Download Influence of congenital human cytomegalovirus infection and the

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Daniel E. Noyola et al.
DOI: 10.1002/eji.201242752
Eur. J. Immunol. 2012. 42: 3256–3266
Influence of congenital human cytomegalovirus
infection and the NKG2C genotype on NK-cell subset
distribution in children
Daniel E. Noyola1 , Claudia Fortuny2 , Aura Muntasell3 ,
Antoni Noguera-Julian2 , Carmen Muñoz-Almagro4 , Ana Alarcón5 ,
Teresa Juncosa4 , Manuela Moraru6 , Carlos Vilches6
and Miguel López-Botet3,7
1
Department of Microbiology, Universidad Autónoma de San Luis Potosı́, San Luis Potosı́,
México
2
Infectious Diseases Unit, Pediatrics Department, Hospital Sant Joan de Déu, Universitat de
Barcelona, Barcelona, Spain
3
IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain
4
Department of Molecular Microbiology, Hospital Sant Joan de Déu, Universitat de Barcelona,
Barcelona, Spain
5
Department of Neonatology, Hospital Sant Joan de Déu, Universitat de Barcelona, Barcelona,
Spain
6
Immunogenetics-HLA, Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain
7
Immunology Unit, Universitat Pompeu Fabra, Barcelona, Spain
Human cytomegalovirus (HCMV) has been reported to reshape the NK-cell receptor (NKR)
distribution, promoting an expansion of CD94/NKG2C+ NK and T cells. The role of NK
cells in congenital HCMV infection is ill-defined. Here we studied the expression of NKR
(i.e., NKG2C, NKG2A, LILRB1, CD161) and the frequency of the NKG2C gene deletion in
children with past congenital infection, both symptomatic (n = 15) and asymptomatic
(n = 11), including as controls children with postnatal infection (n = 11) and noninfected
(n = 20). The expansion of NKG2C+ NK cells in HCMV-infected individuals appeared particularly marked and was associated with an increased number of LILRB1+ NK cells in cases
with symptomatic congenital infection. Increased numbers of NKG2C+ , NKG2A+ , and
CD161+ T cells were also associated to HCMV infection. The NKG2C deletion frequency
was comparable in children with congenital HCMV infection and controls. Remarkably,
the homozygous NKG2C+/+ genotype appeared associated with increased absolute numbers of NKG2C+ NK cells. Moreover, HCMV-infected NKG2C+/+ children displayed higher
absolute numbers of NKG2A+ and total NK cells than NKG2C+/− individuals. Our study
provides novel insights on the impact of HCMV infection on the homeostasis of the NK-cell
compartment in children, revealing a modulatory influence of NKG2C copy number.
Keywords: Congenital infection
r
Cytomegalovirus
r
NK cells
r
NKG2C
See accompanying Commentary by Malmberg et al.
Supporting Information available online
Introduction
Correspondence: Prof. Miguel López-Botet
e-mail: [email protected]
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Human cytomegalovirus (HCMV) infection is highly prevalent worldwide (50–100%), and usually follows a subclinical course in healthy individuals. The virus remains in a
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Eur. J. Immunol. 2012. 42: 3256–3266
lifelong latent state, occasionally undergoing reactivation, but
may have a pathogenic role in immunodeficient and immunosuppressed patients [1–3]. Moreover, HCMV has been associated with
atherosclerosis, lymphoproliferative disorders, and glioblastoma,
as well as with an accelerated immunosenescence and a shorter
lifespan [4–7].
Vertical transmission of HCMV during pregnancy is considered the most common cause of congenital infection worldwide,
affecting ∼0.2–2% of infants and potentially causing fetal lesions
[8–10]. Though most infected newborns are asymptomatic, ∼10%
display a variety of clinical disorders [8, 11] potentially leading to important sequelae such as mental retardation and deafness. The type of maternal infection (i.e., primary versus reactivation/reinfection) conditions the risk of congenital infection and
the pregnancy stage at which transmission occurs is related to clinical severity [12–16]. Maternal antibodies with neutralizing activity are transferred to the fetus predominantly during the third
trimester of gestation and may prevent congenital CMV disease
[17]. Among other factors, fetal immune immaturity may determine the outcome of congenital infection [18, 19]. An effective
defense against HCMV requires the participation of T and NK cells,
and the virus has developed different immune evasion strategies
[20]. Patients with congenital HCMV infection have been shown
to display mature CD8+ T-cell responses [21, 22], and an expansion and differentiation of a specific TcR γδ+ cell subset has been
recently reported [23]. In contrast, information on the role of NK
cells in this context is rather limited [24, 25].
HCMV infection stably alters the distribution of NK-cell receptors (NKRs) in healthy adult blood donors and children. A positive serology for HCMV was associated to increased proportions of NK and T cells expressing CD94/NKG2C, an activating
killer lectin-like receptor (KLR) specific for the HLA-E class Ib
molecule, as well as LILRB1 (ILT2, LIR-1, CD85j) [4, 26, 27], an
inhibitory receptor which interacts with HLA class I molecules
and the UL18 HCMV glycoprotein [28, 29]. The association of
HCMV infection with increased proportions of NKG2C+ cells has
been reported in chronic lymphocytic leukaemia patients [30],
solid organ and hematopoietic transplant recipients [31–33], a primary T-cell immunodeficiency [34], as well as in individuals coinfected by other pathogens, for example, HIV-1 [35–37], hantavirus
[38], chikungunya [39], HBV, and HCV [40]. Moreover, NKG2C+
NK cells expanded in response to HCMV-infected fibroblasts in
vitro, and it was hypothesized that the CD94/NKG2C activating
KLR might recognize HCMV-infected cells [41]. Altogether, these
observations are reminiscent of the pattern of response to murine
CMV (MCMV) specifically mediated by the Ly49H+ NK-cell subset [42] and, on that basis, it has been speculated that the CD57+
NKG2C+ subset might represent “memory” NK cells [32]. Interestingly, a complete deletion of the NKG2C gene has been reported in
Japanese and European blood donors with ∼4% homozygosity and
32–34% heterozygosity rates [43, 44]; yet, whether this genetic
trait may influence the NK-cell response to HCMV is unknown.
In the present study, the relationship between congenital HCMV
infection, NKG2C genotype, and NKR distribution was addressed.
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Immunity to infection
Results
NKR distribution in NK and T-cell populations from
children with congenital HCMV infection
An immunophenotypic study was carried out in blood samples
from children with evidence of past HCMV infection, either congenital symptomatic (n = 15), asymptomatic (n = 11), or postnatal (n = 11), and from noninfected children (n = 20). NKR expression (i.e., NKG2C, NKG2A, LILRB1, and CD161) was assessed by
flow cytometry in NK (CD56+ CD3− ) and T cells (CD3+ ). Despite
some differences in age distribution, both the proportions and the
absolute numbers of NK and T cells were comparable in all four
study groups (Table 1).
Children with symptomatic congenital infection displayed
higher proportions of NKG2C+ and lower percentages of NKG2A+
NK cells than asymptomatic or noninfected groups (Fig. 1). In
contrast, the distributions of NKG2C+ and NKG2A+ NK cells were
comparable in children with congenital symptomatic and postnatal infection. Remarkably, both the relative and absolute numbers
of LILRB1+ NK cells were markedly increased in symptomatic congenital infection, whereas no significant differences in the proportions of CD161+ NK cells were perceived (Fig. 1). Age, clinical
features, and the proportions of NKG2C+ and LILRB1+ NK cells
corresponding to cases of symptomatic congenital infection are
displayed as Supporting Information Table 1. Multivariate analysis indicated that the immunophenotypic differences observed
were independent of age.
Studies in dizygotic twins further illustrated the impact of congenital symptomatic infection on the NKR repertoire (Table 2). In
a first pair (TP1, 22 months old), only the HCMV-positive symptomatic boy displayed a marked increase of NKG2C+ and LILRB1+
NK cells as well as reduced proportions of NKG2A+ cells, compared
to his noninfected sister. In a second set of infected twin males
(TP2, 6 years old), the HCMV-associated immunophenotype was
only evident in the case with symptomatic infection compared to
the asymptomatic sibling. In both cases, CD161 expression levels
appeared lower in NK cells from individuals with symptomatic
HCMV infection, an effect that was not perceived when groups
were compared (Fig. 1).
The NKR distribution pattern associated to HCMV infection
in T lymphocytes resembled only partially that observed in NK
cells (Fig. 2). Overall, the absolute numbers of NKR+ T cells
were increased in HCMV+ children, particularly in the congenital symptomatic group. In fact, the proportions of NKG2C+ ,
LILRB1+ , and CD161+ T cells were significantly higher in congenitally infected than in noninfected children. In addition,
NKG2A+ T cells appeared also higher in children with congenital symptomatic infection, at variance with the reduced
proportions of NKG2A+ NK cells in the same group. Altogether, these results point out that marked changes in NKR
distribution, particularly an increase of NKG2C+ and LILRB1+
NK cells, are associated with congenital symptomatic HCMV
infection.
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Table 1. Age, gender, and lymphocyte counts in the study groups
Age (years)a)
Gender
Male
Female
% NK cellsa)
NK cells/mm3a)
% T cellsa)
T cells/mm3a)
Congenital
symptomatic
n = 15
Congenital
asymptomatic
n = 11
Postnatal
n = 11
Noninfected
n = 20
pb)
2.3 (0.05–6.9)
0.25 (0.1–6.9)
1.9 (0.4–4.7)
0.75 (0.1–3.6)
c)
8 (53.3%)
7 (46.7%)
6.5
(2–31)
343
(75–1733)
75
(54–84)
3240
(1065–8721)
4 (36.4%)
7 (63.6%)
5.6
(2–8)
260
(79–1327)
75
(56–86)
3807
(2417–13 075)
5 (45.5%)
6 (54.5%)
6.5
(2–14)
226
(126–786)
74
(63–87)
2565
(1978–5690)
7 (35%)
13 (65%)
6.8
(3–17)
373
(151–1423)
72
(62–89)
4285
(2029–6729)
NS
NS
NS
NS
NS
a)
Values are expressed as median (range).
Statistical analyses were performed using the Mann–Whitney U test (for continuous variables) or the chi-square test (for gender). NS indicates
no significant differences between any of the groups.
c)
Significant differences were observed comparing symptomatic congenital infection with asymptomatic (p = 0.027) and noninfected groups
(p = 0.013), as well as noninfected with postnatal infection groups (p = 0.036).
b)
The NKG2C genotype modulates the HCMV influence
on the NK-cell compartment
The putative implications of the NKG2C deletion on the response
to HCMV infection are uncertain. On that basis, a genotypic
analysis of NKG2C was conducted in children with symptomatic
(n = 15) and asymptomatic (n = 11) congenital infection, as well
as in a control group including children with postnatal infection
(n = 11) and noninfected (n = 19). The homozygous NKG2C
deletion was found in a single uninfected control individual. In
addition, no significant differences were found between the frequencies of the heterozygous NKG2C+/− genotype detected in
uninfected controls and children with congenital infection (42.1%
versus 34.6%; p = 0.61). Altogether these results argue against a
direct relation of the NKG2C deletion with the incidence of congenital HCMV infection in newborns.
In line with previous reports [26, 27, 32], individual differences in NKG2C surface staining intensity were noticed (Supporting Information Fig. 1). The NKG2Cbright/intermediate expression
pattern was generally associated to HCMV infection, whereas all
noninfected and ∼43% of infected children displayed a predominant NKG2Cdim phenotype. The proportions of NKG2C+ cells correlated significantly (r = 0.74; p < 0.001) with the KLR surface
expression levels (MFI). The possibility that NKG2C copy number might influence the expansion of NKG2C+ cells and/or the
expression levels of the receptor was addressed. To this end, the
proportions and absolute numbers of NK cells bearing NKG2C, as
well as its surface staining intensity, were compared after stratification for HCMV infection and the NKG2C genotype. As expected,
increased proportions of NKG2C+ NK cells and higher surface levels of the KLR were detected in HCMV-positive children (Table 3);
though less marked, a significant association of both parameters
with the NKG2C genotype was also noticed. On the other hand,
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the absolute numbers of NKG2C+ NK cells appeared significantly
higher in NKG2C+/+ than in NKG2C+/- children (Table 3). Regression analysis confirmed an age-independent association between
HCMV infection and the proportions of the NKG2C+ subset
(p < 0.001), as well as between the NKG2C genotype and absolute
numbers of NKG2C+ cells (p = 0.003) (Supporting Information
Table 2).
Stratification for both HCMV infection and NKG2C genotype
further supported a relationship of the latter with the absolute
numbers of NKG2C+ cells (Fig. 3A). The possibility that these
results might be explained by age differences or a skewed distribution of cases with congenital symptomatic and asymptomatic
infection, displaying different levels of NKG2C+ cells (Fig. 1), was
ruled out by multivariate analyses.
Unexpectedly, NKG2C+/+ children were observed to display as
well higher proportions (median 7.2% versus 4.6%; p = 0.003)
and absolute numbers (median 359 versus 215 cells/mm3 ;
p = 0.008) of total NK cells than NKG2C+/− children. This finding
was not simply explained by the expansion of the NKG2C+ subset,
as the numbers of NKG2A+ , CD161+ , and total NK cells appeared
also higher in HCMV-positive NKG2C+/+ children compared to
NKG2C+/− individuals (Fig. 3B–D). Multivariate regression analysis confirmed the relation of the NKG2C genotype with both the
proportions (p = 0.001) and total numbers (p = 0.014) of NK cells,
independently of age as a putative confounding variable [45, 46]
(Supporting Information Table 2).
Discussion
In the present study, increased proportions of NKG2C+ NK
cells were detected in children with past congenital HCMV infection; this immunophenotypic feature was particularly marked in
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Eur. J. Immunol. 2012. 42: 3256–3266
Immunity to infection
Figure 1. NKR expression in NK cells from children with congenital HCMV infection. Blood
samples from children with past symptomatic
congenital (SC), asymptomatic congenital (AC),
and postnatal (PN) HCMV infection, as well as
from noninfected children (NI), were analyzed
by multicolor flow cytometry for the expression of (A) NKG2C, (B) NKG2A, (C) LILRB1, and
(D) CD161 in CD56+ CD3− NK cells. The absolute numbers of cells expressing each marker
were calculated on the basis of the numbers
of total lymphocytes/mm3 (right). Each symbol represents the result obtained in a single
test from an individual donor. Comparisons
were performed between all study groups. Only
statistically significant differences are shown
*p < 0.05; **p < 0.01; ***p < 0.001; Mann–Whitney
U test.
symptomatic cases, as further illustrated by studies in twins.
The detection in older patients of high proportions of circulating
NKG2C+ cells years after symptomatic congenital HCMV infection
(Table 2 and Supporting Information Table 1) highlighted the persistence of the NK-cell subset redistribution, consistent with observations in healthy adults (Muntasell and López-Botet, unpublished
data). Though the proportions of NKG2C+ NK cells appeared unrelated to age, the cross-sectional design of this study did not discriminate whether the increase of NKG2C+ cells resulted from a
progressive cumulative process, as reported in cord blood transplantation recipients [31, 33]. Prospective longitudinal studies of
the NK-cell immunophenotype in congenital and early postnatal
HCMV infection are warranted to approach the dynamics of these
events.
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
We previously reported that CD94/NKG2C+ cells expanded in
vitro in response to HCMV-infected fibroblasts, an effect that was
prevented by early treatment with a blocking anti-CD94 mAb [41].
Based on these studies, we hypothesized that a cognate interaction of the activating KLR with HCMV-infected cells might drive
a preferential proliferation, differentiation, and/or survival of the
NKG2C+ NK-cell subset in response to cytokines (i.e., IL-15). The
NKG2C+ population has been compared to murine Ly49H+ NK
cells, which specifically recognize the m157 viral glycoprotein on
MCMV-infected cells. After sequential expansion and contraction
phases in response to MCMV infection, Ly49H+ NK cells tend to
persist in the circulation, accounting for a more efficient response
to reinfection [42, 47]. By analogy with the adaptive immune
response, the term “memory NK cell” was coined to define this
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Table 2. NKR expression in dizygotic twins discordant for the presence or severity of past congenital HCMV infectiona)
Twin pair (TP)
HCMV infection
% NKG2C
% NKG2A
% LILRB1
% CD161
1
1
2
2
Symptomatic
Noninfected
Symptomatic
Asymptomatic
38
8
30
14
49
68
46
50
44
16
24
9
46
82
56
79
a)
NKR expression was analyzed by flow cytometry in NK cells from a child (22 months old) with symptomatic congenital HCMV infection and his
noninfected sibling (TP1), as well as in siblings with congenital symptomatic and asymptomatic infection (TP2, 6 years old). The percent of NK
cells expressing NKR are displayed.
pattern of response, and it has been speculated that NKG2C+ NK
cells might be a human counterpart of Ly49H+ murine NK cells
[32, 41]. Nevertheless, despite that circumstantial observations
support that NKG2C+ NK cells might contribute to controlling
HCMV viremia [34], as yet there is no formal evidence supporting
that they specifically exert their effector functions against HCMVinfected cells, protecting against viral reactivation or reinfection
[48]. Restrictions in sample volume did not allow to perform functional studies of NKG2C+ NK cells, as those reported in adult
HCMV-infected individuals [31].
Studies in immunodeficiencies and immunosuppressed
patients indirectly suggest that the magnitude of the NKG2C+
expansion may be inversely related to the effectiveness of the
T-cell mediated response to HCMV infection [31, 32, 34–36]. As
shown for other pathogens (e.g., HBV), we hypothesized that vertical HCMV transmission might favor the establishment of partial
tolerance, impairing an effective T-cell-mediated control of the
infection, and promoting in this case the expansion of NKG2C+
NK cells. Nevertheless, the minimal phenotypic changes detected
in asymptomatic cases is consistent with the view that, irrespective of the time of infection and immune immaturity, an effective
control of the pathogen may limit its impact on the NKR distribution. These observations, together with the expansion of NKG2C+
cells observed in postnatal infection and in healthy adults, point
out that other factors (e.g., viral load, virus and host genetics, frequency of viral reactivation) determine the magnitude of HCMV
impact on the NK-cell compartment. In this regard, differences
in viral exposure might explain why the expansion of NKG2C+
cells appeared more marked in children with postnatal infection than in the group with congenital asymptomatic infection.
Early postnatal infection often occurs along breastfeeding due to
viral excretion in maternal milk, causing symptomatic disease in
some newborns particularly in premature infants. By contrast,
transplacental transmission is restricted to the time window of
maternal viremia, and appears a relatively unpredictable infective pathway, as illustrated by the identification of twins with
discordant infection. Whether the response of NK cells to HCMV
may contribute to the immunopathogenesis of clinical disorders
along acute congenital symptomatic infection remains an open
issue.
The NKG2C+ NK-cell population appears to be phenotypically
heterogeneous according to the KLR staining intensity and coexpression of other NKR. HCMV infection was associated to an
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
increase of NKG2Cbright NK cells [26] shown to display a CD57+
phenotype [32]. We originally reported that, as compared to the
NKG2A+ NK-cell subset, this population contained higher proportions of LILRB1+ and KIR+ cells, but displayed lower surface levels
of NKp46 and NKp30 NCR [26]. Studies in several samples confirmed this immunophenotypic pattern in children with congenital
HCMV infection (data not shown); to what extent the persistent
NKR redistribution might condition the innate response to other
infections and tumors deserves attention.
A marked increase of LILRB1+ NK cells was also observed
in symptomatic congenital HCMV infection, as compared to the
other groups. The LILRB1 inhibitory receptor is expressed at
late differentiation stages by cytotoxic T lymphocytes specific for
different microbial pathogens [49–52]. Similarly to T lymphocytes, activated NK cells undergo clonal expansions, experiencing
differentiation events that modify their phenotype and survival
[42, 53]. In this regard, LILRB1 is displayed by a variable
fraction of CD56dim NK cells [4], whereas it appears virtually
undetectable in the CD56bright subset, which was shown to bear
longer telomeres [54]. In the same line, most LILRB1+ cells were
predominantly found among the CD27-negative cell population
[4], corresponding to late NK differentiation stages [55]. Recent
studies indicate that LILRB1 expression may be also upregulated
in NK cells upon in vitro exposure to cytokines [56]. Hence,
the marked increase of LILRB1+ NK populations in symptomatic
congenital HCMV infection likely reflects the accumulation of
cells activated/differentiated under the pressure of the pathogen.
HCMV congenital symptomatic infection was also associated
to higher proportions and absolute numbers of NKG2C+ and
LILRB1+ T cells. Yet, the pattern was different to that observed
in NK cells, as NKG2A+ and CD161+ T lymphocytes were also
increased. NKR expression has been associated to late differentiation stages of TcRαβ+ CD4+ and CD8+ T cells, modulating
their Ag-specific response [51, 57]. NKR may be also expressed
by TcRγδ+ T cells and were detected in a subset of TcRγδ+ T cells
specifically responding to congenital HCMV infection [23]. Further studies are required to more precisely define the NKR distribution in different T-cell subsets and their functional implications
in congenital HCMV infection.
The frequency of the NKG2C gene deletion appeared comparable in children with congenital infection and controls. Further studies in a larger cohort are required to address whether
the NKG2C genotype might have a more subtle influence on the
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Eur. J. Immunol. 2012. 42: 3256–3266
Immunity to infection
Figure 2. NKR expression in T cells from children with congenital HCMV infection. Blood
samples from children with past symptomatic
congenital (SC), asymptomatic congenital (AC),
and postnatal (PN) HCMV infection, as well as
from noninfected children (NI), were analyzed
by multicolor flow cytometry for the expression of (A) NKG2C, (B) NKG2A, (C) LILRB1, and
(D) CD161 in CD3+ T cells. The absolute numbers of T cells expressing each marker were calculated on the basis of the numbers of total
lymphocytes/mm3 (right). Each symbol represents the result obtained in a single test from an
individual donor. Comparisons were performed
between all study groups. Only significant differences are shown *p < 0.05; **p < 0.01; ***p <
0.001; Mann–Whitney U test.
pathogenesis and/or clinical outcome of congenital HCMV infection. Remarkably, HCMV-infected NKG2C+/+ children exhibited
greater numbers of circulating NKG2C+ cells than heterozygous
individuals. Multivariate analysis ruled out the effect of two possible confounding factors, that is, age and skewed distribution of
cases with symptomatic congenital infection. These results suggested that the NKG2C genotype might modulate the proliferation
and/or survival of circulating NKG2C+ cells, ultimately influencing the magnitude and/or persistence of the NKG2C+ expansion.
Functional consequences of gene copy number variation have been
reported for some immunoreceptors [58, 59]. This view would
indirectly reinforce the hypothesis of an active involvement of the
activating KLR in this process. On the other hand, the basis for the
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
association of the NKG2C genotype with the absolute numbers of
NKG2A+ , CD161+ , and total NK cells, that appeared reduced in
NKG2C+/− as compared to NKG2C+/+ children, is uncertain.
In summary, the opportunity of studying this rather exceptional
cohort, despite its limitations (e.g., cross-sectional study, small
size, and restricted sample volumes), provides novel insights on
the influence of HCMV on the homeostasis of the NK-cell compartment in children, particularly in congenital infection. Further
studies are warranted to confirm these observations in a larger
cohort, to assess whether they stand in HCMV-positive adults and,
eventually, to identify the mechanisms underlying the influence
of the NKG2C genotype on the dynamics of the NK-cell response
to HCMV infection.
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Table 3. Relationship of HCMV infection and NKG2C genotype with the numbers of NKG2C+ NK cells and surface levels of the receptor in
childrena)
HCMV+b)
d)
(N = 37)
HCMV− (N = 19)
pe)
% NKG2C+ NK cells
NKG2C+ NK cells / mm3
NKG2C MFI
26.1 (8.9–57.4)
85.9 (10.12–635.9)
168 (45–474)
12.2 (0.1–22.3)
44.4 (0.5–207.74)
74 (46–146)
< 0.001
0.18
< 0.001
% NKG2C+ cells
NKG2C+ NK cells/mm3
NKG2C MFI
NKG2C+/+ c) d) (N = 34)
22.1 (7.8–54)
93.6 (10.12–635.9)
163 (45–474)
NKG2C +/− (N = 20)
14.3 (7.4–57.4)
31.3 (11.23–192.6)
116 (46–197)
pe)
0.023
0.001
0.042
a)
NKG2C expression was analyzed by flow cytometry as described in Figure 1; mean fluorescence intensity (geo MFI) is expressed in arbitrary units.
The NKG2C deletion was determined as described in Material and methods.
b)
Children with past HCMV infection, either congenital symptomatic (n = 15), asymptomatic (n = 11), and postnatal (n = 11) were compared to
noninfected children (n = 19).
c)
NKG2C homozygous and heterozygous children were compared.
d)
Values are expressed as median (range).
e)
Statistical analysis was based on the Mann–Whitney U test.
Materials and methods
Subjects and samples
Children participating in this study were enrolled at the Pediatric Infectious Diseases Unit at Hospital de Sant Joan de Déu
(Barcelona, Spain). Congenital HCMV infection was defined by
the detection of HCMV DNA (either from urine, blood, and/or
neonatal dried blood samples), except for a single case defined by
detection of CMV-specific IgM antibodies within the first 3 weeks
of life. A control group of healthy children without known congenital HCMV infection and referred to the laboratory for presurgical routine blood analysis were recruited. The study population included four pairs of dizygotic twins: Two with congenital
infection, one with a single infected sibling, and a fourth pair
noninfected. The study was approved by the Research and Ethics
Committee at Hospital de Sant Joan de Déu and informed consent
was obtained from parents prior to inclusion.
Study groups
Children with congenital HCMV infection were divided by conventional clinical criteria in symptomatic and asymptomatic. In
our series, clinical manifestations at birth associated to symptomatic congenital HCMV infection included: intracranial calcifications (53.3%), sensori-neural hearing loss (53.3%), microcephaly (46.7%), splenomegaly (40%), thrombocytopenia (40%),
hepatomegaly (33.3%), petechiae (33.3%), purpura (26.7%),
jaundice (20%), intrauterine growth restriction (20%), and chorioretinitis (13.3%) (Supporting Information Table 1).
The study included two groups of controls: (i) children with
postnatal HCMV infection, defined as individuals aged >12
months with positive HCMV-specific IgG antibodies, or children
of <12 months with a positive PCR assay performed in urine
and/or blood; in the latter, HCMV infection had been clinically
unnoticed and neonatal dried blood spots were tested by PCR to
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
rule out congenital infection; (ii) noninfected children, defined as
those seronegative for HCMV-specific IgM and IgG antibodies, or
individuals aged <12 months with HCMV-specific IgG, but negative for HCMV-specific IgM and PCR assay in urine and/or blood.
Infants younger than 12 months with a positive serology in whom
a urine or blood PCR test could not be performed were excluded
from the study, since it was not possible to ascertain their HCMV
infection status.
HCMV Ab and PCR assays
Detection of anti-HCMV antibodies was carried out by the clinical
laboratory using standard diagnostic tests. Detection of HCMV
genome was performed by using Q-CMV Real Time Complete
Kit (Nanogen Advanced Diagnostics, Torino, Italy), a nucleic
R
-MGB (Minor Groove
acid amplification assay based on TaqMan
Binder) technology for detection and quantification of CMV DNA.
The amplification reaction targets the gene region that encodes
the Major Immediate Early Antigen (MIEA) of HCMV as well as a
region of the human beta globin gene, which is amplified simultaneously with the target sequence to verify successful DNA isolation
in order to exclude false-negative results.
Antibodies and flow cytometry
Anti-NKG2C was from R&D Systems (Minneapolis, MN). AntiNKG2A (clone Z199, kindly provided by Dr. A. Moretta, University of Genova), anti-LILRB1 (clone HP-F1), anti-CD161 (clone
HP-3G10), and the anti-Myc (clone 9E10) negative control, were
directly produced in our laboratory. Indirect immunofluorescence
staining with these reagents was carried out with a phycoerythrin
(PE)-labeled F(ab )2 rabbit anti-mouse Ig (Dako, Glostrup, Denmark). Anti-CD3-peridin-chlorophyll-protein (PerCP) and antiCD56-allophycocyanin were from BD Biosciences (San Diego, CA);
anti-CD45-allophycocyanin-Cy7 was from BioLegend (San Diego,
CA).
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Eur. J. Immunol. 2012. 42: 3256–3266
Immunity to infection
Figure 3. Relationship between the NKG2C
genotype, NK-cell phenotype, and the absolute
numbers of NK cells in HCMV-positive children.
Immunophenotypic analyses and calculation
of the absolute cell numbers were performed as
described in Figure 1. The NKG2C genotype was
determined as described in the Materials and
methods. Data from children with past HCMV
infection, either congenital symptomatic
(n = 15), asymptomatic (n = 11), and postnatal
(n = 11) were pooled and compared with
that of noninfected children (n = 16). The
proportions and absolute numbers of (A)
NKG2C+ , (B) NKG2A+ , (C) CD161+ , and (D)
total NK cells are displayed. Each symbol
represents the result obtained in a single test
from an individual donor. Comparisons were
performed between all study groups. Only
significant differences are shown *p < 0.05;
**p < 0.01; ***p < 0.001; Mann–Whitney U test.
The expression of NKG2C, NKG2A, LILRB1, and CD161 by NK
and T cells was analyzed by multicolor flow cytometry in fresh
peripheral blood samples, obtained by venous puncture in EDTA
tubes. Whole blood samples were pretreated with human aggregated Ig (30 μg/mL) to block Fc receptors, incubated with individual NKR-specific mAbs, washed and further incubated with a
PE-tagged F(ab )2 rabbit anti-mouse Ig. Washed samples were
incubated with anti-CD3-PerCP, anti-CD56-allophycocyanin, and
anti-CD45-allophycocyanin-Cy7. Erythrocytes were lysed using BD
PharmLyse lysing buffer (BD Biosciences). Samples were analyzed
in a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). BD
FACSDiva software (BD Biosciences) was used for data analysis
and calculation of the MFI values.
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Results from hemograms, obtained in parallel to the samples
used for immunophenotypic analysis, were used to calculate the
absolute numbers of NK and T-cell populations. Some data could
not be obtained from all participating children due to technical
reasons; missing data included: hemogram (n = 1); NKG2C+ cells
(n = 1); LILRB1+ cells (n = 2); CD161+ cells (n = 2); NKG2C
genotype (n = 1); NKG2C MFI (n = 7).
NKG2C genotype analysis
CNV of NKG2C was assessed by a PCR method based on the
approach used by Miyashita et al. [43], with modifications. Briefly,
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Daniel E. Noyola et al.
homo- and heterozygosity for the NKG2C gene and its deletion
were determined by a single PCR that yields amplicons of different lengths in each genotype. This is achieved by means of
two primer pairs recognizing sequence motifs specific for, either
NKG2C, or the gene arrangement resulting from its deletion [60].
In one child participating in the study this analysis could not be
performed.
Eur. J. Immunol. 2012. 42: 3256–3266
monoclonal TCR-alphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood 2008. 112: 4609–4616.
6 Mitchell, D. A., Xie, W., Schmittling, R., Learn, C., Friedman, A.,
McLendon, R. E. and Sampson, J. H., Sensitive detection of human
cytomegalovirus in tumors and peripheral blood of patients diagnosed
with glioblastoma. Neuro. Oncol. 2008. 10: 10–18.
7 Koch, S., Solana, R., De la Rosa, O. and Pawelec, G., Human
cytomegalovirus infection and T-cell immunosenescence: a mini review.
Mech. Ageing Dev. 2006. 127: 538–543.
Statistical analysis
8 Stagno, S., Cytomegalovirus. In Remington, J. S. and Klein, J. O. (Eds.),
Infectious diseases of the fetus and newborn infant. W.B. Saunders Company,
Comparisons of categorical variables among study groups were
performed using the chi-square or Fisher exact test, as appropriate.
Continuous variables were compared using the Mann–Whitney
U test. A p value <0.05 was considered statistically significant.
Spearman’s rank correlation coefficient was used to evaluate the
association between continuous variables. Multivariate analysis
was carried out using linear regression analysis; the dependent
variables were subjected to logarithmic transformation prior to
inclusion in the regression model.
Philadelphia, 2001, pp 389–424.
9 Dollard, S. C., Grosse, S. D. and Ross, D. S., New estimates of the prevalence of neurological and sensory sequelae and mortality associated with
congenital cytomegalovirus infection. Rev. Med. Virol. 2007. 17: 355–363.
10 Ludwig, A. and Hengel, H., Epidemiological impact and disease burden
of congenital cytomegalovirus infection in Europe. Euro. Surveill. 2009. 14:
26–32.
11 Demmler, G. J., Congenital cytomegalovirus infection and disease. Semin.
Pediatr. Infect. Dis. 1999. 10: 195–200.
12 Fowler, K. B., Stagno, S., Pass, R. F., Britt, W. J., Boll, T. J. and Alford,
C. A., The outcome of congenital cytomegalovirus infection in relation to
maternal antibody status. N. Engl. J. Med. 1992. 326: 663–667.
13 Boppana, S. B., Rivera, L. B., Fowler, K. B., Mach, M. and Britt, W. J.,
Intrauterine transmission of cytomegalovirus to infants of women with
preconceptional immunity. N. Engl. J. Med. 2001. 344: 1366–1371.
Acknowledgments: This work was supported by grants from
Plan Nacional de I+D (SAF2010–22153-C03), and EU SUDOE
program (SOE2/P1/E341). A.M. is supported by Asociación
Española Contra el Cáncer (AECC). We thank Gemma Heredia and
Marı́a Cañizares for their excellent technical assistance, and Joan
Vila for his expert advice in statistical analysis. We are grateful to
patients and their families for generously accepting to participate
in this study.
14 Pass, R. F., Fowler, K. B., Boppana, S. B., Britt, W. J. and Stagno, S.,
Congenital cytomegalovirus infection following first trimester maternal
infection: symptoms at birth and outcome. J. Clin. Virol. 2006. 35: 216–220.
15 Lilleri, D., Fornara, C., Furione, M., Zavattoni, M., Revello, M. G. and
Gerna, G., Development of human cytomegalovirus-specific T-cell immunity during primary infection of pregnant women and its correlation with
virus transmission to the fetus. J. Infect. Dis. 2007. 195: 1062–1070.
16 Adler, S. P., Nigro, G. and Pereira, L., Recent advances in the prevention
and treatment of congenital cytomegalovirus infections. Semin. Perinatol.
2007. 31: 10–18.
Conflict of interest: The authors declare no financial or commercial conflict of interest.
17 Nozawa, N., Fang-Hoover, J., Tabata, T., Maidji, E. and Pereira, L.,
Cytomegalovirus-specific, high-avidity IgG with neutralizing activity in
maternal circulation enriched in the fetal bloodstream. J. Clin. Virol. 2009.
46(Suppl 4): S58–S63.
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Abbreviations: HCMV: human cytomegalovirus · KLR: killer lectin-like
receptor · NKR: NK-cell receptor
Full correspondence: Prof. Miguel López-Botet, IMIM (Hospital del Mar
Medical Research Institute), Doctor Aiguader 88, 08003 Barcelona,
Spain
Fax: +34-93-3160410
e-mail: [email protected]
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See accompanying Commentary:
http://dx.doi.org/10.1002/eji.201243050
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C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 17/6/2012
Revised: 11/8/2012
Accepted: 6/9/2012
Accepted article online: 11/9/2012
www.eji-journal.eu