi-squared analysis unless otherwise stated. The
Bonferroni correction was applied where relevant. Bino-
17 using the ENTER method.
Group 1 HLA-C and KIR2DL3 Is Overrepresented
in Exposed Seronegative Aviremic Individuals. Forty-
eight individuals with apparent resistance to HCV infec-
tion from injection drug use were typed for HLA-C and
for KIR2DL2 and KIR2DL3. Twenty-four of these had
been tested for T-cell responses by ELISPOT assay,24of
whom 15 had a positive response to at least one of the
HCV antigens studied. The KIR genotypes in these 48
cases were compared to the 257 individuals with chronic
HCV acquired from IDU. Both groups had similar fre-
difference in the frequency of KIR2DL3 or KIR2DL2 in
combination with one group 1 HLA-C allele. However,
the combination of homozygosity for KIR2DL3 and 2
frequency in the exposed seronegative aviremic individu-
als as compared to chronically infected individuals
(25.0% versus 9.7%, P ? 0.003, odds ratio [OR] ? 3.1,
95% confidence interval [CI] ? 1.3-7.1). This recessive
model of protection from HCV infection is similar to
that observed for antibody-positive HCV-exposed in-
Group 1 HLA-C in Combination with KIR2DL3 Is
Beneficial in Chronic HCV Infection. In all, 165 of the
IDU with chronic HCV infection and 43 additional in-
Table 2. Demographics of the Interferon-Treated Cohort
SVR n (%) No SVR n (%)
Number of patients
43.6 ? 9.2
69.8 ? 60.0
48.0 ? 8.2
79 ? 54.8
Table 3. KIR and HLA-C Associations in the Exposed Seronegative Aviremic Individuals
P Value (Pc) OR (95% CI)
Number of patients 48257
Group 1 HLA-C
Group 2 HLA-C
0.10 0.59 (0.30-1.16)
HEPATOLOGY, Vol. 51, No. 4, 2010KNAPP ET AL. 1171
dividuals with chronic HCV infection had been treated
with IFN-?-based therapy in our outpatient clinics. The
frequencies of KIR2DL2 and KIR2DL3 were similar be-
tween those with and those without a sustained virologi-
cal response (SVR). Individuals who had an SVR to
75.2%, P ? 0.02, OR ? 2.4, 95% CI ? 1.1-5.3) (Table
4). Consideration of HLA-C with their cognate KIR re-
ceptors demonstrated that the beneficial effect of group 1
HLA-C was mediated in combination with KIR2DL3
(82.2% versus 67.3%, P ? 0.01, OR ? 2.3, 95% CI ?
1.2-4.3), but not KIR2DL2 (43% versus 39.6%, P ?
considering group 1 HLA-C in the presence or the ab-
with group 1 HLA-C only in combination with
KIR2DL3 (P ? 0.016, OR ? 2.49, 95% CI ? 1.19-
ative aviremic individuals, we observed no statistically
significant association with homozygosity of KIR2DL3:
HLA-C group 1 being protective (P ? 0.1). Logistic re-
gression analysis demonstrated that the effect of
age, ALT, dose modifications, duration, cirrhosis, and
gender within our cohort (Table 5). It was also indepen-
dent of whether the individual had received the current
gold standard of therapy (pegylated interferon and riba-
CI ? 1.01-5.00), the use of pegylated versus standard
interferon (P ? 0.033, OR ? 2.27, 95% CI ? 1.07-
4.82), and more weakly independent of the use of ribavi-
rin (P ? 0.057, OR ? 2.13, 95% CI ? 0.98-4.66).
Standardized viral load data on the cohort was not avail-
able and therefore could not be included in this analysis.
Allelic Specificity of Protection by HLA-C and KIR.
Although the specificity of KIR2DL3 for HLA-C is de-
termined by residue 80 of the MHC class I heavy chain,
the binding of KIR to HLA-C is determined by several
additional residues. Recent work has shown that HLA-C
allelic diversity can affect binding of KIR to HLA-C, and
hence different HLA-C alleles within the C1 grouping
may have different protective effects in HCV infection.22
To test this model we determined the frequency with
which specific group 1 HLA-C alleles were beneficial in
combination with KIR2DL3 in the IFN-treated popula-
tion. HLA-Cw*03 alleles were found more frequently in
combination with KIR2DL3 in individuals with SVR
than in those without SVR (22.4% versus 7.9%, P ?
0.004, OR ? 3.4, 95% CI ? 1.5-8.7) (Table 6). This
trend was observed in individuals treated with pegylated
IFN-? and ribavirin (21.8% with SVR versus 11.3%
without SVR, P ? 0.06), and also in those with other
regimens (3 out 11 with SVR versus 1 out of 39 without
SVR P ? 0.03, Fisher’s exact test). By logistic regression
independent of genotype, age, ALT, dose modifications,
duration, cirrhosis, and gender and the following treat-
ment regimens: pegylated interferon and ribavirin versus
Table 4. KIR and HLA-C Associations in the Interferon-Treated Individuals
(Pc) OR (95% CI)
Number of patients 107101
Group 1 HLA-C
Group 2 HLA-C
0.01 (0.1)2.3 (1.2-4.3)
Table 5. Logistic Regression Analysis of the Association of
KIR2DL3-C1 with the Outcome of Interferon Treatment
Pegylated IFN ?Ribavirin
Duration 24 versus 48
1172KNAPP ET AL.HEPATOLOGY, April 2010
other protocols (P ? 0.028, OR ? 3.06, 95% CI ?
1.13-8.31), the use of pegylated versus standard inter-
feron (P ? 0.032, OR ? 2.94, 95% CI ? 1.10-7.84),
and the use of ribavirin (P ? 0.019, OR ? 3.41, 95%
CI ? 1.23-9.49). Overall, HLA-Cw*12 in combination
with KIR2DL3 was also more common in those with
SVR (12.1% versus 4.0%, P ? 0.03, OR ? 3.4, 95%
CI ? 1.0-14.6). The most frequent group 1 HLA-C al-
lele, HLA-Cw*07 (27% of all HLA-C alleles in this pop-
As both treatment-induced and spontaneously resolv-
ing HCV infection are associated with KIR2DL3 and
group 1 HLA-C alleles, we determined if the same
HLA-C alleles were associated with spontaneously resolv-
ing HCV infection in our previously described UK co-
hort.19Data were available on 341 individuals (122 with
those remaining persistently infected, were more likely to
have the combination of HLA-Cw*03 and KIR2DL3
(31.2% versus 19.2%, P ? 0.01 OR ? 1.9, 95% CI ?
1.1-3.2) (Table 7). Furthermore, this was only beneficial
in individuals homozygous for KIR2DL3 (18.9% versus
9.1%, P ? 0.01, OR ? 2.3, 95% CI ? 1.2-4.4), indicat-
to be due to linkage disequilibrium with another gene at
the MHC. HLA-Cw*12 in combination with KIR2DL3
was also found more frequently in those with resolved
HCV infection, but this did not reach statistical signifi-
the exposed seronegative aviremic cohort, with the fre-
quency of HLA-Cw*03 in combination with KIR2DL3
being 16.7% in the exposed seronegative aviremic indi-
viduals as compared to 18.3% in the chronically infected
IDU. The increase in group 1 HLA-C allotypes as com-
pared to the chronically infected individuals is thus re-
lated to small changes across the range of group 1 HLA-C
This work shows that KIR2LD3 and its group 1
HLA-C ligand are associated with favorable outcomes
across a spectrum of clinical profiles following HCV ex-
posure. These data, combined with our previous work
and that of Romero et al.,20show a beneficial influence of
of individuals: those having an apparent degree of resis-
who resolve chronic infection with IFN-?-based treat-
detectable anti-HCV Ab or RNA, but up to 60% of these
individuals will have detectable T-cell responses to HCV
KIR2DL3 and group 1 HLA-C alleles implies that they
are a population selected out by exposure to HCV. In-
deed, the frequency of this combination of genes is re-
markably similar to that found for Ab-positive RNA-
Table 6. KIR2DL3 and Group 1 HLA-C Associations in
P Value (Pc) OR (95% CI)
Number of patients107 101
Table 7. KIR2DL3 and Group 1 HLA-C Associations in the Spontaneously Resolving Cohort
(Pc) OR (95% CI)
Number of patients
KIR2DL3 and HLA-C alleles
KIR2DL3 number and HLA-Cw*03
0.01 (0.02)2.3 (1.2-4.4)
HEPATOLOGY, Vol. 51, No. 4, 2010 KNAPP ET AL.1173
negative individuals.19Thus, they appear to be distinct
from this subgroup of individuals on the basis of a lack of
anti-HCV Ab, but not KIR:HLA genotype. This implies
that that they do not have an additional NK cell protec-
tion above that observed with the Ab-positive, RNA-neg-
ative group of individuals. Consistent with the beneficial
advantageous effect of the KIR:HLA genotype in sponta-
neous clearers was strongest in those who acquired HCV
infection from IDU, suggesting that the size of inoculum
is important. The chronically infected IDU comparator
uals had a frequency of KIR2DL3-C1 similar to that of a
US cohort of Caucasians with chronic HCV.36This im-
plies that the KIR:HLA genotypes in our population are
representative of Caucasians with chronic HCV infec-
tion, and not due to a selection bias.
KIR2DL3-C1 was associated with SVR in our IFN-?-
treated individuals, but homozygosity for these genes was
not. One possibility is that the activation of NK cells by
pharmacological doses of IFN-? can overcome the inhi-
cells that coexpress KIR2DL3 and KIR2DL1 may be an-
tiviral in this context, but not in spontaneously resolving
infection. Alternatively, KIR protection could also be re-
lated to an effect on KIR-positive T cells in IFN-?-in-
as individuals who are homozygous for KIR2DL3-C1
would be predicted to have more NK cells that expressed
KIR2DL3 than those who were heterozygous for either
gene. However, we cannot rule out that in all stages of
chronic infection it is related to an effect of KIR2DL3 on
the KIR-positive T cells, which are usually of the effector
similar numbers to healthy controls.37Furthermore, the
odds ratio is smaller for this group of individuals than for
the exposed seronegative aviremic cohort, implying that
although these alleles are associated with viral clearance
they may be less important than in spontaneously resolv-
ing infection. Thus, they are one of several factors that
determine outcome following IFN-?-based therapy, of
which the most important is viral genotype. One compli-
cating factor in the interpretation of the data is that cir-
rhosis is an adverse factor in response to antiviral therapy
for HCV. Recent work has suggested that homozygosity
for KIR2DL3-C1 is associated with the development of
cirrhosis in chronic HCV infection and that KIR and
HLA-C may affect the severity of liver disease following
transplantation for HCV infection.36,38Thus, the benefi-
cial effect of KIR2DL3-C1 homozygosity on viral clear-
ance may be balanced by the adverse effect that this gene
combination has on disease stage. This could be one ex-
planation as to why we have not observed KIR2DL3-C1
homozygosity as an advantageous factor in these individ-
Our observations of KIR:MHC extend to the allelic
level for HLA-C for the IFN-treated patients and also
those resolving infection. Overall, across all groups we
found a trend toward most group 1 HLA-C alleles apart
from Cw*07 being beneficial. There is no clear data that
shows that HLA-Cw*07 is a stronger binder of KIR than
other HLA-C allotypes, but it may be crossreactive with
KIR2DL1 and this crossreactivity may be relevant as a
and function40and NK cells from HLA-Cw*07-positive
individuals make significantly more IFN-? when chal-
lenged with an MHC class I-negative target than those
with other HLA-C allotypes.40These data suggest that
NK cells from HLA-Cw*07-positive individuals may be
“licensed” differently from those with other group 1
HLA-C allotypes. The effect of this may be to respond to
activating signals with a different magnitude of IFN-?
secretion. One effect of this could be a change in the NK
cell response to HCV infection. Furthermore, KIR2DL2
and KIR2DL3 segregate as alleles at a single locus and
protection in HCV is only observed with KIR2DL3.
is sensitive to the peptide bound by HLA-C this raises the
cell reactivity. Although functional work is required to
tease out the precise mechanisms of inhibitory KIR-me-
diated protection in viral infections, our data underscore
the relevance of this gene family in the immune response
hepatology specialist nurses at Southampton General
Hospital for their assistance in performing this study.
We thank Karen Parker and all the
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