HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells

Non-classical MHC class I molecule HLA-E is the ligand for CD94/NKG2 NK cell receptors. Surface expression of HLA-E requires binding of specific HLA class I leader sequences. The uterine mucosa in early pregnancy (decidua) is infiltrated by large numbers of NK cells, which are closely associated with placental trophoblast cells. In this study we demonstrate that trophoblast cells express HLA-E on their cell surface in addition to the previously reported expression of HLA-G and HLA-C. Furthermore, we show that the vast majority of decidual NK cells bind to HLA-E tetrameric complexes and this binding is inhibited by mAb to CD94. Thus, recognition of fetal HLA-E by decidual NK cells may play a key role in regulation of placentation. The functional consequences of decidual NK cell interaction were investigated in cytotoxicity assays using polyclonal decidual NK cells. The overall effect of CD94/NKG2 interaction with HLA-E is inhibition of cytotoxicity by decidual NK cells. However, since decidual NK cells are unable to kill trophoblast even in the presence of mAb to MHC class I molecules and NK cell receptors, HLA-E interaction with CD94/NKG2 receptors may regulate other functions besides cytolysis during implantation.     

The immune potential of decidua-resident CD16+CD56+ NK cells in human pregnancy

Human CD56⁺CD3^? NK cells can be subdivided into two different subsets according to the expression pattern of CD56 and CD16. CD56^+/brightCD16^? (CD16^?) NK cells are prominently cytokine producers with little cytotoxicity whereas CD56^+/dimCD16⁺ (CD16⁺) NK cells are efficient killers with poorer cytokine production potential. In human pregnancy, CD56⁺ decidual (d)NK cells accumulate in the maternal fetal interface to regulate placental immunity and development. Unlike peripheral blood (pb)NK cells, the majority of dNK cells are CD56 positive with limited CD16 reactivity. Our results demonstrated that in normal and pathological pregnancies, CD16⁺ dNK cells are a unique population in comparison to CD16^? dNK subset. The expression of NK activation receptors CD335, CD336, CD244 and CD314 on CD16⁺ dNK subpopulation was lower than that on CD16^? dNK cells. Upon cytokine stimulation with rhIL-12/15/18 or TGFβ blockade, the CD16⁺ dNK subset exhibited more robust response on the expression of IFNG, IL-8 and CD107a, compared to that of the CD16^? dNK subpopulation. Functions of the CD16⁺ dNK subset were shown to be independent of cellular interaction with trophoblast cells. Studies of preeclamptic patients revealed lower proportions of CD16⁺ dNK cells, suggesting potential protective roles of these cells during normal gestations.. Therefore, we suggest that the CD16⁺ dNK subset, through compensating CD16^? dNK cell function, is an indispensable component to regulate decidual immune response and to support placentation.     

Interaction of Decidual CD56+ NK with Trophoblast Cells during Normal Pregnancy and Recurrent Spontaneous Abortion at Early Term of Gestation

The immunological paradox of pregnancy, when maternal immune system recognizes but does not reject the semiallogenic foetus, is not yet fully understood. The aim of this work was to detail the mechanisms of the interaction of decidual CD56+ NK, infiltrating the maternal part of placenta, and trophoblast cells of foetal origin. Samples of the endometrial tissue from 13 healthy non‐pregnant women, 37 placentas, obtained after medical abortion of viable pregnancy at 7–10 weeks of gestation, and 26 samples of placentas from first‐trimester recurrent spontaneous abortion (RSA) were used as the material for investigation. Phenotype of NK was assessed by flow cytometry. The influence of trophoblast cells upon IFNγ and GrB mRNAs expression by dNK was investigated by RT‐PCR. The influence of dNK upon trophoblast cells migration and invasion was studied using collagen and Matrigel systems. In RSA group comparing to the normal pregnancy, the decrease of dNK with inhibitory receptors (NKG2A) and elevation of activated dNK were seen. In normal pregnancy, but not in RSA, trophoblast cells increased the expression of IFNγ and GrB mRNAs by CD56+ dNK. Both in normal and RSA pregnancy, dNK inhibited the migration and invasion of trophoblast cells. Initially, low invasive and migration capacities of trophoblast cells were seen during RSA. Thus, unbalanced activation of dNK can lead to the impairment of dNK and trophoblast cells interaction during RSA.     

人早孕期蜕膜基质细胞对蜕膜NK细胞分泌IL-22的调节作用

目的:探讨蜕膜基质细胞(Decidual stromal cells,DSCs)与蜕膜NK细胞(dNK)共培养后IL-22的分泌水平。方法:收集早孕蜕膜组织,分离蜕膜基质细胞(DSCs)及蜕膜免疫活性细胞(Decidual immunocytes,DICs),磁珠分选蜕膜CD56brightCD3-NK细胞,再与DSC按不同比例直接接触共培养(dNK∶DSC为1∶1、1∶2、1∶3)24小时,收集上清。ELISA检测上清中IL-22的表达。结果:与对照组相比,DSC能上调dNK分泌IL-22。结论:蜕膜基质细胞可以促进蜕膜NK细胞分泌IL-22。     

Surface NK receptors and their ligands on tumor cells

The identification of MHC-class I-specific inhibitory receptors in humans and mice provided a first explanation of why NK cells can kill target cells that have lost or underexpress MHC-class I molecules but spare normal cells. However, the molecular basis of NK-mediated recognition and tumor cell killing revealed a higher degree of complexity. Thus, under pathological conditions, NK cells may express insufficient amounts of triggering receptors and target cells may or may not express ligands for such receptors. Here we briefly illustrate the main NK receptors and their cellular ligands and we delineate the major receptor/ligands interactions leading to NK cell activation and tumor cell lysis.     

人早孕期蜕膜基质细胞下调蜕膜NK细胞杀伤活性

通过分析人早孕期蜕膜基质细胞(decidual stromal cell,DSC)对蜕膜NK细胞(dNK)表面趋化因子受体CXCR4与细胞内颗粒酶B表达水平的影响,研究早孕蜕膜基质细胞对局部NK细胞的训导作用。收集早孕蜕膜组织,分离DSC及蜕膜免疫活性细胞,进一步通过磁珠分选蜕膜CD3-CD56bright NK细胞,将分离的蜕膜NK细胞与DSC按1∶1比例共培养24h,收集蜕膜NK细胞,流式细胞仪检测其表面趋化因子受体CXCR4和细胞内颗粒酶B(granzyme B)的表达水平。结果显示,与对照组相比,在与DSC细胞共培养之后,趋化因子受体CXCR4+NK细胞的百分率明显上升,而蜕膜NK细胞内颗粒酶B阳性率显著下降(P<0.05)。结果表明,人早孕母-胎界面DSC细胞上调蜕膜NK细胞表面趋化因子受体CXCR4的表达,下调NK细胞内颗粒酶B的表达水平,可能抑制其杀伤活性。     

IL-22 secreted by decidual stromal cells and NK cells promotes the survival of human trophoblasts

Interleukin-22 (IL-22) has been implicated as an important immune regulator in many physiologic and pathological processes, but little is known about the IL-22 in the fetal-maternal interface. In this study, we demonstrated that co-culture of decidual stromal cells (DSCs) and decidual natural killer (dNK) cells resulted in increased secretion of IL-22, compared to culture of DSCs or dNK cells alone. The trophoblast cell line, HTR8/SVneo, expresses IL-22 receptor α1 (IL-22R1). Combinant human (rh) IL-22 significantly promoted the proliferation and viability, and inhibited the apoptosis of HTR8/SVneo cells. By Western blotting and immunohistochemistry, we confirmed that villi expressed IL-22R1, and the villi from unexplained spontaneous miscarriage patients expressed reduced levels of IL-22R1 than those from normal early pregnancy. These findings indicate that the IL-22 secreted by DSCs and dNK might promote the survival of trophoblasts and participate in the maintenance of pregnancy by binding to the IL-22R1. The reduced level of IL-22/IL-22R1 in villi might be involved in the occurrence of spontaneous miscarriage.     

Toxoplasma gondii Infection of Decidual CD1c+ Dendritic Cells Enhances Cytotoxicity of Decidual Natural Killer Cells

There is crosstalk between decidual natural killer (dNK) cells and decidual dendritic cells (dDCs) that promotes tolerance of trophoblast cells carrying paternally derived antigens. In the present study, we report that infection of CD1c⁺ dDCs with Toxoplasma gondii enhanced gamma interferon (IFN-γ) production by dNK cells in co-culture. The enhancement of IFN-γ production was induced by cytokine IL-12 which increased obviously in co-culture of dDCs with dNK cells following T. gondii infection, and this enhancement largely abrogated when cells were cultured in the presence of an anti-IL-12 antibody. The expression of KIR2DL4 and NKG2D on dNK cells was increased after T. gondii infection, and higher expression of NKG2D was induced by co-cultured dDCs. Neutralization of IL-12 decreased NKG2D expression on dNK cells. Furthermore, dDCs with T. gondii infection increased the cytotoxicity of co-cultured dNK cells against K562 target cells, which was mediated by activating receptor of NKG2D. Thus, T. gondii infection of dDCs enhanced dNK cell IFN-γ production and NKG2D expression, and then led to increased cytotoxicity of dNK cells. The up-regulated dNK cell cytotoxicity at the maternal–fetal interface may contribute to abnormal pregnancy outcomes caused by T. gondii infection in early pregnancy.     

The effect of pregnancy on the uterine NK cell KIR repertoire

The major leukocyte population in the decidua during the first trimester of pregnancy consists of NK cells that express receptors capable of recognizing MHC class I molecules expressed by placental trophoblast. These include members of the killer immunoglobulin-like receptor (KIR) family, the two-domain KIR (KIR2D), which recognize HLA-C. Interactions between decidual NK (dNK) cell KIR2D and placental HLA-C contribute to the success of pregnancy and dNK cells express KIR2D at higher frequency than peripheral NK (pNK) cells. Thus, they are biased toward recognizing HLA-C. In order to investigate when this unusual KIR repertoire appears, we compared the phenotype of NK cells isolated from non-pregnant (endometrium) and pregnant (decidua) human uterine mucosa. Endometrial NK (eNK) cells did not express KIR2D at a higher level than matched pNK cells, so the bias toward HLA-C recognition occurs as a response to pregnancy. Furthermore, HLA-C expression was upregulated on uterine stromal cells as the mucosa transformed from endometrium to decidua at the onset of pregnancy. As uterine NK (uNK) cells can mature from NK precursors and acquire KIR expression in utero, the pregnancy-specific bias of uNK cells toward HLA-C recognition could arise as developing uNK cells interact with uterine stromal cells, which express higher levels of HLA-C during pregnancy.     

Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines

The lack of classical HLA-class I molecules on trophoblast is necessary to prevent allorecognition by maternal CTL, but may induce activation of NK cells. A protective role against NK cells equipped of suitable inhibitory receptors has been proposed for nonclassical HLA-class I molecules including HLA-E and HLA-G. In the present study we show that the NK-mediated killing of two choriocarcinoma cell lines, JAR and JEG3, is induced upon engagement of natural cytotoxicity receptors (NCR) with their specific ligands. In particular, we show that NKp44, a triggering receptor expressed at the NK cell surface only after in vitro culture in the presence of IL-2, plays a central role in triggering NK cytotoxicity against trophoblast cells. Also NKp46 appear to contribute to this function by cooperating with NKp44. On the other hand, other triggering receptors such as NKp30, 2B4, and NKG2D are not involved in killing of choriocarcinoma. Our findings suggest that resistance of trophoblast to NK-mediated cytotoxicity is the result of insufficient activating interactions between the various triggering NK receptors and their target cell ligands. On the other hand, the interaction of nonclassical HLA class I molecules with inhibitory NK receptors appears to play only a marginal role in regulating the susceptibility of choriocarcinoma to NK mediated cytotoxicity.     

Apoptosis induced by NK cells is modulated by the NK-active cytokines IL-2 and IL-12

NK cells kill sensitive targets via exocytosis of cytoplasmic granules containing membrane damaging perforins and DNA damaging granzymes. Therefore, the target cell can either die by necrosis or by apoptosis. A third, non-secretory, mechanism of killing mediated by Fas-FasL interaction can be used by activated NK cells to kill Fas+ targets. Here, we have studied the modulation exerted by two NK active cytokines, IL-2 and IL-12, on the apoptosis-inducing activity of NK cells in NK-sensitive Fas- K562 and NK-insensitive Fas+ Raji cells. Our results show that apoptosis is preferentially induced at low target:effector ratios. Resting NK cells virtually do not induce apoptosis in target cells while IL-2 stimulation endows NK cells with the capacity of inducing apoptosis and IL-12 further enhances this effect against both targets. Finally, we demonstrate that cytokine- stimulated NK cells use both granzyme and Fas-FasL pathways of apoptosis induction.      

不明原因自然流产与蜕膜NK细胞表面活化性受体表达的相关性研究

探讨不明原因自然流产患者蜕膜NK细胞杀伤活性与其细胞表面活化性受体NKp46、NKp44、NKp30和NKG2D表达的相关性。选取21例早孕不明原因自然流产患者为病例组,25例正常早孕人流妇女为对照组,收集两组的蜕膜组织,Ficoll密度梯度离心分离淋巴细胞,MACS磁珠分选CD3-CD56+NK细胞。以K562细胞为靶细胞,用细胞染色及流式细胞技术检测两组蜕膜NK细胞杀伤活性,用流式细胞技术检测两组蜕膜CD56brightCD16-NK和CD56dimCD16+NK细胞上活化性受体NKp46、NKp44、NKp30和NKG2D的表达,并与NK细胞杀伤活性进行相关性分析。结果:(1)早孕蜕膜NK细胞具有杀伤活性;(2)病例组蜕膜NK细胞的杀伤活性较正常对照组显著增强(P=0.014);(3)病例组蜕膜CD56brightCD16-NK细胞中NKp44的表达比正常对照组显著升高(P=0.021);病例组蜕膜CD56dimCD16+NK细胞中NKp46和NKp44的表达比正常对照组显著升高(分别P=0.026,P=0.041);其余活化性受体的表达两组未见明显差异;(4)蜕膜NK细胞杀伤功能与蜕膜CD56brightCD16-NK细胞中NKp44的表达呈显著正相关(r=0.677,P<0.05),和蜕膜CD56dimCD16+NK细胞中NKp46的表达呈显著正相关(r=0.634,P<0.05)。蜕膜NK细胞活化性受体NKp46和NKp44表达增加,从而使蜕膜NK细胞的杀伤功能增强可能在不明原因自然流产的发病中起重要作用。     

Letter From the Editor

ABSTRACT: The NK-susceptibility of trophoblast cells to allogeneic and autologous intraplacental natural killer (NK), antibody-dependent (K), and mitogen-induced cell-mediated cytotoxicity was studied, using untreated and neuraminidase-treated trophoblast cells from normal, full-term deliveries. The work was preceded by systematic studies of placental cell separation and labelling techniques, and the effects of these techniques on the NK target, K562. The results indicated that maternal NK cells are present among intraplacental lymphocytes, but that their activity is lower than that of peripheral blood lymphocytes and they are not stimulated by interferon to the same extent as peripheral blood lymphocytes (PBL). Trophoblast cells were rarely susceptible to allogeneic NK cells, with low cytotoxicity at high effector-target cell ratios in only two of five experiments. Interferon (IF)-boosted NK cells mediated some cytolysis of trophoblasts in three of four experiments, but high effector/target cell ratios were also required for the effect to be observed. The trophoblast cells could be lysed, however, by K cells and lectin-induced cytotoxicity. Removal of surface sialic acid by neuraminidase treatment of the trophoblast cells had little effect on the susceptibility of these cells to unstimulated NK cells (one of four experiments), but resulted in susceptibility to IF-boosted NK cells in four of four experiments. Normal trophoblast cells did not compete in IF-NK(K562) assays and neuraminidase-treated cells competed weakly in only one of three such experiments, indicating that the NK “target structure” is only weakly expressed on human trophoblast cells. Intraplacental lymphocytes lysed autologous trophoblast cells to a lower extent than allogeneic PBL. This lysis was markedly increased if antibody against the target cells was present in the assay. These data indicate that a) the trophoblast cell is susceptible to maternal cell-mediated lysis by several mechanisms that could potentially be activated in vivo, b) NK cells are present in the intraplacental lymphocyte pool, and c) the access of NK cells and interferon activated NK cells to the NK cell target structure is blocked by cell surface sialic acid residues. This target structure may be similar to that found on other susceptible cells, and in similarity to the tumor—NK interaction, the cell surface sialic acid is ineffective in blocking cytotoxicity if the appropriate antibody is present. Assuming NK cells mediate ADCC, this indicates that sialic acid does not mask the target site of the lytic molecule. These data are relevant to the understanding of the NK– target interaction in a situation where it is known that the target is nonself.     

Identification of NKp80, a novel triggering molecule expressed by human NK cells

The ability of NK cells to kill a wide range of tumor or virally infected target cells as well as normal allogeneic T cell blasts appears to depend upon the concerted action of multiple triggering NK receptors. In this study, using two specific monoclonal antibodies [(mAb) MA152 and LAP171], we identified a triggering NK receptor expressed at the cell surface as a dimer of approximately 80 kDa (NKp80). NKp80 is expressed by virtually all fresh or activated NK cells and by a minor subset of T cells characterized by the CD56 surface antigen. NKp80 surface expression was also detected in all CD3- and in 6 / 10 CD3+ large granular lymphocyte expansions derived from patients with lymphoproliferative disease of granular lymphocytes. In polyclonal NK cells, mAb-mediated cross-linking of NKp80 resulted in induction of cytolytic activity and Ca2+ mobilization. A marked heterogeneity existed in the magnitude of the cytolytic responses of different NK cell clones to anti-NKp80 mAb. This heterogeneity correlated with the surface density of NKp46 molecules expressed by different NK clones. The mAb-mediated masking of NKp80 led to a partial inhibition of the NK-mediated lysis of appropriate allogeneic phytohemagglutinin-induced T cell blasts, while it had no effect on the lysis of different tumor target cells, including T cell leukemia cells. These data suggest that NKp80 recognizes a ligand on normal T cells that may be down-regulated during tumor transformation. Molecular cloning of the cDNA coding for NKp80 revealed a type II transmembrane molecule of 231 amino acids identical to the putative protein encoded by a recently identified cDNA termed KLRF1.     

Uterine NK Cells and Trophoblast HLA Class I Molecules

PROBLEM: To investigate the proposal that NK cells in decidua may control trophoblast migration during implantation of the human placenta. METHOD: Use Mab specific for HLA-G and for HLA-C in association with flow cytometry and immunoprecipitation to determine the expression of these HLA molecules by trophoblast. Expression of Killer inhibitory/activatory receptors (KIR/KAR) and the CD94 receptor by decidual NK cells was also studied. RESULTS: Extravillous trophoblast expressed HLA-G and HLA-C in both β₂m-associated form and as free heavy chains. KIR and KAR are expressed by decidual NK cells. The repertoire of receptors varied between different women and also between blood and decidual NK cells from the same women. The expression of CD94 was also different between blood and decidual NK cells. CONCLUSION: The recognition of HLA-G/HLA-C by KIR/KAR and CD94 could provide a mechansm by which decidual NK cells control trophoblast migration.     

CYR61 modulates the vascular endothelial growth factor C expression of decidual NK cells via PI3K/AKT pathway

Citation Zhang X, Ding L, Diao Z, Yan G, Sun H, Hu Y. CYR61 modulates the vascular endothelial growth factor C expression of decidual NK cells via PI3K/AKT pathway. Am J Reprod Immunol 2012; 67: 216–223 Problem Either vascular endothelial growth factor C (VEGFC) or CYR61 plays an important role in placental development and may be involved in pre‐eclampsia. Decidual natural killer (dNK) cells are the main source of VEGFC in the maternal–fetal interface. However, it is unclear about CYR61 on the regulation of VEGFC secretion in dNK cells. Method of study Decidual natural killer cells were isolated from decidual tissues of first trimester of pregnancy with anti‐human CD56–conjugated microbeads. Integrin αvβ3 was detected using immunofluorescent staining. dNK cells were cultured in the presence of CYR61, anti‐human αvβ3 integrin antibody (LM609), PI3K inhibitor (LY294002), or MEK inhibitor (U0126). VEGFC mRNA and protein were evaluated by real‐time PCR and ELISA, respectively. Results Exogenous CYR61 induced the expression of VEGFC in dNK cells in both mRNA and protein levels. Integrin αvβ3 was strongly expressed on dNK cell surface. Anti‐αvβ3 integrin antibody inhibited the effect of CYR61 on VEGFC expression. LY294002, but not U0126, significantly reduced this promotion effect of CYR61 on dNK cells. Conclusions The upregulation of VEGFC secretion mainly depends on CYR61 binding with integrin αvβ3 on the surface of dNK cells. PI3K/AKT, rather than the ERK/MAPK signal, is involved in the regulation.     

Regulation of CD69 expression on human natural killer cells: differential involvement of protein kinase C and protein tyrosine kinases

Human peripheral blood natural killer (NK) cells (CD56⁺, CD16⁺, CD3_ε^? lymphocytes) express CD69 after their stimulation by interleukin-2 (IL-2) or interferon-α (IFN-α). This activation antigen represents a triggering surface molecule in NK cell clones as its stimulation triggers the cytolytic machinery of these cells. However, the mechanisms regulating the expression of CD69 in NK cells are unknown despite the functional relevance of CD69 in NK cell activation. Thus, we have analyzed the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the expression of CD69 on purified NK cells activated by IL-2, IFN-α, anti FcγRIII (CD16) monoclonal antibodies or by K562 target cells. We found that CD69 is induced on NK cells not only by IL-2 and IFN-α but also by activation of the CD 16 pathway, the interaction with NK target cells and the direct activation of PKC by phorbol 12-myristate 13-acetate (PMA), indicating that CD69 induction is associated to different NK activation pathways. The treatment with the PKC inhibitor staurosporine abolished the induction of CD69 induced by PMA or K562. However, it did not significantly affect CD69 induction by IL-2, IFN-α or CD 16 cross-linking. This demonstrates that whereas PKC can play a central role in the regulation of CD69 expression in some instances (response to K562 cells or PMA), it does not participate in others (response to IL-2, IFN-α or anti CD16 monoclonal antibodies). On the other hand genistein, a competitive inhibitor of PTK enzymes, blocked the expression of CD69 induced by activation of NK cells via IL-2 or IFN-α receptors, CD16 and K562 receptor(s), indicating that stimulation of PTK is a common step in the signal transduction events leading to the induction of CD69 antigens after the activation of NK cells via these receptors.     

ORIGINAL ARTICLE: Peripheral Blood NK Cells Reflect Changes in Decidual NK Cells in Women With Recurrent Miscarriages

Citation Park DW, Lee HJ, Park CW, Hong SR, Kwak‐Kim J, Yang KM. Peripheral blood NK cells reflect changes in decidual NK cells in women with recurrent miscarriages. Am J Reprod Immunol 2010; 63: 173–180 Problem We aimed to investigate if peripheral blood natural killer (pNK) cell levels are correlated with decidual NK (dNK) cell levels, and if chemokine expression has any role in dNK cell regulation. Method of study Decidual tissues of women having two or more miscarriages with normal karyotype were collected after miscarriage and an immuno‐histochemisty study was made. pNK cells were evaluated using flow cytometric analysis. Results The %CD3^?/56⁺ and %CD3^?/56⁺/16⁺ pNK cells showed a significant correlation with mean number of CD56⁺ dNK cells. The number of decidual CD16⁺ cells was significantly higher in women with elevated pNK (≥15%) than that of normal pNK (<15%). The %CD3^?/56⁺ and %CD3^?/56⁺/16⁺ pNK cells showed an inverse correlation with duration of gestation. The CCL3⁺ and CXCL12⁺ cells were present in the decidua; however, staining intensity was not correlated with number of dNK cells. Conclusion The pNK cell levels reflect changes in dNK cell levels. This implicates that pNK cell level is a clinically useful marker to predict pregnancy outcome. Further study is needed to examine if elevated pNK cells enhance recruitment of dNK cells in the decidua.