HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy

OBJECTIVE: The aim of this study was to assess the use of circulating trophoblast cells in maternal peripheral blood for noninvasive prenatal diagnosis of numeric chromosomal aberrations. STUDY DESIGN: A combined procedure for immunocytochemical identification and deoxyribonucleic acid fluorescence in situ hybridization was used after a single enrichment step consisting of density gradient centrifugation. A specific HLA-G monoclonal antibody was used in combination with X and Y chromosome specific probes in deoxyribonucleic acid fluorescence in situ hybridization to confirm fetal identity of cells bearing HLA-G in the case of a male fetus. RESULTS: We detected fetal trophoblast cells expressing HLA-G in maternal blood starting at 9 weeks' gestation. In addition to fetal sex prediction with X and Y chromosome-specific probes, fetal aneuploidy was confirmed in peripheral blood from a pregnancy complicated by trisomy 21. CONCLUSION: Although the numbers of fetal cells were extremely low, the proof of concept was demonstrated. Early noninvasive prenatal screening for numeric chromosomal abnormalities with fetal trophoblast cells is feasible.     

Immune interactions at the maternal-fetal interface

Models of murine allogeneic pregnancy have established that maternal T cells recognize fetal alloantigens and are normally suppressed or deleted. While the precise cellular interactions and mechanisms involved in maternal lymphocyte tolerance are not yet clear, the identity of some of the critical factors are beginning to be uncovered. Signals that have been shown in mice to have an obligatory role in immunological survival of the semiallogeneic fetus include, but are probably not limited to, indoleamine-2,3-dioxygenase and the newly discovered B7 family protein, B7-H1. Whether these proteins have intersecting functions is unknown, but it is possible that both are involved in the control of maternal T regulatory cells, which are also strictly required for successful allogeneic pregnancy in mice. Additional factors that are involved include trophoblast and/or maternally derived FasL, and in humans, class Ib HLA molecules. The potency of these mechanisms in protecting the fetal allograft is underscored by the scarcity of knockout and transgenic models in which pregnancy is immunologically compromised. Here, the current understanding of mechanisms of specific suppression of maternal lymphocytes is reviewed.     

HLA-G inhibits the allogeneic proliferative response.

HLA-G is a non-classical MHC class I molecule expressed at the feto/maternal interface where it plays a role in materno-fetal tolerance by inhibiting NK cells. Expression of killing inhibitory receptors capable of interacting with HLA-G on T lymphocytes led us to hypothesize that HLA-G molecules could also modulate T cell responses, analyzed here in the context of the allogeneic proliferative response. Using LCL-HLA-G transfectants as stimulators of T cells present among peripheral mononuclear cells and K562-HLA-G1 transfectants as inhibitors in a classical mixed lymphocyte reaction, we showed that HLA-G is able to inhibit T cell allo-proliferation. These findings provide new insight into the role of HLA-G in preventing allograft rejection.     

Antibodies to trophoblast antigens HLA-G, placenta growth factor, and neuroD2 do not improve detection of circulating trophoblast cells in maternal blood

OBJECTIVES: Non-invasive prenatal diagnosis using circulating fetal trophoblast cells has been challenging due to lack of a reproducible trophoblast-specific antibody. We investigated the use of three trophoblast cell-specific antibodies, HLA-G, placenta growth factor, and neuroD2, for the isolation of trophoblast cells from the maternal circulation. METHODS: Trophoblast cells were isolated by density centrifugation from maternal blood samples (gestational age 10-20 weeks, n = 9). All women were carrying a male fetus. Following immunocytochemical staining with the trophoblast-specific antibodies, fluorescent in situ hybridization was performed, to verify whether any stained cells were indeed fetal. RESULTS: The HLA-G antibody had a ubiquitous staining pattern, which was not specific for trophoblast cells. Neither the placenta growth factor nor the neuroD2 antibodies were able to identify any trophoblast cells. Following fluorescent in situ hybridization, no male cells were detected on any of the slides. CONCLUSION: The antibodies used in this study were unable to improve detection of trophoblast cells in the maternal circulation.     

Cell surface detection of HLA-E gene products with a specific monoclonal antibody.

An HLA-E-specific monoclonal antibody (mAb) was obtained by immunization of human beta2-microglobulin transgenic mice (M-TGM) with spleen cells from double transgenic mice expressing HLA-E molecules (EM-TGM). This mAb, designated V16, specifically recognizes in flow cytometry analysis the HLA-E expressing mouse cells, whereas it does not bind to mouse cells expressing various HLA class I molecules (HLA-A2, -A3, -A11, -A26, A29, -B7, -B27, -Cw3, -Cw7, and HLA-G). V16 mAb binds efficiently to human EBV-infected B lymphocytes, PHA blasts and PBL, thus establishing the surface expression of HLA-E in vivo on these cells.     

How Does the Maternal Immune System Contribute to the Development of Pre-eclampsia?

An immunological aura has hovered over the study of pre-eclampsia for many years but there has still been little progress in explaining the various ‘immune’ phenomena associated with this elusive disease. When considering the primary defect of placentation that leads to pre-eclampsia the focus should be on the intermingling of the invasive placental trophoblast cells with maternal leukocytes in the uterine wall. The MHC status of trophoblast cells is a crucial factor to be considered, as these molecules can act as ligands for uterine immune cells, including T cells, NK cells and myelomonocytic cells. Extravillous trophoblast cells express an unusual combination of HLA-C, HLA-G and HLA-E molecules and only one of these HLA molecules, HLA-C, shows any appreciable polymorphism. In humans, uNK cells express an array of receptors, some of which are known to bind to the HLA class I molecules expressed by extravillous trophoblast cells. HLA-C is the dominant ligand for killer immunoglobulin-like receptors (KIR) expressed by uterine NK cells that may deliver an inhibitory or activating signal. KIR haplotypes comprise two groups, A and B; these differ principally by having additional activating receptors in the B haplotype. In any pregnancy, the maternal KIR genotype could be AA (no activating KIR) or AB/BB (presence of between one and five activating KIRs). The HLA-C ligands for KIR on trophoblast cells may belong to two groups, C1 and C2 that are defined by a dimorphism at position 80 of the α1 domain. This maternal–fetal immunological interaction, occurring at the site of placentation, therefore involves two polymorphic gene systems, maternal KIRs and fetal HLA-C molecules. Uterine NK-cell function is thus likely to vary in each pregnancy. In pre-eclamptic pregnancies we have found that some KIR/HLA-C combinations appear unfavourable to trophoblast-cell invasion due to the overall signals that the NK cell receives. The academic excitement of this work is the realisation that this is a novel form of allorecognition based on NK cells that operates entirely differently from self/non-self discrimination used by T cells.     

Differential down-modulation of HLA-G and HLA-A2 or -A3 cell surface expression following human cytomegalovirus infection

During pregnancy, the non-classical major histocompatibility complex (MHC) class I HLA-G molecule is specifically expressed in trophoblast cells at the materno-fetal interface and may exert a local control of the immune response against viral infections. Human cytomegalovirus (HCMV) infection, which is the major cause of congenital defects, encodes multiple glycoproteins (US2, US3, US6, US10 and US11) that interrupt the MHC class I pathway of antigen presentation. The effect of some of these unique short (US) proteins on HLA-G expression has been previously studied, but little is known about the modulation of HLA-G cell surface expression during the course of HCMV infection which ensures expression of all of these US proteins. Using flow cytometry analysis, HLA-G cell surface expression was evaluated in HCMV-infected U373-HLA-G transfectant cells and compared with the modulation of the endogenous classical HLA-A2 molecules. The results indicated that HCMV infection down-modulated HLA-G cell surface expression, but later after infection and to a lesser extent than HLA-A2. Using various HLA-G/HLA-A2 chimeras, we showed that the unique structure of HLA-G cytoplasmic tail was partly involved in the resistance of HLA-G to viral down-modulation. Such limited down-modulation of HLA-G may have functional consequences in term of innate immunity against congenital HCMV infection.     

HLA-G suppresses proliferation of CD4(+) T-lymphocytes

HLA-G is a non-classical MHC class 1 molecule, expressed primarily on human foetal trophoblast cells, which exhibits almost no genetic polymorphism. Because of these unusual features, HLA-G has been suggested to help prevent maternal immune attack of the semi-allogeneic foetus. The aim of these experiments was to investigate the effects of HLA-G on T-lymphocyte responses by using MHC class II-bearing HLA-G transfectants as stimulators of a mixed lymphocyte reaction. The presence of HLA-G, but not classical HLA class I, on the surface of stimulator cells markedly suppressed thymidine incorporation by peripheral blood mononuclear responder cells from a class I-similar, class II-dissimilar male. The suppressive effect of HLA-G on the mixed lymphocyte reaction persisted after depletion of phagocytes and CD8(+) T-cells from the responder population, but the mixed lymphocyte reaction was entirely abolished by depletion of CD4(+) T-cells. These results suggest that HLA-G exerts a direct suppressive effect on CD4(+) T-lymphocytes, even in the absence of the CD8(+) cells with which other human MHC class I molecules are thought to interact. Thus, HLA-G may allow the foetus to escape maternal immune attack by modulating CD4(+) T-cell activity.     

Less HLA-G expression in Plasmodium falciparum-infected third trimester placentas is associated with more natural killer cells

During pregnancy, maternal immune tolerance of the fetal semi-allogeneic graft is partly the consequence of extravillous trophoblast HLA-G expression and its interaction with natural killer (NK) cells. Plasmodium falciparum malaria is frequently associated with maternal and fetal complications. Local HLA-G expression and the number of NK cells were evaluated immunohistochemically in P. falciparum-infected and uninfected placentas (15 each) collected in a seasonal malaria-hypoendemic area. In control placentas, HLA-G was almost always expressed in extravillous trophoblast whereas, in infected placentas, it was significantly more weakly expressed in extravillous trophoblast but was also detected in intervillous space macrophages. NK cells were evaluated in intervillous and intravillous spaces and in basal plate. NK cells were always more abundant in basal plate than in intervillous and intravillous spaces in infected or control placentas. For each area, more NK cells were seen in infected than control placentas. These data suggest that HLA-G down-regulation and more NK cells in placentas may be among the mechanisms involved in poor birth outcome associated with P. falciparum infection.     

HLA-G,pre-eclampsia,immunity and vascular events

Pre-eclampsia, one of the main complications in pregnancy, is characterised by shallow cytotrophoblast invasion of decidua as well as by vascular endothelial cell dysfunction, leading to a poor perfusion of placenta. A striking feature of pre-eclamptic pregnancies is that expression of HLA-G protein is reduced in term placentas compared with normal pregnancy. How such HLA-G deficient expression may be related to the pre-eclamptic pathology is unknown. Here, we review the major structural characteristics of HLA-G and some of its functions that have been recently characterised. Soluble HLA-G1 isoform down-regulates both CD8(+) and CD4(+) T cell reactivity. HLA-G also modulates innate immunity by binding to several NK and/or decidual receptors, inducing particular cytokine secretion. HLA-G was shown to be less susceptible to human cytomegalovirus-derived US protein down-modulation. Finally, soluble HLA-G1 down-regulates endothelial cell proliferation and migration. In view of these different HLA-G properties, we will briefly discuss how defective HLA-G function may contribute to the low trophoblast invasion and vascular abnormalities observed in pre-eclamptic placentas.     

Inconsistency of fetal trophoblast cells in first trimester maternal peripheral blood prevents non-invasive fetal testing using this cell target

We have investigated whether maternal peripheral blood from the first trimester of pregnancy is a reliable source of identifiable trophoblast cells. The cells were enriched from 30 ml of venous blood, with multiple antibodies shown previously to enrich trophoblasts and a new cocktail based on known trophoblast surface features. Three different magnetic solid phases were tested to enrich trophoblasts, and both positive and negative cell enrichment strategies were examined. The cells were identified as trophoblast by morphology coupled with immunocytochemistry to co-localize cytokeratin with one of three IGF-II, PAI-1 or hPLH proteins or by in-situ hybridization with a mixture of 50 oligos directed to eight different expressed genes, alpha-HCG, IGF-II, PAI-1, HASH2, hPLH, p57(KIP2), PP5, H-19. While these tools worked beautifully in chorionic villi cell/sprout preparations and tissue sections, we could not detect and identify any trophoblasts in maternal peripheral blood even if the maternal peripheral blood was drawn 5-20 min following termination of pregnancy or from individuals maintaining the pregnancy.Based on our own experience and that of some reports in the literature, trophoblasts do not appear to be a viable candidate for fetal screening using maternal peripheral blood as the source. It is important to note that while trophoblast deportation is a biological phenomenon that has been described repeatable, they do not provide a means to perform prenatal genetic diagnosis.     

The short forms of HLA-G are unlikely to play a role in pregnancy because they are not expressed at the cell surface

HLA-G is a nonclassical class I MHC molecule of unknown function expressed on human invasive trophoblast. In trophoblast cells, HLA-G mRNA is alternatively spliced into a variety of forms which are predicted to encode a full length membrane-bound form, three short membrane-bound isoforms and two soluble isoforms. The aim of this study was to determine which of these protein isoforms are translated, which are expressed on the cell surface and which are secreted. Artificial cDNAs encoding the isoforms were generated by PCR mutagenesis, ligated to an epitope tag and transfected into a human cell line capable of expressing MHC class I. Protein products of appropriate sizes were detected in cells transfected with cDNAs encoding all membrane-bound forms, but surface biotinylation studies indicated that only full length membrane-bound HLA-G was present at the cell surface. Full length HLA-G was also detected by surface antibody binding and flow cytometry. Soluble HLA-G1 was detected in cells transfected with the appropriate cDNA only after treatment with monensin, which inhibits transport of glycoproteins through the Golgi apparatus. These results suggest that full length HLA-G, but not short HLA-G isoforms can be expressed on the surface of human cells and that soluble HLA-G is rapidly secreted. Thus, it is likely that the full length membrane-bound and soluble forms of HLA-G are the only biologically active forms to which the mother is exposed.     

人类白细胞抗原G与子痫前期的相关性

人类白细胞抗原G（HLA—G）为非经典的HLA—I类分子,特异性高表达于母胎界面的绒毛外滋养细胞．通过诱导免疫耐受对妊娠和胚胎起到保护作用。子痫前期是产科领域危害母婴健康的主要疾病之一．子痫前期患者血清及胎盘组织HLA—G表达下降,但具体作用机制仍不清楚。随着基础研究的深入,明确了HLA—G基因、蛋白质的结构及遗传方式,在基因多态性方面做了大量研究工作,力求从基因水平阐明其与子痫前期的关系。发现部分子痫前期患者可能存在HLA—G基因缺陷,且HLA—G多态性在子痫前期及正常妊娠者中分布也不同。     

Murine ectoplacental cone-derived trophoblast cells express chemokine receptors

Chemokine receptors (CCRs) have been shown to regulate T cell migration and differentiation as well as the establishment of Th1/Th2 bias. Furthermore, T cells and T cell products are essential to trophoblast development. Thus, postulating that chemokines as well as their receptors may be expressed by trophoblast to move T cells into an interaction with the feto-placental unit, we examined whether CCRs are expressed during the early stages of ectoplacental cone (EPC) formation. For this, murine EPC-derived trophoblast were examined for their ability to express CCRs constitutively or inducible by interferon-γ (IFN-γ). Immunofluorescence experiments on EPC-derived trophoblast cells showed that CCR3, CXCR4 and CCR5 are significantly expressed. IFN-γ accelerated the mobilization of intracellular pools of CCR molecules during early cell culture periods (2–6 h) and, in most cases, increased their expression on EPC-derived trophoblast cells. CCR activity could be detected in the culture supernatants of these cells, inversely proportional to cell surface expression, suggesting the existence of rapid endocytosis and recycling mechanisms. This finding indicates that the level of intracellular CCRs may partly be determined in the extracellular matrix, an event that could play an important role towards neutralization of specific T cell/trophoblast interactions during early stages of pregnancy and protect the fetus against harmful maternal immune responses.     

Immunoregulation in normal pregnancy and pre-eclampsia: an overview

Pre-eclampsia is a major disorder of human pregnancy, which may have an immunological basis. It is a disease of two stages. The first stage concerns the relative failure of early trophoblast invasion and remodelling of the spiral arteries, leading to a poor blood supply to the placenta, exposing it to oxidative stress. The inadequate trophoblast invasion may result from decreased expression of human leukocyte antigen-G (HLA-G) leading to an abnormal interaction with decidual natural killer (NK) cells, which are believed to play a major role in these processes through the production of immunoregulatory cytokines and angiogenic factors. Recent evidence suggests that the interaction between trophoblast human leukocyte antigen-C (HLA-C) molecules and decidual NK cell receptors may be the point at which the apparent partner specificity of the disease originates. The second stage is the maternal syndrome, which is characterized by a generalized systemic inflammatory response involving both leukocytes and endothelium. The inflammatory stimulus is believed to come from the placenta. In pre-eclampsia, placental oxidative stress may lead to increased shedding of apoptotic and/or necrotic syncytiotrophoblast debris into the maternal circulation. There is evidence that such trophoblast debris interacts with maternal leukocytes and endothelial cells to stimulate the release of proinflammatory cytokines, which could then trigger the maternal disease.