Lucy Gardner

Immunocytochemical characterization of the unusual large granular lymphocytes in human endometrium throughout the menstrual cycle.

Leukocyte populations were studied at all stages of the menstrual cycle using a panel of monoclonal antibodies to natural killer cells, T cells, B cells, and macrophages on frozen sections of endometrium. At the time of implantation (mid-secretory phase) the majority of leukocytes appear to be Leu19+, CD16-, Leu7-, CD2+, CD3-, CD5-, CD7-, and HLA DR- with the morphology of large granular lymphocytes. The numbers of these cells showed a marked increase in the mid-secretory phase. These cells exhibited similar phenotypic characteristics to those found in decidua. These findings, therefore, suggest that the recruitment of these large granular lymphocytes to the uterus is under hormonal control and is not a local response to the presence of invading trophoblast.

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.

BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages

Summary BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

Dendritic Cells in the Human Decidua

Abstract Dendritic cells (DCs) in the pregnant human uterine mucosa have been poorly characterized, although they are likely to regulate immune responses to both placental trophoblast cells and uterine infections. In this study an HLA-DR+, CD11c+ lin− (CD3−, CD19−, CD56−, CD14−) population has been identified by three-color flow cytometry. The cell isolates were prepared either by collagenase digestion or mechanically from first-trimester decidual tissue. The decidual DCs comprised ∼1.7% of CD45+ cells in the isolates and had the phenotype of immature myeloid DCs. No CD1a+ Langerhans cells or CD123+ plasmacytoid DCs were detected. The decidual DCs were DC-SIGN−, DEC-205+, CD40+. Two subsets could be distinguished on the basis of relative expression of HLA-DR, which also differed in expression of DC-activation markers. The DCs were identified in situ by immunohistology by DEC-205 staining. Cells with dendritic processes were found scattered through both the decidua basalis (in which trophoblast cells are infiltrating) and the decidua parietalis. They were also visible in endothelial-lined spaces. This is the first study to identify and describe the phenotype and distribution of human decidual DCs.

A critical look at HLA-G

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class-I molecule that was found to be expressed by placental trophoblast cells 20 years ago. Because trophoblast forms the physical interface between fetus and mother, HLA-G might play a role in maternal immunological accommodation of the semi-allogeneic fetus. Despite a mounting number of publications, there is still no consensus on many aspects of HLA-G, including its tissue distribution and receptor binding. Here we critically review the literature and suggest why some of this controversy has arisen and how it might be resolved. We do conclude that the evidence for trophoblast HLA-G stimulating leukocyte immunoglobulin-like receptor B1 receptors on decidual leukocytes is compelling. These findings suggest how a fetal molecule might influence the local maternal immune response.

Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies

Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class‐I (HLA‐I) loci has made it difficult to generate locus‐specific monoclonal antibodies (mAbs). The problem of defining an antibody’s reactivity against the thousands of existing HLA‐I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA‐I to characterize experimentally the reactivity of nine mAb against 96 common HLA‐I allotypes. In conjunction with donor HLA‐I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG‐3 and JAR; and the placental cell lines HTR‐8/SVneo, Swan‐71 and TEV‐1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA‐C, HLA‐G and HLA‐E, but not HLA‐A, HLA‐B or HLA‐DR molecules in normal pregnancy. Tumour‐derived JEG‐3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.

A homodimeric complex of HLA-G on normal trophoblast cells modulates antigen-presenting cells via LILRB1.

In healthy individuals, the non‐classical MHC molecule HLA‐G is only expressed on fetal trophoblast cells that invade the decidua during placentation. We show that a significant proportion of HLA‐G at the surface of normal human trophoblast cells is present as a disulphide‐linked homodimer of the conventional β2m‐associated HLA‐I complex. HLA‐G is a ligand for leukocyte immunoglobulin‐like receptors (LILR), which bind much more efficiently to dimeric HLA‐G than to conventional HLA‐I molecules. We find that a LILRB1‐Fc fusion protein preferentially binds the dimeric form of HLA‐G on trophoblast cells. We detect LILRB1 expression on decidual myelomonocytic cells; therefore, trophoblast HLA‐G may modulate the function of these cells. Co‐culture with HLA‐G+ cells does not inhibit monocyte‐derived dendritic cell up‐regulation of HLA‐DR and costimulatory molecules on maturation, but did increase production of IL‐6 and IL‐10. Furthermore, proliferation of allogeneic lymphocytes was inhibited by HLA‐G binding to LILRB1/2 on responding antigen‐presenting cells (APC). As HLA‐G is the only HLA‐I molecule that forms β2m‐associated dimers with increased avidity for LILRB1, this interaction could represent a placental‐specific signal to decidual APC. We suggest that the placenta is modulating maternal immune responses locally in the uterus through HLA‐G, a trophoblast‐specific, monomorphic signal present in almost every pregnancy.

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.

Paternal MHC expression on mouse trophoblast affects uterine vascularization and fetal growth

The mammalian fetus represents a semiallograft within the maternal uterus yet is not rejected. This situation is particularly pronounced in species with a hemochorial type of placentation, such as humans and rodents, where maternal tissues and blood are in direct contact with fetal trophoblast and thus potentially with paternal antigens. The main polymorphic antigens responsible for graft rejection are MHC antigens. In humans the trophoblast cells invading into the decidua have a unique pattern of MHC class I expression characterized by both classical (HLA-C) and nonclassical (HLA-G and HLA-E) molecules. Whether such an unusual MHC repertoire on the surface of trophoblast is a conserved feature between species with hemochorial placentation has not been resolved. Here we demonstrate, using a range of methods, that C57BL/6 mouse trophoblast predominantly expresses only one MHC class I antigen, H2-K, at the cell surface of giant cells but lacks expression of nonclassical MHC molecules. Antigenic disparity between parental MHCs affects trophoblast-induced transformation of the uterine vasculature and, consequently, placental and fetal gowth. Maternal uterine blood vessels were more dilated, allowing for increased blood supply, in certain combinations of maternal and paternal MHC haplotypes, and these allogeneic fetuses and placentas were heavier at term compared with syngeneic controls. Thus, maternal–fetal immune interactions are instrumental to optimize reproductive success. This cross-talk has important implications for human disorders of pregnancy, such as preeclampsia and fetal growth restriction.

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.