Susan E. Hiby

Combinations of Maternal KIR and Fetal HLA-C Genes Influence the Risk of Preeclampsia and Reproductive Success

Preeclampsia is a serious complication of pregnancy in which the fetus receives an inadequate supply of blood due to failure of trophoblast invasion. There is evidence that the condition has an immunological basis. The only known polymorphic histocompatibility antigens on the fetal trophoblast are HLA-C molecules. We tested the idea that recognition of these molecules by killer immunoglobulin receptors (KIRs) on maternal decidual NK cells is a key factor in the development of preeclampsia. Striking differences were observed when these polymorphic ligand: receptor pairs were considered in combination. Mothers lacking most or all activating KIR (AA genotype) when the fetus possessed HLA-C belonging to the HLA-C2 group were at a greatly increased risk of preeclampsia. This was true even if the mother herself also had HLA-C2, indicating that neither nonself nor missing-self discrimination was operative. Thus, this interaction between maternal KIR and trophoblast appears not to have an immune function, but instead plays a physiological role related to placental development. Different human populations have a reciprocal relationship between AA frequency and HLA-C2 frequency, suggesting selection against this combination. In light of our findings, reproductive success may have been a factor in the evolution and maintenance of human HLA-C and KIR polymorphisms.

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.

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.

Human Decidual Natural Killer Cells Express the Receptor for and Respond to the Cytokine Interleukin 15

Abstract The natural killer (NK) cells that are present in the uterine mucosa (decidua) during early pregnancy have a distinctive phenotype, CD56bright CD16−. These cells have previously been shown to proliferate and be activated by interleukin (IL)-2. However, IL-2 is absent from the decidua and placenta, and we have therefore investigated whether IL-15 is present in the uterus and can act on decidual NK cells. Both IL-15 mRNA and protein were found in a variety of cells but particularly in decidual macrophages. IL-15 induced a proliferative response in decidual NK cells that was blocked by anti-IL-15 and was augmented by stem cell factor. The cytolytic activity of decidual NK cells against K562 was augmented. Interestingly, in contrast to IL-2, although activation with IL-15 resulted in some killing of JEG-3 choriocarcinoma cells, normal trophoblast cells remained resistant to lysis. These findings suggest that IL-15 is a candidate cytokine responsible for NK cell proliferation in vivo in the progesterone-dominated secretory endometrium and early decidua.

Association of maternal killer-cell immunoglobulin-like receptors and parental HLA-C genotypes with recurrent miscarriage

BACKGROUND The natural killer (NK) cells at the site of placentation express killer-cell immunoglobulin-like receptors (KIR) that can bind to human leukocyte antigen (HLA)-C molecules on trophoblast cells. Both these gene systems are polymorphic and an association of particular maternal KIR/fetal HLA-C genotypes has been shown in pre-eclampsia. Pre-eclampsia and recurrent miscarriage (RM) share the pathogenesis of defective placentation and therefore we have now genotyped couples with RM. METHODS AND RESULTS DNA was obtained from the male (n = 67) and female (n = 95) partners of couples with three or more spontaneous miscarriages and genotyped for HLA-C groups and 11 KIR genes using the PCR-sequence-specific primer method (SSP). The frequency of the HLA-C2 group was increased in both parents (reaching significance only in the male partners, P = 0.018) compared with a parous control population. The KIR gene frequencies of the male partners were similar to controls, but the women had a high frequency of KIR AA haplotypes that lack activating KIR. In particular, the activating KIR for HLA-C2 groups (KIR2DS1) was significantly lower in these women (P = 0.00035, odds ratio 2.63, confidence interval 1.54-4.49). CONCLUSIONS This is the first report to identify a genetic male factor that confers risk in RM. These findings support the idea that successful placentation depends on the correct balance of NK cell inhibition and activation in response to trophoblast.

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.

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.

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.

Human uterine NK cells have a similar repertoire of killer inhibitory and activatory receptors to those found in blood, as demonstrated by RT-PCR and sequencing.

The expression of natural killer (NK) cell receptors specific for HLA class I molecules has been studied in CD56bright, CD3- NK cells isolated from the pregnant uterine mucosa, the decidua. RT-PCR was performed on cDNA from uterine NK cells with primers designed to amplify members of the killer inhibitory receptor (KIR)/killer activatory receptor (KAR) gene family. Sequencing of the PCR products revealed that uterine NK cells express KIR/KAR which have two or three extracellular immunoglobulin superfamily (Ig-SF) domains. NK receptors for both groups of HLA-C alleles were found. KIR, characterised by a long cytoplasmic tail containing the immune receptor tyrosine-based inhibitory motif (ITIM), and KAR, characterised by a short cytoplasmic domain with a transmembrane region containing a charged lysine, were both identified. Different individuals appear to have a distinct but overlapping repertoire of KIR/KAR. No new members of this NK receptor gene family were identified in the uterine CD56bright NK cells. Similar findings were obtained from non-pregnant endometrial tissues representative of different stages of the menstrual cycle. Immunohistology confirmed that the KIR protein products were expressed by decidual NK cells. These results reveal that NK receptors for trophoblast HLA class I molecules are present in maternal uterine NK cells. Fetal trophoblast cells infiltrating the decidua express HLA-G and HLA-C gene products. This suggests that maternal recognition of the fetus may be mediated by an NK allorecognition system.

A KIR B centromeric region present in Africans but not Europeans protects pregnant women from pre-eclampsia.

Significance Pre-eclampsia is especially common in women of African ancestry and a major cause of maternal death. The killer-cell immunoglobulin-like receptor (KIR) genes that we analyzed are expressed by natural killer cells—immune cells that populate the uterus and are essential for successful pregnancy. KIR proteins bind HLA ligands on the implanting placental trophoblast cells. African and European women share similar risk associations for pre-eclampsia, but protection is associated with different KIR genes. African women are protected by a combination of KIR B haplotype genes that is present almost exclusively in Africans. This study emphasizes the importance of studying diseases in Africans, where the KIR/HLA genetic system is at its most diverse and maternal mortality rates are the highest in the world. In sub-Saharan Africans, maternal mortality is unacceptably high, with >400 deaths per 100,000 births compared with <10 deaths per 100,000 births in Europeans. One-third of the deaths are caused by pre-eclampsia, a syndrome arising from defective placentation. Controlling placentation are maternal natural killer (NK) cells that use killer-cell immunoglobulin-like receptor (KIR) to recognize the fetal HLA-C molecules on invading trophoblast. We analyzed genetic polymorphisms of maternal KIR and fetal HLA-C in 484 normal and 254 pre-eclamptic pregnancies at Mulago Hospital, Kampala, Uganda. The combination of maternal KIR AA genotypes and fetal HLA-C alleles encoding the C2 epitope associates with pre-eclampsia [P = 0.0318, odds ratio (OR) = 1.49]. The KIR genes associated with protection are located in centromeric KIR B regions that are unique to sub-Saharan African populations and contain the KIR2DS5 and KIR2DL1 genes (P = 0.0095, OR = 0.59). By contrast, telomeric KIR B genes protect Europeans against pre-eclampsia. Thus, different KIR B regions protect sub-Saharan Africans and Europeans from pre-eclampsia, whereas in both populations, the KIR AA genotype is a risk factor for the syndrome. These results emphasize the importance of undertaking genetic studies of pregnancy disorders in African populations with the potential to provide biological insights not available from studies restricted to European populations.