KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C.

Abstract

Natural killer (NK) cells play pivotal roles in immune responses against infection with viruses, such as hepatitis C virus (HCV), and killer cell immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells. The aim of this study was to investigate the possibility that KIR genes and their human leukocyte antigen (HLA) ligands influence progression to cirrhosis in patients infected with genotype 1 of HCV. A total of 145 Brazilian patients with confirmed chronic hepatitis C grouped from F0 to F4 according to fibrosis progression to cirrhosis were evaluated. Genotyping of KIR and HLA genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes. The HLA-C2 KIR ligand was more frequent in patients than in healthy controls (74.5% vs 64.3%, p = 0.04, odds ratio (OR) = 1.6, 95% confidence interval (CI) = 1.03-2.52). Moreover, the HLA-C1C2 genotype was more frequent in patients with advanced fibrosis or cirrhosis (F3-F4 group) than in patients in the F0-F2 group (61.6% vs 44.7%, p = 0.06) and in the F4 group compared with the F0-F3 group (65.7% vs 46.7%, p = 0.05, OR = 2.19, 95% CI = 1.01-4.73). NK and KIR ligands may contribute to the development of liver damage in patients chronically infected by HCV.

Full text

Page 1

KIR genes and their human leukocyte antigen ligands in the progression to
cirrhosis in patients with chronic hepatitis C
Amanda Vansan Marangona,*, Giovanni Faria Silvab, Camila Fernanda Verdichio de Moraesc,
Rejane Maria Tomasini Grottoc, Maria InËs de Moura Campos Pardinic, Dayse Sousa de Paulid,
Ana Maria Sella, Jeane Eliete Laguila Visentainera, Ricardo Alberto Moliternoa
aImmunogenetics Laboratory, Department of Basic Sciences, Maringà State University, UEM, Maringà, Paranà, Brazil
bGastroenterology Division, Internal Medicine Department, Botucatu Medical School, Sâo Paulo State University, UNESP, Botucatu, Sâo Paulo, Brazil
cMolecular Biology Laboratory of Blood Transfusion Center, Botucatu Medical School, Sâo Paulo State University, UNESP, Botucatu, Sâo Paulo, Brazil
dLondrina Clinical Hospital, Londrina State University, UEL, Londrina, Paranà, Brazil
A R T I C L EI N F O
Article history:
Received 5 January 2011
Accepted 25 August 2011
Available online 28 August 2011
Keywords:
NK cells
HLA
KIR
Hepatitis C
Liver fibrosis
A B S T R A C T
Natural killer (NK) cells play pivotal roles in immune responses against infection with viruses, such as
hepatitis C virus (HCV), and killer cell immunoglobulin-like receptors (KIRs) are related to the activation and
inhibition of NK cells. The aim of this study was to investigate the possibility that KIR genes and their human
leukocyte antigen (HLA) ligands influence progression to cirrhosis in patients infected with genotype 1 of
HCV. A total of 145 Brazilian patients with confirmed chronic hepatitis C grouped from F0 to F4 according to
fibrosis progression to cirrhosis were evaluated. Genotyping of KIR and HLA genes was performed by
polymerase chain reaction with sequence-specific oligonucleotide probes. The HLA-C2 KIR ligand was more
frequent in patients than in healthy controls (74.5% vs 64.3%, p ? 0.04, odds ratio (OR) ? 1.6, 95% confidence
interval (CI) ? 1.03–2.52). Moreover, the HLA-C1C2 genotype was more frequent in patients with advanced
fibrosis or cirrhosis (F3–F4 group) than in patients in the F0–F2 group (61.6% vs 44.7%, p ? 0.06) and in the F4
group compared with the F0–F3 group (65.7% vs 46.7%, p ? 0.05, OR ? 2.19, 95% CI ? 1.01–4.73). NK and KIR
ligands may contribute to the development of liver damage in patients chronically infected by HCV.
? 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.
1. Introduction
Natural killer cells (NK) are crucial components of the innate
response and provide defense mainly against viral infections and
cancer. They also provide a link to priming of adaptive immunity.
The function of NK cells is regulated by a fine balance of inhibitory
and activating signals, which are mediated by a diverse array of
cell-surface receptors including killer cell immunoglobulin-like re-
ceptors (KIR). These receptors can either inhibit or activate NK cells
andthedifferencebetweenthemliesmainlyintheirintracytoplas-
matic tail [1–3].
In humans, KIRs are encoded by a cluster of genes located in the
leukocyte receptor complex on chromosome 19q13.4. At least 14
KIR genes have thus far been characterized, of which 8 are NK cell
inhibitors (KIR2DL1, ?2DL2, ?2DL3, ?2DL4, ?2DL5, ?3DL1, ?3DL2,
and ?3DL3), 6 are activating (KIR2DS1, -2DS2, -2DS3, -2DS4, -2DS5,
and -3DS1), and 2 are pseudogenes (KIR2DP1 and -3DP1) [4,5].
Based on the genetic content and pattern of segregation at the
population level, KIR haplotypes are divided into 2 groups, A and B,
which differ in the number and kind of KIR genes. Common to both
groups of haplotypes are the “framework genes”: KIR3DL2, -3DP1,
-2DL4, and -3DL3 [4–6].
KIRs specifically recognize some human leukocyte class I anti-
gens (HLA-I) present on target cells. HLA-Bw4 and distinct allo-
types of HLA-C (C1 and C2 groups) are the predominant ligands for
most KIRs [7]. The HLA-C alleles were classified as C1 or C2 KIR
ligand groups, depending upon 2 amino acid positions encoded in
exon 2. The HLA-C1 allotypes contain serine at position 77 and
asparagine at position 80 and are ligands for the inhibitory recep-
tors KIR2DL2 and KIR2DL3. The HLA-C2 allotypes contains aspara-
gine and lysine at those positions and are ligands for the inhibitory
receptor KIR2DL1 and for the activating receptor KIR2DS1 [8,9].
HLA-Bw4allotypesarecharacterizedbyatleast5differentpatterns
of amino acid exchanges at positions 77 and 80–83 and serve as
ligands for KIR3DL1. In addition, HLA-A3 and HLA-A11 are ligands
of KIR3DL2 [10–12].
Accumulated evidence suggests that NK cells play an important
role not only in the host defense against pathogens in the liver but
also in liver injury and repair [2,3]. Because of the important role of
KIR genes in the control of NK cell activation, investigations have
been conducted on different populations to evaluate the associa-
tionsbetweenKIRandliverdiseasessuchasviralhepatitis[13–15].
* Corresponding author.
E-mail address: amanda_vansan@hotmail.com (A.V. Marangon).
Human Immunology 72 (2011) 1074-1078
Contents lists available at SciVerse ScienceDirect
0198-8859/11/$32.00 - see front matter ? 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.humimm.2011.08.017

Page 2

Most patients with hepatitis C virus (HCV) infection progress to
chronic disease, which can lead to liver fibrosis and the subsequent
occurrence of cirrhosis and hepatocellular carcinoma [16]. How-
ever, the mechanisms responsible for the onset and progression of
hepatic lesions during chronic hepatitis C are not fully understood.
Hence, the aim of the present study was to investigate the possible
association of KIR genes and their HLA ligands with fibrosis pro-
gression to cirrhosis in patients with chronic hepatitis C.
2. Subjects and methods
2.1. Studied population
Between September 2004 and January 2009 a total of 761 pa-
tientsreceivedadiagnosisofHCVinfectionattheInternalMedicine
Department, Gastroenterology Division, Botucatu Medical School.
Of these, 145 unrelated patients with infection only by the HCV
genotype1wereincludedinthisstudy.Atotalof616patientswere
excluded based on the following criteria: human immunodefi-
ciency virus–positive serology, hepatitis B virus coinfection, other
hepatic diseases, presence of non-1 HCV genotype, and history of
antiviral therapy before liver biopsy. In addition, 289 unrelated
healthyindividualsfromSouthernBrazil[17]wereusedascontrols
and compared with HCV patients.
The studied protocol was approved by the Ethics Committee for
ResearchInvolvingHumanBeingsofSÄoPauloStateUniversityand
all subjects provided written consent to participate in the study.
2.2. Liver biopsy
Biopsies in all patients were carried out using the technique of
Menghini or a Tru-Cut needle to determine the degree of fibrosis
andprogressionofinfection.Thefragmentswereanalyzedwhenat
least 8 portal spaces were present and stained with hematoxylin
and eosin, Masson trichrome, and reticulin stain. The hepatic biop-
sies were analyzed by a pathologist using the Metavir score: F0, no
fibrosis;F1,portalfibrosiswithoutsepta;F2,portalfibrosisandfew
septa; F3, numerous septa without cirrhosis; and F4, cirrhosis [18].
The patients were stratified into subgroups according to fibrosis
stage:absenceoffibrosisormildfibrosis(F0–F2,n?85),advanced
fibrosis or cirrhosis (F3–F4, n ? 60), without cirrhosis (F0–F3), and
with cirrhosis (F4).
2.3. HCV genotyping
The HCV genotype was defined by the reverse line probe assay
(Innolipa v. 1.0, Innogenetics, Ghent, Belgium) according to the
manufacturer’s instructions. Genotyping was preceded by the ex-
traction of HCV viral RNA present in the patient’s plasma, followed
byreverse-transcriptionpolymerasechainreaction(PCR;Amplicor
HCV test, v. 2.0, Roche Diagnostic Systems, Branchburg, NJ) accord-
ing to the manufacturer’s instructions.
2.4. DNA extraction
Genomic DNA was isolated from 250 ?L of peripheral blood
using a NeoIsocolumn kit (One Lambda, Inc., San Diego, CA) or
purelink(Invitrogen,Carlsbad,CA)accordingtothemanufacturer’s
instructions.
2.5. KIR and HLA-I genotyping
KIR (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1,
KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5,
KIR3DS1, KIR2DP1, and KIR3DP1) and HLA-I (HLA-A, -B, and -C)
genes were genotyped according to the manufacturer’s instruc-
tions by PCR with sequence-specific oligonucleotide probe proto-
cols using commercial KIR and HLA genotyping test kits (One
Lambda, Inc.) [19].
ThesequencesofallalleleswereanalyzedonebyoneintheHLA
database (http://www.ebi.ac.uk/imgt/hla/) by the presence of a
certain amino acid in the adequate positions and classified into C1,
C2, and Bw4 groups. The C1 group of alleles observed in patients
were C*01, C*03, C*07, C*08, C*12, C*14, C*15:07, and C*16:01. The
C2 group of alleles were C*02, C*04, C*05, C*06, C*07:07, C*12:05,
C*15, C*16:02, C*17, and C*18. The group of alleles that belongs to
the Bw4 group were B*13, B*27:05, B*27:09, B*37:01, B*38, B*44,
B*47, B*49, B*51, B*52, B*53, and B*58. Only alleles observed in the
patients were described.
2.6. KIR2DS4
The 2 major full-length subtype and deleted forms of KIR2DS4
[20] were also analyzed by PCR with sequence-specific oligonucle-
otide probe protocols. The samples with doubtful results were
confirmed by in-house PCR with sequence specific primers accord-
ing to Martin [21] and optimized by Rudnick et al. [17].
2.7. Statistical analysis
KIR and KIR-HLA ligand frequencies were obtained by direct
counting. The ?2test with Yates’ correction or Fisher’s exact tests
was applied to compare the distribution of the frequencies of al-
leles and genes between the groups. Odds ratios (OR) with 95%
confidence intervals (CI) were also calculated. The analyses were
carried out using SISA online statistics (http://www.quantita-
tiveskills.com/sisa/). p values ?0.05 were considered statistically
significant. Because the gametic phase of the alleles from the dif-
ferent loci is not known (segregation analysis into families was not
carriedout),haplotypesandthecalculationoftheirfrequencywere
determined by the likelihood-ratio test using the Arlequin statisti-
cal software program according to Excoffier et al. [22].
3. Results
3.1. Patient characteristics
Patient characteristics are summarized in Table 1. According to
the Metavir score, 58.6% of patients presented no or mild fibrosis
(4.8% were F0, 29.7% F1, and 24.1% F2) and 41.8% presented ad-
vancedfibrosisorcirrhosis(15.2%wereF3and26.2%F4).Themean
age of patients with advanced fibrosis or cirrhosis was higher than
thatofpatientswithoutfibrosisormildfibrosis(48.6?9.8yearsvs
39.8 ? 9.3 years, respectively, p ? 0.05). The same trend was seen
when comparing the F4 group versus the F0–F3 group (27.8 ? 9.6
years vs 19.9 ? 7.0 years, respectively, p ? 0.05). With respect to
possibleriskfactorsforacquiringhepatitisC,theuseofintravenous
drugs was the main risk factor associated with the F0–F2 group
(25.8%) compared with only 2 patients (3.3%) in the F3–F4 group
(p ? 0.05%). The use of intravenous drugs was also the main risk
factor associated with the F0–F3 group compared with the F4
group (20.5% vs 5.3%, p ?0.05).
3.2. KIR genes and HLA ligand frequencies
ToevaluatetheroleofKIRgenesandtheirHLAligandsindisease
progression, patients were first stratified into individuals with ab-
sence of fibrosis or mild fibrosis (F0–F2, n ? 85) and individuals
with advanced fibrosis or cirrhosis (F3–F4, n ? 60) Next, patients
withcirrhosis(F4stage,n?38)wereseparatedandcomparedwith
patients without cirrhosis (F0–F3 stage, n ? 107). Furthermore, all
patients were compared with unrelated healthy controls.
The HLA epitopes (HLA-C1, -C2, -Bw4 and -A3/A11) were ana-
lyzed in all groups. The gene and genotype frequencies of the HLA
ligands are presented in Table 2. HLA-C2 was more frequent in
patientsthaninhealthycontrols(74.5%vs64.3%,p?0.04,OR?1.6,
95% CI ? 1.03–2.52). The heterozygote genotype HLA-C1C2 exhib-
ited a trend of being more frequent in the F3–F4 group compared
withtheF0–F2group(61.6%vs44.7%;p?0.06,OR?1.98,95%CI?
1.02–3.9). The HLA-C1C2 genotype was also more frequent in pa-
tients with liver cirrhosis (F4) compared with patients without
A.V. Marangon et al. / Human Immunology 72 (2011) 1074-1078
1075

Page 3

cirrhosis (F0–F3; 65.7% vs 46.7%, p ? 0.05, OR ? 2.19, 95% CI ?
1.0–4.7).
HLA-Bw4 molecules were divided into 2 groups based on
whether isoleucine or threonine was present at position 80 (Bw4-
80Ile and Bw4-80Thr, respectively). The Bw4-80Ile exhibited a
trend of being less frequent in the group with liver cirrhosis (F4)
comparedwithpatientswithoutcirrhosis(39.5%vs56.1%,p?0.09,
OR ? 0.51, 95% CI ? 0.24–1.0; Table 2).
With respect to KIR genes, no association between the 16 KIR
genesandsusceptibilitytoHCV,progressiontofibrosis,orcirrhosis
was observed (Table 3). Nevertheless, a lower frequency of the
homozygotegenotypeKIR2DL3/KIR2DL3wasobservedintheF3–F4
group(45.4%)comparedwiththeF0–F2group(62.0%;Table4).The
deleted variant of KIR2DS4 was the most frequent form in all
groups, but the distribution of the KIR2DS4 forms (the full-length
gene, the deletion variant, or both) was similar in all groups.
Inanalysisoftheinteractionsbetweenactivatingandinhibitory
KIR and HLA epitopes (KIR2DL1 and its ligand C2; KIR2DL2 with C1;
KIR2DL3 with C1; KIR3DL1 with Bw4; KIR3DL1 with Bw4-80Ile;
KIR3DL1withBw4-80Thr;KIR3DL2withHLA-A3and-A11;KIR2DS1
with C2; KIR2DS2 with C1; KIR3DS1 with Bw4; KIR3DS1 with Bw4-
80Ile; and KIR3DS1 with Bw4-80Thr), none of the receptor–ligand
pairs was associated with susceptibility or progression of disease
(Table 4).
Furthermore, patients were identified as KIR AA, AB, or BB hap-
lotype based on the multiple KIR genes they possessed. KIR BB
haplotypes were characterized by the presence of one or more of
the following genes:KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5,
and KIR3DS1. Conversely, KIR AA haplotypes were characterized by
both the absence of any 1 of the preceding genes and the presence
of the genes KIR3DL3, KIR2DL3, KIR2DL1, KIR3DP1, KIR2DL4,
KIR3DL1, KIR2DS4, and KIR3DL2. The AB haplotype was character-
ized by the presence of genes of the 2 groups [4,23,24]. The KIR AA
haplotype was the most frequent haplotype in all groups (33.1% of
all patients, 39.2% in the F3–F4 group, 33.3% in the F0–F2 group,
34.2% in the F4 group, 32.7% in the F0–F3 group, and 33.2% in
unrelated healthy controls). No significant difference in the distri-
bution of KIR haplotypes was observed.
4. Discussion
NK cells represent a fundamental component of the innate im-
munesystem.TherepertoireofNKcellsisextensiveintheliver[25]
and cytolytic activity seems to have an inverse association with
liver fibrosis stage, suggesting that the presence of cytolytic active
Table 1
Clinical and general features of patients with chronic hepatitis C attended in the Department of Internal Medicine, Gastroenterology Division, Botucatu Medical School,
from September 2004 to January 2009 (n ? 145)
Characteristics Patients
(n ? 145)
F0–F2
(n ? 85)
F3–F4
(n ? 60)
F4
(n ? 38)
p
Age (mean ? SD)a
Gender, n (%)
Male
Female
Risk factor, n (%)
Blood transfusion
Intravenous druga
Gluconergan
Nasal drug
Othersb
Duration of infectionc(mean ? SD)
METAVIR stage,dn (%)
F0
F1
F2
F3
F4
43.4 ? 10.4 39.8 ? 9.348.6 ? 9.850.0 ? 9.53
?0.05
112 (0.77)
33 (0.22)
67 (0.78)
18 (0.21)
45 (0.75)
15 (0.25)
17 (0.71)
11 (0.28)
0.73
0.73
36 (0.24)
24 (0.16)
22 (0.15)
5 (0.03)
58 (0.40)
21.8 ? 8.3
17 (0.20)
22 (0.25)
10 (0.11)
2 (0.02)
34 (0.40)
19.0 ? 6.9
19 (0.31)
2 (0.03)
12 (0.20)
3 (0.05)
24 (0.40)
25.7 ? 8.7
10 (0.26)
2 (0.05)
6 (0.15)
3 (0.08)
17 (0.44)
27.8 ? 9.6
0.15
?0.05
0.25
0.64
0.97
0.10
7 (0.04)
43 (0.29)
35 (0.24)
22 (0.15)
38 (0.26)
7 (0.08)
43 (0.50)
35 (0.41)
—
—
—
—
—
22 (0.36)
38 (0.63)
—
—
—
—
38 (1.00)
—
—
—
—
—
ap ? 0.05 when comparing F0–F2 with F3–F4 and F0-F3 with F4.
bTattoo, surgeries, occupational exposure, homosexuals or promiscuous sexual behavior, and unknown risk factors.
cDuration of infection was calculated only for 90 patients (F0 ? 4, F1 ? 25, F2 ? 24, F3 ? 16, and F4 ? 21). The duration of infection for 55 patients is unknown.
dMetavir score: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis and few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis.
Table 2
Distribution of gene and genotype frequencies of KIR-HLA ligands in controls and in patients chronically infected by the HCV-1 genotype, classified according to their liver
biopsies Metavir score
HLA ligandsMetavir score groupsa
All patientsControls
p
F3–F4
n (f)
F0–F2
n (f)
p
F4
n (f)
F0–F3
n (f)
p
A3 and A11
Bw4
C1
C2b
C1C1
C1C2c
C2C2
Bw4-80Iled
Bw4-80Thr
19 (0.31)
40 (0.66)
49 (0.81)
48 (0.80)
12 (0.20)
37 (0.61)
11 (0.18)
28 (0.46)
17 (0.28)
18 (0.21)
64 (0.85)
63 (0.74)
60 (0.70)
25 (0.29)
38 (0.44)
22 (0.25)
47 (0.55)
21 (0.24)
0.21
0.90
0.38
0.27
0.27
0.06
0.38
0.39
0.76
11 (0.29)
24 (0.63)
31 (0.81)
32 (0.84)
6 (0.15)
25 (0.65)
7 (0.18)
15 (0.39)
13 (0.34)
25 (0.23)
80 (0.74)
81 (0.75)
76 (0.71)
31 (0.29)
50 (0.46)
26 (0.24)
60 (0.56)
25 (0.23)
0.64
0.24
0.50
0.13
0.1
0.05
0.60
0.09
0.27
37 (0.25)
104 (0.71)
112 (0.77)
108 (0.74)
37 (0.25)
75 (0.51)
33 (0.22)
75 (0.51)
39 (0.26)
72 (0.24)
218 (0.75)
223 (0.77)
186 (0.64)
92 (0.31)
131 (0.45)
55 (0.19)
141 (0.48)
77 (0.26)
0.98
0.47
0.91
0.04
0.21
0.24
0.40
0.63
0.95
aMetavir score: F3–F4, advanced fibrosis or cirrhosis (n ? 60); F0–F2, without fibrosis or mild fibrosis (n ? 85); F4, with cirrhosis (n ? 38); F0–F3, without cirrhosis (n ? 107).
bAll patients (n ? 145) versus controls (n ? 289): odds ratios (OR) ? 1.61; 95% confidence interval (CI) 1.03–2.52.
cF3–F4 versus F0–F2, OR ? 1.98, 95% CI ? 1.02–3.9; and F4 versus F0–F3, OR ? 2.19, 95% CI ? 1.01–4.7.
dF4 versus F0–F3: OR ? 0.51, 95% CI ? 0.24–1.0.
A.V. Marangon et al. / Human Immunology 72 (2011) 1074-1078
1076

Page 4

peripheralNKcellsmaybeprotectiveagainstliverdiseaseprogres-
sion [26]. NK cells likely play pivotal roles in immune responses
against HCV infection and KIRs are the major group of receptors
that modulate NK function.
It has been observed that KIR genes and HLA–KIR interactions
can influence disease susceptibility and various works suggest that
NK activation enhances HCV clearance. An important association
seems to involve the KIR2DL3 gene. Romero et al. [27] reported an
association between the KIR2DL3/2DL3 genotype and its ligand
HLA-C1C1 with spontaneous HCV clearance. Khakoo et al. [13] also
suggested that the genotypic combination of KIR2DL3 with HLA-C1
was protective in terms of spontaneous clearance of the virus in
individuals infected with low-dose viral inoculums. They hypothe-
sized that this KIR–ligand pair could be protective and improve
resistance to viral infection because it has a weak inhibitory func-
tion that could be easily overridden by signals through activating
receptors upon appropriate ligand binding, resulting in lysis of the
target. In the hierarchy of KIR2DL molecule–mediated inhibition,
KIR2DL3/HLA-C1C1 appears to confer weaker inhibitory responses,
whereas KIR2DL1 with C2C2 appears to have the highest inhibitory
power [28].
Khakoo et al. [13] also suggested that KIR2DL2 seems to coun-
teract KIR2DL3/HLA-C1C1 protection once they determined that
only individuals homozygous for KIR2DL3, and not KIR2DL2/
KIR2DL3 heterozygotes or KIR2DL2 homozygotes, were associated
with HCV clearance. Nonetheless, another study reported an asso-
ciation of KIR2DL2 with viral clearance [29]. A recent study with
intravenous drug users observed a significant association between
the presence of the inhibitory receptors KIR2DL2 and/or KIR2DL3
andtheHLA-C1homozygousgenotypetogetherwiththeactivating
receptor KIR2DS4 in protection from HCV infection [30].
In the present study patients were carefully selected to form a
homogeneous group in terms of HCV genotype and absence of viral
coinfections. The data did not indicate any significant association
between inhibitory or activating KIR genes and susceptibility to
HCVinfection,progressiontofibrosis,orcirrhosis.Nonetheless,the
frequency of the genotype KIR2DL3/KIR2DL3 was higher in the
F0–F2 group (62.0%) compared with the F3–F4 group (45.4%). Kak-
hoo et al. [13] attempted to explain the finding of protection of
KIR2DL3/KIR2DL3genotypebasedontheclonalexpressionofKIRon
NK cells. Within an individual, a KIR gene is expressed on some, but
not all NK cell clones [28]. In this way, homozygotes for KIR2DL3
will have more NK cells solely under the inhibitory control of
KIR2DL3 than will KIR2DL2/KIR2DL3 heterozygotes [13]. Although
the statistical power of the study was insufficient to confirm this
association,theresultmightindicatethattheKIR2DL3homozygote
genotype can be important not only for spontaneous HCV clear-
ance, as previously observed, but also for protection against liver
disease injury and is deserving of further investigation.
With respect to the distribution of HLA-C ligands, HLA-C2 was
more frequent in patients than in healthy controls, suggesting that
thepresenceofHLA-C2isassociatedwithsusceptibilitytoinfection
by the HCV-1 genotype. Because KIR2DL1 binds the C2 HLA ligand,
it is possible that this very strong inhibitory interaction could
account, in part, for the susceptibility to HCV-1 genotype infection,
and the HLA ligand genotype could determine the relative effect of
the KIR genotype. Accordingly, Khakoo et al. [13] reported a higher
frequency of HLA-C2C2 genotype among patients with persistent
infection and a higher frequency of HLA-C1C1 in individuals with
resolved infection. Additionally, Lopez-Vazquez et al. [15] reported
a positive association between HLA-C2 homozygosis and clinical
evolutiontohepatocellularcarcinomainpatientswithchronicHCV
infection. According to the authors, the associations with HLA class
IligandsmayhaveaneffectonthephysiopathologyofHCVbecause
Table 3
Distribution of KIR genes in controls and in patients chronically infected by the HCV-1 genotype, classified according to their liver biopsy Metavir score
KIR genesMetavir score groupsa
All patientsb
Controlsb
p
F3–F4
n (f)
F0–F2
n (f)
p
F4
n (f)
F0–F3
n (f)
p
KIR2DL1
KIR2DL2
KIR2DL3
KIR2DL4
KIR2DL5
KIR2DP1
KIR2DS1
KIR2DS2
KIR2DS3
KIR2DS4
KIR2DS5
KIR3DL1
KIR3DL2
KIR3DL3
KIR3DP1
KIR3DS1
58 (0.96)
32 (0.53)
54 (0.90)
60 (1.00)
28 (0.46)
58 (0.96)
25 (0.41)
32 (0.53)
16 (0.26)
53 (0.88)
23 (0.38)
53 (0.88)
60 (1.00)
60 (1.00)
60 (1.00)
23 (0.38)
84 (0.98)
36 (0.42)
82 (0.96)
85 (1.00)
50 (0.59)
84 (0.98)
35 (0.41)
39 (0.46)
30 (0.35)
79 (0.93)
30 (0.35)
78 (0.92)
85 (1.00)
85 (1.00)
85 (1.00)
37 (0.44)
0.56
0.23
0.76
0.99
0.17
0.56
0.99
0.40
0.28
0.38
0.72
0.57
0.99
0.99
0.99
0.60
38 (1.00)
17 (0.45)
36 (0.95)
38 (1.00)
18 (0.47)
38 (1.00)
17 (0.45)
18 (0.47)
9 (0.24)
32 (0.84)
16 (0.42)
32 (0.84)
38 (1.00)
38 (1.00)
38 (1.00)
17 (0.45)
104 (0.97)
51 (0.48)
100 (0.93)
107 (1.00)
60 (0.56)
104 (0.97)
42 (0.39)
53 (0.50)
37 (0.35)
100 (0.93)
37 (0.35)
99 (0.93)
107 (1.00)
107 (1.00)
107 (1.00)
43 (0.40)
0.60
0.90
1.00
0.45
0.46
0.60
0.68
0.96
0.29
0.16
0.52
0.24
0.45
0.76
0.52
0.24
143 (0.99)
68 (0.47)
134 (0.92)
145 (1.00)
78 (0.54)
142 (0.98)
59 (0.41)
71 (0.49)
46 (0.32)
132 (0.91)
53 (0.37)
131 (0.90)
145 (1.00)
145 (1.00)
145 (1.00)
60 (0.41)
281 (0.97)
136 (0.47)
258 (0.89)
289 (1.00)
152 (0.53)
280 (0.97)
118 (0.41)
136 (0.47)
77 (0.27)
271 (0.94)
100 (0.35)
272 (0.94)
289 (1.00)
289 (1.00)
289 (1.00)
113 (0.39)
0.56
0.94
0.38
0.21
0.89
0.75
0.93
0.78
0.31
0.39
0.76
0.21
0.80
0.80
0.80
0.72
aMetavir score: F3–F4, advanced fibrosis or cirrhosis (n ? 60); F0–F2, without fibrosis or mild fibrosis (n ? 85); F4, with cirrhosis (n ? 38); F0–F3, without cirrhosis (n ? 107).
bAll patients (n ? 145) versus controls (n ? 289).
Table 4
KIR genotypes and KIR-HLA ligand frequencies in patients chronically infected by
the HCV-1 genotype
KIR genotypes and KIR with HLA
ligands
F3–F4a
n (f)
F0–F2a
n (f)
p
KIR2DL2/2DL2
KIR2DL2/2DL3
KIR2DL3/2DL3b
KIR2DL2 ? HLA-C1
KIR2DL3 ? HLA-C1
KIR2DL1 ? HLA-C2
KIR3DL1 ? HLA-Bw4
KIR3DL2 ? HLA-A3 and ? A11
KIR2DS1 ? HLA-C2
KIR2DS2 ? HLA-C1
KIR2DL2/2DL2 ? HLA-C1
KIR2DL2?KIR2DL3 ? HLA-C1
KIR2DL3/2DL3 ? HLA-C1
KIR2DL2/2DL2 ? HLA-C1C1
KIR2DL2?2DL3 ? HLA-C1C1
KIR2DL3/2DL3 ? HLA-C1C1
5 (0.15)
27 (0.45)
27 (0.45)
25 (0.41)
45 (0.75)
45 (0.75)
32 (0.53)
18 (0.30)
17 (0.28)
25 (0.41)
4 (0.12)
21 (0.35)
24 (0.43)
1 (0.03)
7 (0.11)
4 (0.06)
6 (0.16)
30 (0.35)
52 (0.62)
28 (0.32)
57 (0.67)
59 (0.69)
49 (0.57)
16 (0.18)
24 (0.28)
32 (0.37)
6 (0.16)
22 (0.25)
35 (0.41)
2 (0.05)
10 (0.11)
13 (0.15)
1.000
0.314
0.070
0.368
0.397
0.583
0.729
0.172
0.862
0.752
0.739
0.318
0.910
0.999
0.990
0.1911
aF3–F4, advanced fibrosis or cirrhosis (n ? 60); F0–F2,without fibrosis or mild
fibrosis (n ? 85) according to the Metavir score.
bOR ? 0.51, 95% CI ? 0.25–1.02.
A.V. Marangon et al. / Human Immunology 72 (2011) 1074-1078
1077

Page 5

of the capacity of these molecules to present antigens that have
different affinities for cytotoxic T lymphocytes, which may also be
related to their ability to interact with KIR receptors. In this study,
anassociationwasobservedonlywiththeHLA-C2,butnotwiththe
HLA-C1 or HLA-C2C2 genotype.
The heterozygote genotype HLA-C1C2 was more frequent in
individuals with advanced fibrosis or cirrhosis compared with pa-
tients without fibrosis or mild fibrosis and was also more frequent
in cirrhotic than in noncirrhotic patients. As previously discussed,
HLA-C1C2 heterozygosis may have a worse effect on the physiopa-
thology of HCV because of the ability of these molecules to interact
with more inhibitory KIR receptors, leading to a higher NK hypore-
sponsiveness. In other words, HLA-C1C2 patients will probably
have a higher proportion of NK cells that will be inhibited by
KIR2DL1 [13].
With respect to the HLA-Bw4 ligands, we observed a trend
toward a higher frequency of Bw4-80Ile in patients without liver
cirrhosis. Recently, Lopez-Vazquez et al. [15] reported that the
HLA-B Bw4-80Ile epitope and the KIR3DS1 gene had a protective
effect against the development of hepatocellular carcinoma in pa-
tients infected with HCV, especially in those with minimal liver
damage. In contrast, Paladino et al. [14] reported an increased
frequency of 2 copies of KIR3DS1 and HLA-Bw4 in cirrhotic patients
infected with HCV.
5. Conclusion
A significant association with KIR genes to cirrhosis progression
in patients with chronic HCV was not observed, although the
KIR2DL3/KIR2DL3 genotype was more frequent in F0–F2 patients,
HLA-C2wasmorefrequentinpatientsthanhealthycontrols,HLAB
Bw480Ile was more frequent in noncirrhotic patients, and the HLA
C1C2 genotype was more frequent in F3–F4 and F4 patients. NK
cells and KIR genes may contribute to the development of liver
damage in HCV-1 chronically infected patients, although further
investigations in other homogeneous patient samples must be per-
formed to confirm our data.
Acknowledgments
The authors thank all volunteers who took part in this study.
This work was supported in part by Capes, CNPq, and The Brazil
Health Ministry.
References
[1] Orange JS, Fassett MS, Koopman LA, Boyson JE, Strominger JL. Viral evasion of
natural killer cells. Nat Immunol 2002;3:1006–12.
[2] Yamagiwa S, Kamimura H, Ichida T. Natural killer cell receptors and their
ligands in liver diseases. Med Mol Morphol 2009;42:1–8.
[3] Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immu-
nol 2001;1:41–9.
[4] Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Corliss
B, et al. Human diversity in killer cell inhibitory receptor. Genes Immun
1997;7:753–63.
[5] Hsu KC, Liu XR, Selvakumar A, Mickelson E, O’Reilly RJ, Dupont B. Killer Ig-like
receptor haplotype analysis by gene content: evidence for genomic diversity
with a minimum of six basic framework haplotypes, each with multiple sub-
sets. J Immunol 2002;169:5118–129.
[6] MartinAM,FreitasEM,WittCS,ChristiansenFT.Thegenomicorganizationand
evolution of the natural killer immunoglobulin-like receptor (KIR) gene clus-
ter. Immunogenetics 2000;51:268–80.
[7] Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N. Direct binding and
functional transfer of NK cell inhibitory receptors reveal novel patterns of
HLA-C allotype recognition. J Immunol 1998;161:571–7.
[8] Colonna M, Borsellino G, Falco M, Ferrara GB, Strominger JL. HLA-C is the
inhibitory ligand that determines dominant resistance to lysis by NK1- and
NK2-specific natural killer cells. Proc Natl Acad Sci U S A 1993;90:12000–4.
[9] Wagtmann N, Rajagopalan S, Winter CC, Peruzzi M, Long EO. Killer cell inhib-
itory receptors specific for HLA-C and HLA-B identified by direct binding and
by functional transfer. Immunity 1995;3:801–9.
[10] O’Connor GM, Guinan KJ, Cunningham RT, Middleton D, Parham P, Gardiner
CM. Functional polymorphism of the KIR3DL1/S1 receptor on human NK cells.
J Immunol 2007;178:235.
[11] Martin MP, Gao XJ, Lee JH, Nelson GW, Detels R, Goedert JJ, et al. Epistatic
interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat
Genet 2002;31:429–34.
[12] Mu ¨ller CA, Engler-Blum G, Gekeler V, Steiert I, Weiss E, Schmidt H. Genetic
heterogeneity of the HLA-Bw4 and -Bw6 specificities lmmunogenetics 1989;
30: 200–7.
[13] Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, et al. HLA and
NK cell inhibitory receptor genes in resolving hepatitis C virus infection.
Science 2004;305:872–4.
[14] Paladino N, Flores AC, Marcos CY, Fainboim H, Theiler G, Arruvito L, et al.
Increased frequencies of activating natural killer receptors are associated with
liver injury in individuals who do not eliminate hepatitis C virus. Tissue
Antigens 2007;69 (suppl 1):109–11.
[15] Lopez-Vazquez A, Rodrigo L, MartÎnez-Borra J, PÊrez R, RodrÎguez M, Fdez-
Morera JL, et al. Protective effect of the HLA-Bw4I80 epitope and the killer cell
immunoglobulin-like Receptor 3DS1 gene against the development of hepa-
tocellular carcinoma in patients with hepatitis C virus infection. J Infect Dis
2005;192:162–5.
[16] Thomas DL, Seeff LB. Natural history of hepatitis C. Clin Liver Dis 2005;9:
383–98.
[17] Rudnick CCC, Franceschi DSA, Marangon AV, Guelsin GAS, Sell AM, Visentainer
JEL. Killer cell immunoglobulin-like receptor gene diversity in a Southern
Brazilian population from the state of ParanÂ. Hum Immunol 2008;69:872–6.
[18] Bedossa P. Presentation of a grid for computer analysis for complication of
hispathologic lesions in chronic viral hepatitis C. Ann Pathol 1993;13:260–5.
[19] FultonRJ,McDadeRL,SmithPL,KienkerLJ,KettmanJR.Advancedmultiplexed
analysis with the FlowMetrix? system. Clin Chem 1997;43:1749–56.
[20] MaxwellLD,WallaceA,MiddletonD,CurranMD.AcommonKIR2DS4deletion
variant in the human that predicts a soluble KIR molecule analogous to the
KIR1D molecule observed in the rhesus monkey. Tissue Antigens 2002;60:
254–8.
[21] Martin MP. Tipagem de genes KIR e sua aplicaÈÄo em estudos de populaÈÖes,
associaÈÄo com doenÈas e transplantes. In: VIII Congresso da Sociedade Brasil-
eira de Transplante De Medula ssea, 2004. Curitiba, ParanÂ, Brazil.
[22] Excoffier LG, Laval G, Schneider S. Arlequin Ver. 3.0: An integrated software
package for population genetics data analysis. Evol Bioinform Online 2005;1:
47–50.
[23] UhrbergM.TheKIRgenefamily:lifeinthefastlaneofevolution.EurJImmunol
2005;35:10–5.
[24] MartinAM,KulskiJK,GaudieriS,etal.Comparativegenomicanalysis,diversity
and evolution of two KIR haplotypes A and B. Gene 2004;335:121–31.
[25] Crispe IN, Mehal WZ. Strange brew: T cells in the liver. Immunol Today 1996;
17:522–5.
[26] Morishima C, Paschal DM, Wang CC, Yoshihara CS, Wood BL, Yeo AET, et al.
Decreased NK cell frequency in chronic hepatitis C does not affect ex vivo
cytolytic killing. Hepatology 2006;43:573–80.
[27] Romero V, Azocar J, ZÛÒiga J, Clavijo OP, Terreros D, Gu X, et al. Interaction of
NK inhibitory receptor genes with HLA-C and MHC class II alleles in hepatitis C
virus infection outcome. Mol Immunol 2008;45:2429–36.
[28] Kulkarni S, Martin MP, Carrington M. The yin and yang of HLA and KIR in
human disease. Semin Immunol 2008;20:343–52.
[29] Montes-Cano MA, Caro-Oleas JL, Romero-GÔmez M, Diago M, Andrade R,
carmona I, et al. HLA-C and KIR genes in hepatitis C virus infection. Hum
Immunol 2005;66:1106–9.
[30] ZÛÒigaJ,RomeroV,AzocarJ,TerrerosD,Vargas-RojasMI,Torres-GarcÎaD,etal.
Protective KIR–HLA interactions for HCV infection in intravenous drug users.
Mol Immunol 2009;46:2723–7.
A.V. Marangon et al. / Human Immunology 72 (2011) 1074-1078
1078