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3256 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 www.eji-journal.eu 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. www.eji-journal.eu 3257 3258 Daniel E. Noyola et al. Eur. J. Immunol. 2012. 42: 3256–3266 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 www.eji-journal.eu 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 www.eji-journal.eu 3259 3260 Daniel E. Noyola et al. Eur. J. Immunol. 2012. 42: 3256–3266 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 www.eji-journal.eu 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. www.eji-journal.eu 3261 3262 Daniel E. Noyola et al. Eur. J. Immunol. 2012. 42: 3256–3266 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). www.eji-journal.eu 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, www.eji-journal.eu 3263 3264 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. 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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. 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See accompanying Commentary: http://dx.doi.org/10.1002/eji.201243050 59 González, E., Kulkarni, H., Bolivar, H., Mangano, A., Sánchez, R., Catano, G., Nibbs, R. J. et al., The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. Science 2005. 307: 1434–1440. 60 Moraru, M., Cañizares, M., Muntasell, A., de Pablo, R., López-Botet, M. and Vilches, C., Assessment of copy-number variation in the NKG2C 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