NK Cells Detect Changes in Adaptive Immunity within Mouse Decidua from Gestation Day Eight

Viable human CD56+ CD16? peripheral blood Natural Killer (NK) cells show specific in vitro binding under shear forces to ligands expressed by endothelial cells in cryostat sections of gestation day (gd)7 mouse decidua basalis. In serial assays, numbers of cells adhering to gd7 tissue are constant for men but have cyclical variation for fertile women, suggesting a brief gain in functional decidual homing potential of this NK cell subset during the menstrual cycle. Regardless of gender, numbers of adhering cells from an individual donor, increase dramatically when the substrate is decidua basalis from a later gestational timepoint. Here, we report that human blood CD56+ CD16? NK cells which adhere as single cells over gd7 decidua basalis, adhere as large clusters over gd8 and gd9 tissues, suggestive of antigen recognition and lymphocyte activation. We asked which cells within mouse decidua basalis trigger this response in CD56+ CD16? cells. Using decidua from mice transgenic for myeloid dendritic cell (mDC) expression of enhanced yellow fluorescent protein (eYFP), we found cluster formation was independent of mDC contact. Use of decidua from alymphoid mice showed clustering behavior required substrate lymphocytes. By use of decidua containing NK cells but lacking T and B cells, decidual T and/or B lymphocytes were identified as the cells altered after gd7 in a manner that activates CD56+ CD16? cell clustering. This timepoint is just prior to mouse spiral arterial modification and its detection by these indicator cells implicates adaptive, decidual immune responses in the regulation of NK cell function.     

Studies on the interaction between trophoblastic invasion and maternal immune cell infiltration at the implantation site in early human pregnancy by means of double immunoperoxidase technic using monoclonal antibodies

Interaction between trophoblastic invasion and maternal immune cell infiltration at the implantation sites in early human pregnancy was analyzed by means of a double immunoperoxidase technique using Troma-1, a rat monoclonal antibody, which recognizes trophoblastic cells and a set of mouse monoclonal antibodies to react with various immune cells. The results were as follows. The most prominent immune cells in the implantation sites were monocytes/macrophages, which were positive for HLA-DR. These cells were adjacent to trophoblastic cells which were infiltrating into the decidua basalis. It therefore appeared that these cells function as "antigen presenting cells" which recognize and present the processed fetal information to maternal T cells. A small number of cells with mature T cell markers were found to be infiltrating around the anchoring villi and the extra-villous trophoblastic cells in the decidua compacta. But a larger number of T cells were adjacent to the villi in the decidua spongiosa and the extra-villous trophoblastic cells invading the decidua spongiosa and the myometrium. These cells may therefore play a role in preventing trophoblastic cells from invading the myometrium in the implantation sites. A relatively large number of cells with E rosette receptors but without mature T cell markers were observed in the decidua basalis, but few were found in the myometrium, into which a larger number of mature T cells were infiltrating. The distribution of particular cells was similar to that of endometrial granulocytes studied in our laboratory. There were thus likely to be immune cells in humans equivalent to non-T granulated suppressor cells in mice, which have been shown to suppress the generation of cytotoxic T cells (Clark et al.).     

Possible role of natural killer and natural killer T-like cells in implantation failure after IVF

During implantation, maternal immunoactivation and tolerance are not only limited to the decidua but are also observed in the periphery, predominantly affecting the innate immune system. Since unexplained female infertility, as well as recurrent spontaneous abortion and implantation failure, are thought to be associated with pathological maternal immunotolerance mechanisms, this study focused on immune profile analysis of IVF candidates. Previous studies on peripheral natural killer (NK) cell characteristics of IVF patients have been limited to the comparison of blood samples taken prior to the IVF procedure. This study performed a follow-up study and compared patient's data obtained on the day of oocyte collection with the data 1 week after embryo transfer. The aim was to investigate phenotypic (subpopulations, CD69, T-cell immunoglobulin mucin 3 and NK-activating receptor expression) and functional (perforin and CD107a expression) changes in the peripheral NK and NK T (NKT)-like cell populations. During this short period of time around the IVF procedure, women with failed IVF reflected unfavourable Th1-oriented changes of NK and NKT-like cells. In comparison the follow-up data for women with successful conception remained principally constant. The observed peripheral changes during early pregnancy in the same individual may also have importance in successful embryo implantation.     

Comparison of decidual leukocytes following spontaneous vaginal delivery and elective cesarean section in uncomplicated human term pregnancy

The aim of this study was to quantify and compare leukocyte populations in term decidua basalis and parietalis obtained after spontaneous vaginal delivery (SVD) or elective cesarean section (CS) without labor. Decidua basalis and parietalis samples were obtained from placentas after SVD (n = 20) and after CS (n = 30). Following mechanical disaggregation, leukocytes were purified and stained with monoclonal antibodies. Percentages of leukocyte subclasses within the CD45(+) cell fraction and activated T cells were determined by flow cytometry. No differences were found in the percentages of CD45(+) cells or CD56(bright)CD16(-) uterine natural killer (NK) cells between decidua basalis from SVD and CS or between decidua parietalis from SVD and CS. In decidua basalis and parietalis from SVD, a significantly higher number of CD56(dim)CD16(+) NK cells was found compared to CS. In decidua basalis from SVD, there was a significantly lower percentage of CD14(+) cells and higher percentage of CD19(+) cells compared to CS. The percentage of CD3(+) T cells expressing CD25 or human leukocyte antigen (HLA)-DR was significantly decreased in decidua basalis and parietalis from SVD compared to CS. Comparison of decidua collected after SVD or CS suggests that labor is associated with dynamic changes in the distribution of decidual leukocytes, specifically NK and T cell subpopulations. In particular, the disappearance of the CD4(+)CD25(+) T cell population, which possibly contains a subpopulation of regulatory T cells, may contribute to the initiation of labor. Further investigation into factors affecting decidual leukocytes may expand our understanding of the immunological events at the maternal-fetal interface.     

Implantation aus molekularer Sicht

Implantation is an extraordinary process in nature that involves the embryo with trophoblastic cells and the maternal endometrial epithelium. The trophoblast forms vascular connections necessary for nutrient transport to the fetus. The molecular dialogue that occurs between the implanting conceptus and the uterine decidua involves cell-cell and cell-extracellular matrix interactions, mediated by lectins, integrins, matrix degrading enzymes and their inhibitors, and a variety of cytokines. The decidua is infiltrated by a population of CD56⁺CD16^? natural killer (NK) cells with a distinctive phenotype. These cells are particularly numerous in the decidua at the implantation site where they come into close contact with invading trophoblast cells. Selective expression of the unusual class I human leukocyte antigen (HLA) molecule, HLA-G, by extravillous trophoblast may assist in protecting invasive cytotrophoblast from potential maternal NK cell attack. The purpose of the present review is to describe briefly our present state of knowledge of embryo implantation at the cellular and molecular level.     

Antigen presenting capacity of murine decidual tissue in vivo

Antigen presenting cells (APC) within murine decidual tissue in vivo have been shown to process the soluble antigen ovalbumin after intravenous administration and to present it in a form recognizable by immune T lymphocytes. In vivo antigen pulsed decidual APC stimulated T cell proliferation as efficiently as splenic APC and in an MHC restricted manner. In addition, anti-class II antibody plus complement treatment significantly reduced decidual antigen presenting capacity in vitro. These findings show that class II positive cells within the decidua can present antigen effectively in vivo and may therefore serve as APC for the presentation of fetal antigens to the maternal immune system during pregnancy.     

Elsevier Trophoblast Research Award Lecture: Unique Properties of Decidual T Cells and their Role in Immune Regulation during Human Pregnancy

Maternal lymphocytes at the fetal–maternal interface play a key role in the immune acceptance of the allogeneic fetus. Most studies focus on decidual NK cells and their interaction with fetal trophoblasts, whereas limited data are available on the mechanisms of fetus specific immune recognition and immune regulation by decidual T cells at the fetal–maternal interface. The aim of this review is to describe the phenotypic characteristics of decidual T cell subsets present at the fetal–maternal interface, their interaction with HLA-C expressed by fetal trophoblasts and their role in immune recognition and regulation at the fetal–maternal interface during human pregnancy.     

The balance between cytotoxic NK cells and regulatory NK cells in human pregnancy

NK cells kill tumor cells and virus-infected cells as well as secrete a variety of cytokines. These effector functions are regulated by the balance between activating receptor signals and inhibitory receptor signals which are triggered by specific major histocompatibility complex (MHC) or non-MHC ligands. It is thought currently that the balance between immunostimulation and immunoregulation in T cell immunity is achieved by a Th1/Th2/Th3/Tr1 and CD4(+)CD25(+) regulatory T (Treg) cell paradigm. Here, we discuss the cytokine paradigm of NK cells in human pregnancy. During normal, intact pregnancy, peripheral blood NKr1 cells and decidual NK3 cells increase, while these NK cell populations decrease significantly in miscarriage cases, suggesting that an imbalance in NK1/NK2/NK3/NKr1 is correlated with miscarriage. Recent investigations have shown that not only Treg cells, but also regulatory NK (NK reg) cells, play very important roles in the maintenance of pregnancy. We summarize the progress in studying NK reg cells and focus on how NK reg cells and cytotoxic NK cells affect the reproductive immune response.     

Natural Killer Cells and Dendritic Cells at the Human Feto-maternal Interface: an Effective Cooperation?

Human endometrium and in particular decidua, harbours a considerable population of immunocompetent cells. The most prominent of these are uterine natural killer (uNK) cells, which differ considerably from their peripheral blood counterparts in terms of both gene expression and function. Recently, the existence of DC-SIGN positive immature dendritic cells (DCs) in human decidua has been demonstrated. Evidence exists that immature DCs are required for the initiation and maintenance of peripheral tolerance, whereas mature DCs, which are only found in minimal amounts in human decidua, are associated with a Th1 polarization of T cells. Although the study of uNK-DC cross-talk is only beginning, it may in the future provide important insights into how acceptance of the fetus by the maternal immune system is mediated.     

Distribution of Th1 and Th2 cell populations in human peripheral and decidual T cells from normal and anembryonic pregnancies

OBJECTIVE: To examine whether maternal immune responses during normal pregnancy are Th2 biased and whether there are specific changes when anembryonic pregnancy occurs. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology at a university hospital. PATIENT(S): We studied 32 pregnant women receiving elective abortions of normal pregnancies and 35 women with anembryonic pregnancies between 6 weeks and 10 weeks of gestational age. INTERVENTION(S): Using the multilabeling capability of three-color flow cytometry, it is possible to measure intracellular cytokines and cell surface markers simultaneously to determine which cells are the cytokine-producing cells. MAIN OUTCOME MEASURE(S): We examined the extent and proportion of mononuclear cells expressing specific T-cell surface markers and cytokines, interferon gamma, and interleukin 4 in the peripheral blood and deciduae. Secreted cytokines in the supernatants after 24-hour culture were also compared. RESULT(S): During the unstimulated status, the proportion of IL-4-secreting cells significantly exceeded that of IFN-gamma-secreting cells in the peripheral blood and decidua in normal pregnancies and was significantly decreased when anembryonic pregnancies occurred. Consequently, the Th1/Th2 ratios were increased during anembryonic pregnancies. However, after 24-hour culture, only another Th2-type cytokine, IL-10, was markedly increased and exceeded IFN-gamma secretion in cultures from both the peripheral blood and decidua in normal pregnancies. CONCLUSION(S): The decidual T lymphocytes are Th2 predominant. When anembryonic pregnancy occurs, this Th2 predominance disappears.     

Co-ordinate expression of Th1/Th2 phenotypes in maternal and fetal blood: evidence for a transplacental nexus

If maternal atopy and environmental exposure affect prenatal Th cell development, the maternal and fetal immune systems should display common Th1/Th2 phenotypes. To test this hypothesis, we studied maternal and neonatal blood samples from mothers with total serum IgE <300 IU/mL. Basal levels of IFN-γ, IL-4, and eotaxin in paired maternal and fetal sera were tightly correlated. Polyclonal T cell activation in vitro by Staphylococcal exotoxin B induced co-ordinate IFN-γ production from paired maternal and fetal mononuclear cells, accompanied by co-ordinate increases in activated CD4+CD69+ cells that display the CCR4+Th2 and CXCR3+ Th1 phenotypes. Maternal and fetal CD4+CXCR3+ T cells were subsequently identified as the major producers of IFN-γ. The data established that a transplacental nexus exists during normal pregnancy and that fetal Th cell responses may be biased by the maternal immune system.     

The basis and value of currently used immunomodulatory therapies in recurrent miscarriage

Recurrent miscarriage (RM) without an obvious identifiable cause may arise from excessive maternal T and natural killer (NK) cell activity against the trophoblast or early embryo. Impaired regulatory T cell function leading to increased pro-inflammatory Th17 and NK cell cytotoxicity may be central. Ongoing subclinical endometrial infection and/or inflammation with increased secretion of TNFα and stimulation of autoimmunity to heat shock proteins may also be contributory. Therapies with a varying theoretical basis and clinical evidence aimed at reducing excessive endometrial immune activity have been used non-selectively in women with RM with variable success. Recent work has now improved our understanding of the role of the different immune cells and proteins that are important at each stage of a normal pregnancy. The vulnerability of the early embryo to T and NK cell-mediated rejection suggests that immune-based therapies need to be maximally effective during early pregnancy. Targeting RM women with demonstrable T and NK cell activity may improve the overall clinical efficacy of these treatments. It may also prevent costly and possibly harmful use in women who are unlikely to respond, and make better use of scarce resources. This report describes the underlying principles behind the use of the different immune-based therapies. The broad evidence supporting their efficacy is also described, as are the possible adverse consequences. Suggestions are also made on how the maternal immune system may be positively modulated using current, widely available treatments that have minimal or no side effects.     

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.     

Immunodystrophism: evidence for a novel alloimmune hypothesis for recurrent pregnancy loss involving Th1-type immunity to trophoblast

Pregnancy loss is the most common complication of pregnancy. Recurrent pregnancy loss occurs in approximately 1% of pregnant women. Many immunologic theories have been proposed but have not withstood rigorous analysis. A novel alloimmune hypothesis involving T helper (Th) 1-type immunity to trophoblast is the latest theory proposed for recurrent pregnancy loss. The basic hypothesis is, in the decidua there are myriad of antigen presenting cells and other immune response cells. In response to trophoblast invasion, these cells may become activated. A by-product of this activation is the secretion by these cells of either a predominant Th1 or Th2 cytokine profile. In cases where a Th1 cytokine profile predominates, chiefly, interferon-gamma, tumor necrosis factor, or interleukin-12, these cytokines may directly or indirectly be detrimental to early placental cell differentiation and growth and toxic to embryo development. Further evidence for this novel hypothesis comes from recent findings of a genetic predisposition for a vigorous Th1 cytokine response in these women due to a polymorphism in the IL1B promoter region. Further studies are needed to substantiate definitive causative links between Th1-immunity to trophoblast and recurrent pregnancy loss. Clinical trials are also needed to determine the best therapy for this disorder.     

Immunophysiology of normal pregnancy. Part I. Immunoregulatory mechanisms inside the maternal-fetal interface

Contemporary opinions concerning the function of decidual NK cells, T and B lymphocytes as well as macrophages and neutrophils in normal pregnancy were presented in the paper. Maintenance of proper Th1/Th2 balance concerning cytokines produced by trophoblast, decidua and decidual infiltrating immunological cells was also precisely described.     

Immunoactivation in preeclampsia: Vδ2+ and regulatory T cells during the inflammatory stage of disease

Recent data suggest a dominant role of the innate, rather than the adaptive immune system in pregnancy-related immunoregulation. γ/δ T cells, that comprise a minor subpopulation of human peripheral blood lymphocytes, represent a link between the innate and the acquired immune systems. However little is known about how they function in preeclampsia, which is suggested to be associated with a Th1 predominant immune response. The aim of our study was to investigate the presence and phenotype of Vδ2+ cells and of regulatory T cells in the pathogenesis of preeclampsia. Since Vδ2+ T cell function has been shown to be altered in patients with preeclampsia we investigated the expression of perforin, Fas and TIM-3 by Vδ2+ T cells and the possible role of activating and inhibitory NK cell receptors as well as of regulatory T cells. Vδ2+ T cells of preeclamptic patients demonstrated an increased perforin and IFNγ production, which could be explained by dysregulation of NK cell receptor expression. These Th1 polarized cells were less susceptible to apoptosis than Vδ2+ T cells from healthy pregnant women. Our data suggest that activated Vδ2+ T cells of preeclamptic women have an increased cytotoxic potential, which may be due to altered expression of NK cell inhibitory and activating receptors. In this study we report a series of observations, which taken together suggest the role of multiple pathways in generating an exaggerated systemic inflammatory response observed in the clinical stage of preeclampsia.     

穿孔素蛋白及基因在子宫内膜中的表达

目的探讨穿孔素蛋白及基因在不同时期子宫内膜中表达的规律,及其在生殖系统中的作用。方法采用免疫组化与原位杂交技术,观察１０例增殖期、６８例分泌各期、２０例早孕期及４例绝经后子宫内膜中穿孔素蛋白的表达及基因阳性细胞的分布特点、表型特征和数量变化规律。结果在各期子宫内膜中,产生穿孔素蛋白的细胞位于子宫内膜间质,此细胞表型特征为ＣＤ５６＋淋巴细胞（自然杀伤样细胞）。在各期子宫内膜中表达穿孔素蛋白的阳性细胞数量不同,从增殖期、分泌期至早孕期,此阳性细胞数量逐渐增多,各组间差异有显著性（Ｐ＜０．０５）,绝经后子宫内膜中无穿孔素蛋白表达。在增殖期,穿孔素基因阳性细胞大多呈局灶状分布,与同期表达穿孔素蛋白的阳性细胞呈少量单细胞不一致分布,分泌期与早孕期子宫内膜中表达穿孔素基因的阳性细胞呈弥散状分布,并且早孕期此阳性细胞数量增多。结论子宫内膜中穿孔素蛋白和基因表达,可能参与月经来潮的调节,利于胚胎着床和妊娠的顺利进行。     

Gene expression of the constant region of the heavy chain of immunoglobulin G (IgG CRHC) is down-regulated in human decidua in association with preeclampsia

An aberrant interaction at the maternal/fetal interface between the genetically distinct fetal trophoblast cells and cells of the maternal decidua has been proposed as an initiating factor in one of the major complications of human pregnancy, preeclampsia. Biochemical and epidemiological studies suggest that the immune system plays an important role in preeclampsia. Thus, the aim of this study was to determine the decidual gene expression status in preeclampsia of one of the key components of the adaptive immune system. Total RNA was extracted from decidua collected from women with normal pregnancies and those complicated by preeclampsia. Reverse Northern analysis was performed on 72 cDNAs from human decidua and differentially expressed genes identified were analysed further using semi-quantitative RT-PCR and Northern blot analysis. Expression of the gene encoding the constant region of the heavy chain of immunoglobulin G (IgG CRHC) was shown to be down-regulated in association with preeclampsia. These data support the hypothesis that immune maladaptation may play an important role in the pathogenesis of preeclampsia.     

Expression of NK cell receptors on decidual T cells in human pregnancy

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on γδTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.     

分泌期及早期子宫内膜巨噬细胞及自然杀伤细胞的测定

目的 探讨子宫内膜局部免疫微环境对妊娠的影响。方法 对１９例孕６￣９周的正常妊娠和１１例正常分泌期妇女,采用流式细胞技术检测蜕膜及内膜的巨噬细胞及自然杀伤（ＮＫ）细胞。结果 蜕膜组织中巨噬细胞（白细胞分化抗原分化簇（ＣＤ）１４阳性即ＣＤ１４」及ＮＫ细胞（ＣＤ５６阳性即ＣＤ５６）含量较分泌期子宫内膜组织增加,差异有极显著性。ＮＫ细胞的ＣＤ５６ＣＤ１６亚群含量增加,差异有极显著性,而ＣＤ５６ＣＤ１６亚