NKG2C zygosity influences CD94/NKG2C receptor function and the NK‐cell compartment redistribution in response to human cytomegalovirus

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK‐cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV‐seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C⁺ NK‐cell numbers was observed in HCMV⁺ children. We have assessed the influence of NKG2C gene dose on the NK‐cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C⁺ NK‐cell numbers in HCMV⁺ subjects, independently of HLA‐E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa²⁺ influx), degranulation, and IL‐15‐dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C^+/+ and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady‐state NKG2C⁺ NK‐cell numbers, further supporting an active involvement of the receptor in the HCMV‐induced reconfiguration of the NK‐cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention.     

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire

NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A− NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. In conclusion: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.     

Loss of NKG2D in murine NK cells leads to increased perforin production upon long-term stimulation with IL-2

NK cells are innate lymphocytes responsible for lysis of pathogen-infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46⁺ cells (NKG2D^ΔNK). NKG2D^ΔNK NK cells develop normally, have an unaltered IFN-γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long-term stimulation with IL-2, NKG2D-deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL-2 activated NK cells.     

Adaptive reconfiguration of the human NK‐cell compartment in response to cytomegalovirus: A different perspective of the host‐pathogen interaction

As discussed in this review, human cytomegalovirus (HCMV) infection in healthy individuals is associated with a variable and persistent increase of NK cells expressing the CD94/NKG2C activating receptor. The expansion of NKG2C⁺ NK cells reported in other infectious diseases is systematically associated with HCMV co‐infection. The functionally mature NKG2C^bright NK‐cell subset expanding in HCMV⁺ individuals displays inhibitory Ig‐like receptors (KIR and LILRB1) specific for self HLA class I, and low levels of NKp46 and NKp30 activating receptors. Such reconfiguration of the NK‐cell compartment appears particularly marked in immunocompromised patients and in children with symptomatic congenital infection, thus suggesting that its magnitude may be inversely related with the efficiency of the T‐cell‐mediated response. This effect of HCMV infection is reminiscent of the pattern of response of murine Ly49H⁺ NK cells against murine CMV (MCMV), and it has been hypothesized that a cognate interaction of the CD94/NKG2C receptor with HCMV‐infected cells may drive the expansion of the corresponding NK‐cell subset. Yet, the precise role of NKG2C⁺ cells in the control of HCMV infection, the molecular mechanisms underlying the NK‐cell compartment redistribution, as well as its putative influence in the response to other pathogens and tumors remain open issues.     

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity

Human umbilical cord blood （CB） has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL）. It was reported that the activity of CB NK cells was lower than that of adult peripheral blood （PB） NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.     

Spotlight on NKG2C and the human NK‐cell response to CMV infection

Immune responses to cytomegalovirus (CMV) infection in the mouse and human involve the expansion of specific subsets of natural killer (NK) cells with specific phenotypic characteristics and a heightened ability to produce interferon (IFN)‐gamma. In humans, these NK‐cell responses are largely driven by the activating receptor NKG2C, which recognize human leukocyte antigen (HLA)‐E in complex with leader sequence peptides. In this issue of the European Journal of Immunology, Noyola et al. [Eur. J. Immunol. 2012, 42: 3256‐3266] examine NK‐cell responses in a unique cohort of young children with asymptomatic and symptomatic congenital CMV infection. They also address NK‐cell responses to CMV in relation to NKG2C gene copy number. Children with a symptomatic congenital infection exhibited a marked expansion of NKG2C⁺ NK cells. However, despite having slightly lower frequencies of NKG2C⁺ NK cells, children with a heterozygous deletion of the NKG2C gene seemed to control the virus as efficiently as those with two copies of the NKG2C gene. The present studies shed new light on the role of NKG2C copy number variation on the human NK‐cell response to CMV infection.     

MS患者CD94/NKG2A-bright和CD94/NKG2A-dim NK细胞亚群对IFN-β的差异性反应

目的： 分析多发性硬化（MS）进展型患者不同表型NK细胞亚群对临床主要治疗方法的反应性差异.方法： 分离患者外周血中的NK细胞, 以流式细胞术根据表面抑制性受体CD94/NKG2A表达情况分为两个亚群CD94/NKG2A-bright和CD94/NKG2A-dim.分别加入IFN-β, 测定两个亚群表面CD94/NKG2A变化及细胞增殖, 同时检测两种亚群分泌IL-10和TGF-β情况.结果： CD94/NKG2A阳性表达的NK细胞占25.5%, 其中CD94/NKG2A-bright和CD94/NKG2A-dim分别占其中的23.6%和76.4%.加入IFN-β, CD94/NKG2A-bright组增殖率明显低于CD94/NKG2A-dim组, CD94/NKG2A表达峰度变化不大.CD94/NKG2A-dim组中CD94/NKG2A表达显著增加.两个亚群分泌的IL-10和TGF-β与未刺激组相比, 均有明显差异.CD94/NKG2A-bright和CD94/NKG2A-dim组间亦有明显差异.结论： IFN-β通过诱导NK细胞CD94/NKG2A表达在非特异免疫系统中抑制NK细胞; 同时刺激IL-10 和TGF-β分泌进一步发挥对免疫系统的抑制.CD94/NKG2A-bright和CD94/NKG2A-dim对IFN-β反应有差异性.     

Activating and inhibitory receptors on synovial fluid natural killer cells of arthritis patients: role of CD94/NKG2A in control of cytokine secretion

Natural killer (NK) cells are activated early during inflammatory events and contribute to the shaping of the ensuing adaptive immune response. To further understand the role for NK cells in inflammation, we investigated the phenotype and function of synovial fluid (SF) NK cells from patients with chronic joint inflammation, as well as from patients with transient inflammation of the knee following trauma. We confirm that synovial NK cells are similar to the well-characterized CD56(bright) peripheral blood (PB) NK-cell subset present in healthy individuals. However, compared to this PB subset the synovial NK cells express a higher degree of activation markers including CD69 and NKp44, the latter being up-regulated also on CD56(bright) NK cells in the PB of patients. Activated synovial NK cells produced interferon-gamma and tumour necrosis factor, and the production was further up-regulated by antibody masking of CD94/NKG2A, and down-regulated by target cells expressing human leucocyte antigen-E in complex with peptides known to engage CD94/NKG2A. We conclude that synovial NK cells have an activated phenotype and that CD94/NKG2A is a key regulator of synovial NK-cell cytokine synthesis.     

Influence of congenital human cytomegalovirus infection and the NKG2C genotype on NK‐cell subset distribution in children

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.     

c‐Cbl regulates MICA‐ but not ULBP2‐induced NKG2D down‐modulation in human NK cells

The NKG2D activating receptor on human NK cells mediates “altered self” recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane‐bound NKG2DLs can lead to down‐modulation of receptor expression and impairment of NKG2D‐mediated cell functions. Here, we evaluated whether different cell‐bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down‐modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down‐modulation than ULBP2, leading to a severe impairment of NKG2D‐dependent NK‐cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c‐Cbl direct MICA‐induced but not ULBP2‐induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti‐tumor strategies to restore a normal level of NKG2D receptors on human NK cells.     

Association of NKG2A with treatment for chronic hepatitis C virus infection

Natural killer (NK) cells are critical to the immune response to viral infections. Their functions are controlled by receptors for major histocompatibility complex (MHC) class I, including NKG2A and killer‐cell immunoglobulin‐like receptors (KIR). In order to evaluate the role of MHC class I receptors in the immune response to hepatitis C virus infection we have studied patients with chronic HCV infection by multi‐parameter flow cytometry directly ex vivo. This has permitted evaluation of combinatorial expression of activating and inhibitory receptors on single NK cells. Individuals with chronic HCV infection had fewer CD56^dim NK cells than healthy controls (4·9 ± 3·4% versus 9·0 ± 5·9%, P < 0·05). Expression levels of the inhibitory receptor NKG2A was up‐regulated on NK cells from individuals with chronic hepatitis C virus (HCV) (NKG2A mean fluorescence intensity 5692 ± 2032 versus 4525 ± 1646, P < 0·05). Twelve individuals were treated with pegylated interferon and ribavirin. This resulted in a down‐regulation of NKG2A expression on CD56^dim NK cells. Individuals with a sustained virological response (SVR) had greater numbers of NKG2A‐positive, KIR‐negative NK cells than those without SVR (27·6 ± 9·6% NK cells versus 17·6 ± 5·7, P < 0·02). Our data show that NKG2A expression is dysregulated in chronic HCV infection and that NKG2A‐positive NK cells are associated with a beneficial response to pegylated interferon and ribavirin therapy.     

Association of NKG2D genetic polymorphism with susceptibility to chronic hepatitis B in a Han Chinese population

Natural killer (NK) cells are important antiviral effectors of innate immunity because of their contribution to virus elimination. NK cell‐mediated immunological reaction to hepatitis B virus (HBV) infection depends on a fine balance between inhibitory and activating receptors. The aim of the study was to investigate genetic polymorphisms in NK cell receptors (NKR)—KLRD1 (CD94), KLRK1 (NKG2D), KLRC4 (NKG2F), and KLRC1 (NKG2A)—to evaluate the association of NKR genetic polymorphisms with susceptibility to chronic hepatitis B in a Han Chinese population. Twelve single nucleotide polymorphisms (SNPs), including rs2302489 in CD94; rs2255336, rs2617160, rs7980470, rs 2734565, and rs17513986 in NKG2D; rs2617170, rs17549004, and rs3825295 in NKG2F; rs2734414, rs7301582, and rs2734440 in NKG2A, were selected in the present study. SNP genotyping was undertaken in 500 Han Chinese patients (285 patients with chronic hepatitis B and 215 patients who cleared HBV spontaneously) by a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and by the TaqMan method. Single marker association analysis was conducted and the SNP rs2617160 with a TT genotype in NKG2D was associated significantly with an increased risk of chronic hepatitis B (P = 0.044; OR = 1.49; 95% CI = 1.01–2.19). Haplotype analysis with multiple loci indicated that there was no significant association between the haplotypes of the NKR genes and susceptibility to chronic hepatitis B. The SNP rs2617160 in NKG2D associated with susceptibility to chronic hepatitis B in a Han Chinese population. J. Med. Virol. 82:1501–1507, 2010. © 2010 Wiley‐Liss, Inc.     

NK‐cell receptors NKp46 and NCR1 control human metapneumovirus infection

Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK‐cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46‐activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV‐infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK‐cell phenotype in the blood and the lungs of HMPV‐infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1‐deficient mice, that NCR1 plays a critical role in controlling HMPV infection.     

Evidence of NK cell dysfunction in SIV-infected rhesus monkeys: impairment of cytokine secretion and NKG2C/C2 expression

Defects in the adaptive immune response have been extensively characterized in human immunodeficiency virus type-1 (HIV-1)-infected individuals; however, much less is known about the function of natural killer (NK) cells during the course of HIV-1 infection. In the present study, we demonstrate that the NK cells from simian immunodeficiency virus (SIV)-infected rhesus monkeys are significantly impaired in their ability to secrete IFN-gamma, TNF-alpha, and IL-2, while NK cell function in SIV-infected long-term non-progressor monkeys is similar to that of normal monkeys. These findings suggest that abnormal NK cell activity may contribute to the global immune dysfunction observed in HIV-1-infected individuals. NK cell function is modulated by several families of cell surface receptors, including the CD94/NKG2 family. We evaluated the messenger RNA levels of these inhibitory and activating NKG2 molecules in SIV-infected rhesus monkeys. These experiments demonstrate that the activating molecules NKG2C and NKG2C2 are significantly down-regulated in peripheral blood mononuclear cells of SIV-infected rhesus monkeys, suggesting that the dysregulation of these molecules may contribute to the abnormal NK cell function observed in the setting of infection.     

Association of NKG2D gene variants with susceptibility and severity of rheumatoid arthritis

NKG2D (KLRK1) is a C‐type lectin receptor present on natural killer (NK) cells, γδ, CD8⁺ and CD4⁺ T cells. Upon ligand binding, NKG2D mediates activatory and co‐stimulatory signals to NK cells and activated CD4⁺ T cells, respectively. Polymorphisms in NKG2D predispose to infectious diseases, cancer, transplantation and autoimmune disorders. We studied the influence of this NK receptor polymorphism on predisposition to and modification of the disease phenotype in patients with rheumatoid arthritis (RA). Eight different single nucleotide polymorphisms (SNP) in the NKG2 gene were genotyped in 236 patients with RA and 187 controls using Taqman 5' nuclease assays. NKG2D genotype/allele frequency did not differ between patients and controls. Subgroup analysis showed that the frequency of A allele of NKG2D9 and T allele of NKG2D10 was significantly higher in patients with deformities (a marker of severe disease) [11 versus 5%, Pc = 0·03, odds ratio (OR) = 2·44, 95% confidence interval (CI) = 1·09‐5·98 and 10 versus 4%, Pc = 0·04, OR = 2·45, 95% CI = 1·05‐6·39, respectively], while the frequency of alleles G of NKG2D9 and A of NKG2D10 was greater in patients without deformities (Pc = 0·03, OR = 0·41, 95% CI = 0·17‐0·91 and Pc = 0·04, OR = 0·41, 95% CI = 0·16‐0·96). Similar trends of association were observed with deforming phenotype of RA in female patients and deforming young onset RA subgroups. Haplotype analysis revealed that the frequency of haplotype G‐C‐A‐G‐A‐T‐C‐C was higher in patients than in controls (12 versus 8%, P = 0·04, OR = 1·61, 95% CI = 1·01‐2·55), suggesting that it may predispose to RA. Our study suggests that the NKG2D gene polymorphisms may modify the risk of development and severity of RA.     

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus‐mediated inflammatory cardiomyopathy

In enterovirus‐induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b^high NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D‐dependent cytotoxicity compared to CVB3‐susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D‐deficient mice but was shown to be independent of CD8⁺ T‐cell‐dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre‐ (day ?1) as well as post‐infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D‐activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus‐induced myocarditis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

HMBOX1 negatively regulates NK cell functions by suppressing the NKG2D/DAP10 signaling pathway

HMBOX1 is a new member of the homeobox family. Homeobox members have been reported to participate in embryonic development and systemic metabolism, but the function of HMBOX1 remains unclear, especially in the hematopoietic system. Here, we show that HMBOX1 is expressed at a high level in primary human NK cells but is expressed at much lower levels in NK cell lines. Overexpression of HMBOX1 significantly inhibited NK cell activities, including natural cytotoxicity against tumor cells, the level of CD107a (a marker protein for degranulation) and the production of cytolytic proteins (perforin and granzymes). More interestingly, HMBOX1 negatively regulated the expression of NKG2D and the activation of the NKG2D/DAP10 signaling pathway in NK cells. This effect was reversed by knocking down HMBOX1. Taken together, these findings demonstrate that HMBOX1 may act as a negative regulator of NK cell functions via suppressing the NKG2D/DAP10 signaling pathway.