Lydia Farrell

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.

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.

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.

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.

Maternal KIR in Combination with Paternal HLA-C2 Regulate Human Birth Weight

Human birth weight is subject to stabilizing selection; babies born too small or too large are less likely to survive. Particular combinations of maternal/fetal immune system genes are associated with pregnancies where the babies are ≤5th birth weight centile, specifically an inhibitory maternal KIR AA genotype with a paternally derived fetal HLA-C2 ligand. We have now analyzed maternal KIR and fetal HLA-C combinations at the opposite end of the birth weight spectrum. Mother/baby pairs (n = 1316) were genotyped for maternal KIR as well as fetal and maternal HLA-C. Presence of a maternal-activating KIR2DS1 gene was associated with increased birth weight in linear or logistic regression analyses of all pregnancies >5th centile (p = 0.005, n = 1316). Effect of KIR2DS1 was most significant in pregnancies where its ligand, HLA-C2, was paternally but not maternally inherited by a fetus (p = 0.005, odds ratio = 2.65). Thus, maternal KIR are more frequently inhibitory with small babies but activating with big babies. At both extremes of birth weight, the KIR associations occur when their HLA-C2 ligand is paternally inherited by a fetus. We conclude that the two polymorphic immune gene systems, KIR and HLA-C, contribute to successful reproduction by maintaining birth weight between two extremes with a clear role for paternal HLA.

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.

Ex vivo functional responses to HLA-G differ between blood and decidual NK cells

Restricted expression of human leucocyte antigen-G (HLA-G) to fetal extravillous trophoblast cells, which invade the decidua during implantation, suggests a role for HLA-G in placentation. In this study, we have investigated several aspects of HLA-G expression and function. Surface levels of HLA-G expression were measured in 70 normal pregnancies. We show the dimeric conformation that is unique to HLA-G forms after passage through the Golgi apparatus. Differences were found in the receptor repertoire of decidual natural killer (dNK) cells that express the leucocyte immunoglobulin-like receptor B1 (LILRB1), which binds dimeric HLA-G strongly. We then measured functional responses of dNK cells with LILRB1, when stimulated by HLA-G in both monomeric and dimeric conformations. Degranulation, interferon-γ and interleukin-8 production by dNK cells freshly isolated from the first trimester implantation site were either undetected or not affected by HLA-G. These findings should be considered when inferring the activity of tissue NK cells from results obtained with cell lines, peripheral NK or cultured dNK cells.

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.

Distribution of killer cell immunoglobulin-like receptors (KIR) and their HLA-C ligands in two Iranian populations

Killer cell immunoglobulin-like receptors (KIR) gene frequencies vary between populations and contribute to functional variation in immune responses to viruses, autoimmunity and reproductive success. This study describes the frequency distribution of 12 variable KIR genes and their HLA-C ligands in two Iranian populations who have lived for many generations in different environments: the Azerbaijanis at high altitude and the Jonobi people at sea level. The results are compared with those published for other human populations and a large group of English Caucasians. Differences were seen in KIR and HLA-C group frequencies, in linkage disequilibrium and inhibitory/activating KIR ratios between the groups. Similarities with geographically close populations in the frequencies of the KIR A and B haplotypes and KIR AA genotype reflected their common ancestry. The extreme variability of the KIR gene family and their HLA-C ligands is highlighted and their importance in defining differences between geographically and culturally isolated communities subject to different environmental pressures who come from the same ethnic grouping.Killer cell immunoglobulin-like receptors (KIR) gene frequencies vary between populations and contribute to functional variation in immune responses to viruses, autoimmunity and reproductive success. This study describes the frequency distribution of 12 variable KIR genes and their HLA-C ligands in two Iranian populations who have lived for many generations in different environments: the Azerbaijanis at high altitude and the Jonobi people at sea level. The results are compared with those published for other human populations and a large group of English Caucasians. Differences were seen in KIR and HLA-C group frequencies, in linkage disequilibrium and inhibitory/activating KIR ratios between the groups. Similarities with geographically close populations in the frequencies of the KIR A and B haplotypes and KIR AA genotype reflected their common ancestry. The extreme variability of the KIR gene family and their HLA-C ligands is highlighted and their importance in defining differences between geographically and culturally isolated communities subject to different environmental pressures who come from the same ethnic grouping.