How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment.

The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice.

The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education. VIDEO ABSTRACT.

Distinctive phenotypes and functions of innate lymphoid cells in human decidua during early pregnancy.

During early pregnancy, decidual innate lymphoid cells (dILCs) interact with surrounding maternal cells and invading fetal extravillous trophoblasts (EVT). Here, using mass cytometry, we characterise five main dILC subsets: decidual NK cells (dNK)1-3, ILC3s and proliferating NK cells. Following stimulation, dNK2 and dNK3 produce more chemokines than dNK1 including XCL1 which can act on both maternal dendritic cells and fetal EVT. In contrast, dNK1 express receptors including Killer-cell Immunoglobulin-like Receptors (KIR), indicating they respond to HLA class I ligands on EVT. Decidual NK have distinctive organisation and content of granules compared with peripheral blood NK cells. Acquisition of KIR correlates with higher granzyme B levels and increased chemokine production in response to KIR activation, suggesting a link between increased granule content and dNK1 responsiveness. Our analysis shows that dILCs are unique and provide specialised functions dedicated to achieving placental development and successful reproduction.

Tissue stiffness at the human maternal-fetal interface.

STUDY QUESTION: What is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta? SUMMARY ANSWER: The stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta. WHAT IS KNOWN ALREADY: Mechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal-fetal interface. STUDY DESIGN, SIZE, DURATION: An observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Atomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Tissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women. WIDER IMPLICATIONS OF THE FINDINGS: These results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z).

Single-cell reconstruction of the early maternal-fetal interface in humans.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

Molecular definition of group 1 innate lymphoid cells in the mouse uterus.

Determining the function of uterine lymphocytes is challenging because of the dynamic changes in response to sex hormones and, during pregnancy, to the invading foetal trophoblast cells. Here we provide a genome-wide transcriptome atlas of mouse uterine group 1 innate lymphoid cells (ILCs) at mid-gestation. Tissue-resident Eomes+CD49a+ NK cells (trNK), which resemble human uterine NK cells, are most abundant during early pregnancy, and have gene signatures associated with TGF-ß responses and interactions with trophoblast, epithelial, endothelial, smooth muscle cells, leucocytes and extracellular matrix. Conventional NK cells expand late in gestation and may engage in crosstalk with trNK cells involving IL-18 and IFN-γ. Eomes-CD49a+ ILC1s dominate before puberty, and specifically expand in second pregnancies when the expression of the memory cell marker CXCR6 is upregulated. These results identify trNK cells as the cellular hub of uterine group 1 ILCs, and mark CXCR6+ ILC1s as potential memory cells of pregnancy.

High-Resolution Genetic and Phenotypic Analysis of KIR2DL1 Alleles and Their Association with Pre-Eclampsia.

Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A, not KIR2DL1B, associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk.

Effect of randomized serum progesterone concentration on secretory endometrial histologic development and gene expression.

STUDY QUESTION: What doses of secretory phase progesterone (P) in women are associated with altered endometrial structure and/or function? SUMMARY ANSWER: Consistently delayed histological maturation was seen at the lowest tested daily P dose (2.5 mg), whereas consistently altered functional response, as reflected by microarray analysis of gene expression was seen at both the 5 and 2.5 mg doses. WHAT IS KNOWN ALREADY: Progesterone is absolutely required for normal embryo implantation and pregnancy survival. Progesterone supplementation is beneficial in ART cycles. STUDY DESIGN, SIZE, DURATION: In this case-control experimental trial, 46 healthy young female volunteers (age 19-34) underwent a single modeled endometrial cycle after GnRH down-regulation or monitored in natural cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a university hospital, modeled cycles were obtained by GnRH agonist down-regulation, transdermal estradiol (E2) (0.2 mg/d), and daily injections of P in oil for 10 days: 2.5 mg (n = 6), 5 mg (n = 6), 10 mg (n = 12) or 40 mg (n = 12), after the 10th day of E2. Ten healthy, ovulatory women were used as controls. Endometrial biopsies were obtained on the 10th day of P exposure, or urinary LH surge (in controls). Analysis included histological dating, serum progesterone levels, microarray analysis of the whole genome, RT-PCR, western blot and comparison with the GEO database. MAIN RESULTS AND THE ROLE OF CHANCE: In endometrial biopsies, a morphological delay appears in the 2.5 mg/day of P group. Higher sub-physiological levels of P (≥5 mg/day) resulted in normal histology, but aberrant gene expression. P levels required for consistent histological delay were lower than those in all ovulatory women. Gene expression abnormalities occurred at higher sub-physiological P concentrations, without a change in histology, a functional-morphological disassociation. The expression of some endometrial receptivity-associated genes appeared multiphasic, with peak or nadir of mean or median expression levels between the lowest and highest doses, suggesting sustained supraphysiological doses seen in ART treatment cycles may not be optimal. LARGE SCALE DATA: GEO DataSets ID: 200056980; GSE 56980. LIMITATIONS, REASONS FOR CAUTION: These results were obtained in fertile women, who may respond differently from infertile subjects. WIDER IMPLICATIONS OF THE FINDINGS: The dose of P required for normal endometrial structure (5 mg/day) corresponds to a P concentration well below that seen in ovulatory women, suggesting that persistently delayed mid-secretory histology cannot be solely due to inadequate P concentrations in an ovulatory cycle. Endometrial gene expression is differentially regulated by different doses of progesterone. The apparent multiphasic response of some genes to P dose suggests the possibility that P concentration kinetics may play a role in normal endometrial preparation for receptivity. These findings strongly confirm that histologic development is not a reliable measure of endometrial P action. STUDY FUNDING/COMPETING INTEREST(S): Supported by The Eunice Kennedy Shriver National Institute for Child Health and Disease, National Institute of Health, USA (NICHD/NIH) (R01HD067721 and U54HD30476; SLY and BAL) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 240239/2012-1 (RFS). All authors have no competing interests.

Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy.

Tissue-specific NK cells are abundant in the pregnant uterus and interact with invading placental trophoblast cells that transform the maternal arteries to increase the fetoplacental blood supply. Genetic case-control studies have implicated killer cell Ig-like receptor (KIR) genes and their HLA ligands in pregnancy disorders characterized by failure of trophoblast arterial transformation. Activating KIR2DS1 or KIR2DS5 (when located in the centromeric region as in Africans) lower the risk of disorders when there is a fetal HLA-C allele carrying a C2 epitope. In this study, we investigated another activating KIR, KIR2DS4, and provide genetic evidence for a similar effect when carried with KIR2DS1 KIR2DS4 is expressed by ∼45% of uterine NK (uNK) cells. Similarly to KIR2DS1, triggering of KIR2DS4 on uNK cells led to secretion of GM-CSF and other chemokines, known to promote placental trophoblast invasion. Additionally, XCL1 and CCL1, identified in a screen of 120 different cytokines, were consistently secreted upon activation of KIR2DS4 on uNK cells. Inhibitory KIR2DL5A, carried in linkage disequilibrium with KIR2DS1, is expressed by peripheral blood NK cells but not by uNK cells, highlighting the unique phenotype of uNK cells compared with peripheral blood NK cells. That KIR2DS4, KIR2DS1, and some alleles of KIR2DS5 contribute to successful pregnancy suggests that activation of uNK cells by KIR binding to HLA-C is a generic mechanism promoting trophoblast invasion into the decidua.

Composition, Development, and Function of Uterine Innate Lymphoid Cells.

Innate lymphoid cells (ILCs), including NK cells, contribute to barrier immunity and tissue homeostasis. In addition to the role of uterine NK cells in placentation and fetal growth, other uterine ILCs (uILCs) are likely to play roles in uterine physiology and pathology. In this article, we report on the composition of uILCs in the endometrium during the luteal phase and in the decidua during early pregnancy. Whereas nonkiller uILC1s and uILC2s are barely detectable in mouse and not detected in humans, a sizeable population of uILC3s is found in human endometrium and decidua, which are mostly NCR(+) and partially overlap with previously described IL-22-producing uterine NK cells. Development of mouse uILC3 is Nfil3 independent, suggesting unique features of uILCs. Indeed, although the cytokine production profile of mouse uILCs recapitulates that described in other tissues, IL-5, IL-17, and IL-22 are constitutively produced by uILC2s and uILC3s. This study lays the foundation to understand how ILCs function in the specialized uterine mucosa, both in tissue homeostasis and barrier immunity and during pregnancy.

Tissue-Specific Education of Decidual NK Cells.

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self-HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK.

The role of the maternal immune system in the regulation of human birthweight.

Human birthweight is subject to stabilizing selection. Large babies are at risk of obstetric complications such as obstructed labour, which endangers both mother and child. Small babies are also at risk with reduced survival. Fetal growth requires remodelling of maternal spiral arteries to provide an adequate maternal blood supply to the placenta. This arterial transformation is achieved by placental trophoblast cells, which invade into the uterine wall. Under-invasion is associated with fetal growth restriction; but if invasion is excessive large babies can result. A growing body of evidence suggests that this process is controlled by interactions between killer-cell immunoglobulin-like receptors (KIRs) expressed on maternal uterine natural killer cells (uNK) and their corresponding human leukocyte antigen-C (HLA-C) ligands on invading trophoblast. Mothers with the KIR AA genotype and a fetus with a paternal HLA-C2 allele tend to have small babies, because this combination inhibits cytokine secretion by uNK. Mothers with the activating KIR2DS1 gene and an HLA-C2 fetus are more likely to have large babies. When KIR2DS1 binds to HLA-C2 this increases secretion of cytokines that enhance trophoblast invasion. We conclude that specific combinations of the highly polymorphic gene systems, KIR and HLA-C, contribute to successful reproduction by maintaining birthweight between two extremes.

Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation.

Reduced trophoblast invasion and vascular conversion in decidua are thought to be the primary defect of common pregnancy disorders including preeclampsia and fetal growth restriction. Genetic studies suggest these conditions are linked to combinations of polymorphic killer cell Ig-like receptor (KIR) genes expressed by maternal decidual NK cells (dNK) and HLA-C genes expressed by fetal trophoblast. Inhibitory KIR2DL1 and activating KIR2DS1 both bind HLA-C2, but confer increased risk or protection from pregnancy disorders, respectively. The mechanisms underlying these genetic associations with opposing outcomes are unknown. We show that KIR2DS1 is highly expressed in dNK, stimulating strong activation of KIR2DS1+ dNK. We used microarrays to identify additional responses triggered by binding of KIR2DS1 or KIR2DL1 to HLA-C2 and found different responses in dNK coexpressing KIR2DS1 with KIR2DL1 compared with dNK only expressing KIR2DL1. Activation of KIR2DS1+ dNK by HLA-C2 stimulated production of soluble products including GM-CSF, detected by intracellular FACS and ELISA. We demonstrated that GM-CSF enhanced migration of primary trophoblast and JEG-3 trophoblast cells in vitro. These findings provide a molecular mechanism explaining how recognition of HLA class I molecules on fetal trophoblast by an activating KIR on maternal dNK may be beneficial for placentation.

The science of implantation emerges blinking into the light.

Although embryo implantation is essential for human survival, it remains an enigmatic biological phenomenon. Following fertilization, the resulting blastocyst must signal its presence to the mother, attach to the luminal epithelium of the endometrium and embed into the decidualising stroma. Failure to do so results in infertility, which affects around 9% of women. Subsequent placental development requires remodelling of maternal blood vessels by trophoblast cells from the placenta, that invade deep into the decidua. Failure in these very early stages can compromise fetal development, resulting in diseases of pregnancy such as intrauterine growth restriction or pre-eclampsia which can also impact on health in adulthood. Abnormal implantation therefore constitutes a significant disease burden in humans. Although we have known for many years that successful implantation requires an embryo that is competent to implant and an endometrium that is receptive, the molecular basis of these processes remains poorly understood. Our inability to identify implantation-competent embryos or to diagnose/treat the non-receptive endometrium therefore limits our ability to intervene through assisted reproduction techniques. This Implantation Symposium aims to review recent exciting developments in our understanding of the biology of early implantation and to highlight the rapid progress being made to translate these into improved diagnosis and treatment.

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure.

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.

Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2.

Many common disorders of pregnancy are attributed to insufficient invasion of the uterine lining by trophoblast, fetal cells that are the major cell type of the placenta. Interactions between fetal trophoblast and maternal uterine NK (uNK) cells--specifically interactions between HLA-C molecules expressed by the fetal trophoblast cells and killer Ig-like receptors (KIRs) on the maternal uNK cells--influence placentation in human pregnancy. Consistent with this, pregnancies are at increased risk of preeclampsia in mothers homozygous for KIR haplotype A (KIR AA). In this study, we have demonstrated that trophoblast expresses both paternally and maternally inherited HLA-C surface proteins and that maternal KIR AA frequencies are increased in affected pregnancies only when the fetus has more group 2 HLA-C genes (C2) than the mother. These data raise the possibility that there is a deleterious allogeneic effect stemming from paternal C2. We found that this effect also occurred in other pregnancy disorders (fetal growth restriction and recurrent miscarriage), indicating a role early in gestation for these receptor/ligand pairs in the pathogenesis of reproductive failure. Notably, pregnancy disorders were less frequent in mothers that possessed the telomeric end of the KIR B haplotype, which contains activating KIR2DS1. In addition, uNK cells expressed KIR2DS1, which bound specifically to C2+ trophoblast cells. These findings highlight the complexity and central importance of specific combinations of activating KIR and HLA-C in maternal-fetal immune interactions that determine reproductive success.

Killer Ig-like receptor expression in uterine NK cells is biased toward recognition of HLA-C and alters with gestational age.

Immunogenetic studies suggest that interactions between maternal killer Ig-like receptor (KIR) expressed by uterine NK (uNK) cells, and fetal HLA-C molecules on trophoblast, influence the success of human placentation. However, the exact functional response of fresh uNK cells to trophoblast HLA-C molecules is unknown. In this study, we show by quantitative RT-PCR and FACS that both activating and inhibitory KIR specific for HLA-C are expressed at higher levels and on an increased proportion of NK cells in the human decidua compared with blood. In contrast, expression of KIR3DL1/S1, which is specific for HLA-B, is similar in both NK cell populations. Remarkably, there is also a temporal change in the expression pattern of HLA-C-specific KIR, with a decline in both intensity of expression and frequency on uNK cells throughout the first trimester of pregnancy. This selective up-regulation of KIR has functional consequences because uNK cells show increased binding of HLA-C tetramers compared with blood NK cells. Ab cross-linking shows that these KIR are functional and results in increased cytokine secretion. uNK cells, therefore, exhibit a unique KIR profile that enhances their ability to recognize trophoblast cells expressing HLA-C at the materno-fetal interface. This is the first report to demonstrate selective regulation of KIR expression over time in vivo in a normal physiological situation and suggests that KIR expression by uNK cells is regulated by the tissue microenvironment in the decidua.

Conformation of human leucocyte antigen-C molecules at the surface of human trophoblast cells.

Human leucocyte antigen (HLA)-C is expressed at lower levels than other classical HLA-I molecules on somatic cells. Surface HLA-C proteins can occur as conventionally beta(2)-microglobulin (beta2m)-associated complexes or as open conformers dissociated from peptide and/or beta(2)m. We investigated the conformation of HLA-C molecules on normal human trophoblast cells, which invade the maternal decidua during placentation. A panel of monoclonal antibodies to different conformations of HLA-I molecules was used in flow cytometry and surface immunoprecipitation experiments. On the surface of trophoblast cells only beta(2)m-associated complexes of HLA-C molecules were detected. In contrast, both open conformers and beta(2)m-associated HLA-C could be detected on other cells from the decidua, HLA-C-transfectants and cell lines. The levels of HLA-C expressed on primary trophoblast cells could be detected by antibodies specific to non-beta(2)m-associated conformations because binding was seen after acid-induced denaturation of surface proteins. In contrast to HLA-G molecules on trophoblasts, we found no evidence for the presence of disulphide-linked multimers of HLA-C complexes. These results show that most HLA-C molecules present at the trophoblast cell surface are in the conventional beta(2)m-associated conformation. These findings have implications regarding the stability of trophoblast HLA-C molecules and how they interact with receptors on decidual leucocytes during placentation.