Talk:Trophoblast - Protein Expression

Trophoblast Line SGHPL-4

Professor Guy Whitley Position: Professor of cell biology St George's, University of London, Cranmer Terrace, London SW17

trophoblast cell line contacted March 2014
extravillous-like trophoblast cell line, SGHPL-4

<pubmed limit=10>SGHPL-4</pubmed>

Culture

HAMs-F10, Glutamine (2mM), Pen/Strep (1/100)

Media Formulation

PMID 28097431 HAM’s F10 (Biochrom) media containing 10% (v/v) FCS and 1% (v/v) P/S. For expression analysis, starved SGHPL-4 cells were stimulated with 10 ng/ml TNF-α (Sigma-Aldrich).

PMID 19223288 SGHPL-4 cells were cultured in Hams F10 media (Sigma-Aldrich, Dorset, UK) supplemented with 10% (v/v) fetal calf serum (FCS; Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich).

Trophoblast Line TEV-1

Establishment and characterization of a human first-trimester extravillous trophoblast cell line (TEV-1).

J Soc Gynecol Investig. 2005 May;12(4):e21-32.

Feng HC1, Choy MY, Deng W, Wong HL, Lau WM, Cheung AN, Ngan HY, Tsao SW.

Abstract

OBJECTIVE: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. METHODS: Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFbeta1). The responsive gene to TGFbeta1 treatment was confirmed by quantitative real-time PCR. RESULTS: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFbeta1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. CONCLUSION: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies.

PMID 15866109 DOI: 10.1016/j.jsgi.2005.02.008

2017

ABCA1 affects placental function via trophoblast and macrophage

Life Sci. 2017 Dec 15;191:150-156. doi: 10.1016/j.lfs.2017.10.031. Epub 2017 Oct 21.

Chengmao X1, Li L2, Yan L3, Jie Y1, Xiaoju W1, Xiaohui C1, Huimin G1.

Abstract

AIMS: To study the potential impact of ABCA1 on the function of the placenta. MAIN METHODS: Trophoblasts and macrophages were isolated from the placenta with enzymatic digestion; Immunofluorescence assay was used to detect the location of ABCA1 in cells; RT-PCR and Western-blot were used to detect the expression of ABCA1; The cholesterol efflux assays of primary trophoblasts was detected by Amplex Red cholesterol assay kit (Invitrogen);Inflammatory factor secretion from primary macrophages was detected by Elisa. KEY FINDINGS: ABCA1 was mainly located on trophoblast membranes. Decreased ABCA1 expression in trophoblasts reduced the cholesterol efflux of trophoblasts (P<0.01). while increased ABCA1 expression in trophoblasts reduced the cholesterol efflux of trophoblasts (P<0.05). ABCA1 was uniformly expressed on the cell membrane, cytoplasm, and nucleus of macrophages. Decreased ABCA1 expression in macrophages, increased inflammatory factors but reduced IL-10 (P<0.01). While increased ABCA1 expression in macrophages, reduced inflammatory factors but increased IL-10 (P<0.01). SIGNIFICANCE: ABCA1 may be a potential target for the prevention of gestation diseases. Copyright © 2017. Published by Elsevier Inc. KEYWORDS: ABCA1; Anti-inflammatory; Cholesterol; Gestation diseases; Placenta

PMID: 29066252 DOI: 10.1016/j.lfs.2017.10.031

Rab11 family within the human placenta, with novel localization at the maternal-fetal interface. PMID: 28922401 Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-α in a gestational age-dependent manner

Histochem Cell Biol. 2017 Jan 17. doi: 10.1007/s00418-016-1537-1. [Epub ahead of print]

Prokesch A1, Blaschitz A1, Bauer T1, Moser G1, Hiden U2, Zadora J3,4, Dechend R3,4,5, Herse F3,4, Gauster M6.

Abstract

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation. KEYWORDS: Autophagy; Inflammation; Placenta; Preeclampsia; Pregnancy; Trophoblast PMID 28097431

Natural cytotoxicity receptor 1 in mouse uNK cell maturation and function

Mucosal Immunol. 2017 Jan 18. doi: 10.1038/mi.2016.126. [Epub ahead of print]

Felker AM1, Croy BA1.

Abstract

Early and midgestational decidua of mice genetically ablated for expression of the natural killer (NK) cell natural cytotoxicity receptor (NCR; Ncr1Gfp/Gfp mice) shows restricted angiogenesis and atypically small uterine (u)NK cells . We hypothesized that NCR1 inactivation disturbs maturation and angiokine production by uterine natural killer (uNK) cells. Using histological and morphometric approaches, we observed that Ncr1Gfp/Gfp but not control C57BL/6 (B6) implantation sites sustain immature, non-granulated uNK cells into midpregnancy. Mouse uNK cells can be subclassified by their reactivity with Dolichos biflorus agglutinin (DBA) lectin; DBA+ uNK cells with greater Ncr1 expression were investigated. DBA+ uNK cells from Ncr1Gfp/Gfp mice show delayed maturation as indicated by shorter diameters and fewer cytoplasmic granules. Granules in mature Ncr1Gfp/Gfp uNK cells are ultrastructurally abnormal and abundance of granule-associated proteins (perforin, granzyme) and of cytoplasmic proteins (vascular endothelial growth factor; placental growth factor) differs from controls. Leukocyte-leukocyte conjugate formation in gestation day 6.5 and 8.5 intact Ncr1Gfp/Gfp decidua was less frequent than in B6; however, this difference involved leukocytes other than DBA+ uNK cells. These studies strongly support roles for NCR1 and its ligands in normal pregnancy promotion.Mucosal Immunology advance online publication, 18 January 2017; doi:10.1038/mi.2016.126. PMID 28098245 DOI: 10.1038/mi.2016.126

2016

Macrophage polarisation affects their regulation of trophoblast behaviour

Placenta. 2016 Nov;47:73-80. doi: 10.1016/j.placenta.2016.09.004. Epub 2016 Sep 9.

Buckley RJ1, Whitley GS1, Dumitriu IE1, Cartwright JE2.

Abstract

INTRODUCTION: During the first trimester of human pregnancy, fetally-derived extravillous trophoblast (EVT) invade into the uterine decidua and remodel the uterine spiral arteries to ensure that sufficient blood reaches the maternal-fetal interface. Decidual macrophages have been implicated in the regulation of decidual remodelling, and aberrant activation of these immune cells is associated with pre-eclampsia. METHODS: The monocytic cell line THP-1 was activated to induce a classically- or alternatively-activated macrophage phenotype and the conditioned media was used to treat the EVT cell line SGHPL-4 in order to determine the effect of macrophage polarisation on trophoblast behaviour in-vitro. SGHPL-4 cell functions were assessed using time-lapse microscopy, endothelial-like tube formation assays, and western blot. RESULTS: The polarisation state of the THP-1 cells was found to differentially alter the behaviour of trophoblast cells in-vitro with pro-inflammatory classically-activated macrophage conditioned media significantly inhibiting trophoblast motility, impeding trophoblast tube formation, and inducing trophoblast expression of cleaved caspase 3, when compared to anti-inflammatory alternatively-activated macrophage conditioned media. DISCUSSION: Macrophages can regulate trophoblast functions that are critical during decidual remodelling in early pregnancy. Importantly, there is differential regulation of trophoblast function in response to the polarisation state of these cells. Our studies indicate that the balance between a pro- and anti-inflammatory environment is important in regulating the cellular interactions at the maternal-fetal interface and that disturbances in this balance likely contribute to pregnancy disorders associated with poor trophoblast invasion and vessel remodelling. Copyright © 2016 Elsevier Ltd. All rights reserved. KEYWORDS: Decidua; Extravillous trophoblast; Macrophage; Polarisation; THP-1

PMID 27780542 DOI: 10.1016/j.placenta.2016.09.004

2015

Inhibition of DDAH1, but not DDAH2, results in apoptosis of a human trophoblast cell line in response to TRAIL

Hum Reprod. 2015 Aug;30(8):1813-9. doi: 10.1093/humrep/dev138. Epub 2015 Jun 16.

Lumicisi BA1, Cartwright JE1, Leslie K1, Wallace AE1, Whitley GS2.

Abstract

STUDY QUESTION: Does inhibition of dimethylarginine dimethylaminohydrolase (DDAH) increase the sensitivity of trophoblasts to TRAIL-induced apoptosis? SUMMARY ANSWER: Inhibition of DDAH1, but not DDAH2, increases the sensitivity of trophoblasts to TRAIL-induced apoptosis. WHAT IS KNOWN ALREADY: Successful human pregnancy is dependent on adequate trophoblast invasion and remodelling of the maternal spiral arteries. Increased trophoblast apoptosis is seen in pregnancies complicated by pre-eclampsia. The mechanism underlying this increase is unknown. We have previously shown that nitric oxide (NO) is involved in regulating trophoblast motility and invasion, and have also demonstrated an important role for NO in regulating trophoblast sensitivity to apoptotic stimuli. DDAH is an enzyme that metabolizes asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, previously shown to be elevated in the plasma of pre-eclamptic mothers. STUDY DESIGN, SIZE, DURATION: This study used the human extravillous trophoblast-derived cell line SGHPL-4 cells. All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effect of DDAH on trophoblast apoptosis was examined using siRNA and time-lapse microscopy. Changes in the expression of DDAH were followed by PCR and western blot analysis. Receptor expression was followed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Inhibiting the expression of DDAH1, but not DDAH2, resulted in a significant increase in the sensitivity of the EVT cell line SGHPL-4 to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis (P < 0.01). This response could be mimicked by the addition of Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis and the substrate for both isoforms of DDAH. We further showed that this increased sensitivity to apoptosis is accompanied by a significant increase in the expression of TRAIL receptor 2 (TR2; P < 0.05) but not TRAIL receptor 1 (TR1). LIMITATIONS, REASONS FOR CAUTION: This study was performed only in vitro using a well characterized trophoblast cell line, SGHPL-4, derived from first trimester extravillous trophoblasts. WIDER IMPLICATIONS OF THE FINDINGS: This study provides new insight into the role of the DDAH/ADMA pathway in the regulation of trophoblast function. Both dysregulation of DDAH and the accumulation of ADMA have been associated with the development of pre-eclampsia. This is the first study to implicate the DDAH/ADMA pathway as a mechanism that might underlie the poor trophoblast invasion seen in this common pregnancy disorder. STUDY FUNDING/COMPETING INTERESTS: B.A.L. was supported by a grant from Action Medical Research UK (SP4577). A.E.W. was supported by a grant from the Wellcome Trust (091550). There are no competing interests and the authors have no conflict interest to declare. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. KEYWORDS: ADMA; DDAH; TRAIL; apoptosis; extravillous trophoblast PMID 26082478

Human Cytomegalovirus Modulates Expression of Noncanonical Wnt Receptor ROR2 To Alter Trophoblast Migration

J Virol. 2015 Nov 11;90(2):1108-15. doi: 10.1128/JVI.02588-15.

van Zuylen WJ1, Ford CE2, Wong DD1, Rawlinson WD3.

Abstract

Maternal primary cytomegalovirus (CMV) infection, reactivation, or reinfection with a different viral strain may cause fetal injury and adverse pregnancy outcomes. Increasing evidence indicates that fetal injury results not only from direct viral cytopathic damage to the CMV-infected fetus but also from indirect effects through placental infection and dysfunction. CMV alters Wingless (Wnt) signaling, an essential cellular pathway involved in placentation, as evidenced by reduced transcription of canonical Wnt target genes and decreased Wnt3a-induced trophoblast migration. Whether CMV affects the noncanonical Wnt signaling pathway has been unclear. This study demonstrates for the first time that CMV infection inhibits Wnt5a-stimulated migration of human SGHPL-4 trophoblasts and that inhibition of the pathway restores normal migration of CMV-infected cells. Western blot and real-time PCR analyses show increased expression of noncanonical Wnt receptor ROR2 in CMV-infected trophoblasts. Mimicking the CMV-induced ROR2 protein expression via ectopic expression inhibited Wnt5a-induced trophoblast migration and reduced T cell-specific factor (TCF)/lymphoid enhancer-binding factor (LEF)-mediated transcription as measured using luciferase reporter assays. Gene silencing using small interfering RNA (siRNA) duplexes decreased ROR2 transcript and protein levels. In contrast, proliferation of SGHPL-4 trophoblasts, measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was not affected. The siRNA-mediated downregulation of ROR2 in trophoblasts rescued CMV-induced reduction in trophoblast migration. These data suggest a mechanism where CMV alters the expression of the Wnt receptor ROR2 to alter Wnt5a-mediated signaling and inhibit trophoblast motility. Inhibition of this mechanism may be a target for therapeutic intervention for CMV-induced placental damage and consequent fetal damage in congenital CMV infections. IMPORTANCE: Maternal primary cytomegalovirus (CMV) infection, reactivation, or reinfection with a different viral strain may cause fetal injury and adverse pregnancy outcomes. Increasing evidence indicates that fetal injury results not only from direct viral cytopathic damage to the CMV-infected fetus but also from indirect effects through placental infection and placental dysfunction. No effective therapy is currently proven to prevent or treat congenital CMV infection. Understanding the molecular underpinnings of CMV infection of the placenta is essential for therapeutic innovations and vaccine design. CMV alters canonical Wingless (Wnt) signaling, an essential cellular pathway involved in placental development. This study suggests a mechanism in which CMV alters the expression of noncanonical Wnt receptor ROR2 to alter motility of placental cells, which has important implications in the pathogenesis of CMV-induced placental dysfunction. Inhibition of this mechanism may be a target for therapeutic intervention for CMV-induced placental damage and consequent fetal damage in congenital CMV infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved. PMID 26559837

The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142442

2014

Oxygen modulates human decidual natural killer cell surface receptor expression and interactions with trophoblasts

Biol Reprod. 2014 Dec;91(6):134. doi: 10.1095/biolreprod.114.121566. Epub 2014 Sep 17.

Wallace AE1, Goulwara SS1, Whitley GS1, Cartwright JE2.

Abstract

Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P < 0.05) and formed endothelial-like networks to a greater extent (P < 0.05) than SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P < 0.05), and an increased percentage of dNK cells expressed NKG2D at 10% oxygen (P < 0.05) compared to other oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy. © 2014 by the Society for the Study of Reproduction, Inc. KEYWORDS: NKG2D; decidual natural killer cell; oxygen; trophoblast PMID 25232021

Expression and Potential Roles of HLA-G in Human Spermatogenesis and Early Embryonic Development

PLoS One. 2014 Mar 25;9(3):e92889. doi: 10.1371/journal.pone.0092889. eCollection 2014.

Yao GD1, Shu YM2, Shi SL1, Peng ZF1, Song WY1, Jin HX1, Sun YP1. Author information

Abstract

As one of the non-classical major histocompatibility complex(MHC)-1 antigens, Human Leukocyte Antigen G (HLA-G), has been suggested as a prognostic marker to identify the embryo developmental potential. In the present study, we investigated the potential roles of HLA-G in human spermatogenesis and early embryonic development. Quantitative real-time PCR analysis revealed that HLA-G's expression was increased with increased Johnsen score in testicular tissues. There was no significant difference in HLA-G mRNA expression between testicular tissues with Johnsen score of 8-9 and normal sperm from ejaculated semen. HLA-G mRNA expression was detected in human zygotes, embryos and blastocysts but not in unfertilized oocytes. In testicular tissues where sperm was obtained by testicular sperm extraction (Johnsen score was 8 to 9), there were no correlations between HLA-G mRNA expression and fertilization, cleavage and high-quality embryo rates. At 48-72 h post-fertilization, HLA-G expression was higher in fast growing embryos. HLA-G specific siRNA injection into zygotes not only slowed down embryonic cleavage rate at 48 h post-fertilization, but also down-regulated the expression of embryo metabolism related gene (SLC2A1) and cell cycle-regulated gene (CCND2). Taken together, our findings suggested that HLA-G plays significant roles in human spermatogenesis and early embryonic development.

PMID 24667226

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0092889

Mst1 and mst2 are essential regulators of trophoblast differentiation and placenta morphogenesis

PLoS One. 2014 Mar 4;9(3):e90701. doi: 10.1371/journal.pone.0090701. eCollection 2014.

Du X1, Dong Y1, Shi H1, Li J1, Kong S1, Shi D1, Sun LV1, Xu T2, Deng K1, Tao W1. Author information

Abstract

The placenta is essential for survival and growth of the fetus because it promotes the delivery of nutrients and oxygen from the maternal circulation as well as fetal waste disposal. Mst1 and Mst2 (Mst1/2), key components of the mammalian hpo/Mst signaling pathway, encode two highly conserved Ser/Thr kinases and play important roles in the prevention of tumorigenesis and autoimmunity, control of T cell development and trafficking, and embryonic development. However, their functions in placental development are not fully understood, and the underlying cellular and molecular mechanisms remain elusive. Here, we investigated the functions of Mst1/2 in mouse placental development using both conventional and conditional (endothelial) Mst1/2 double knockout mice. We found that the number of trophoblast giant cells dramatically increased while spongiotrophoblast cells almost completely disappeared in Mst1/2 deficient placentas. We showed that Mst1/2 deficiency down regulated the expression of Mash2, which is required for suppressing the differentiation of trophoblast giant cells. Furthermore, we demonstrated that endothelial-specific deletion of Mst1/2 led to impaired placental labyrinthine vasculature and embryonic lethality at E11.5, but neither affected vasculature in yolk sac and embryo proper nor endocardium development. Collectively, our findings suggest that Mst1/2 regulate placental development by control of trophoblast cell differentiation and labyrinthine vasculature at midgestation and Mst1/2 control labyrinth morphogenesis in trophoblast- and fetal endothelial-dependent manners. Thus, our studies have defined novel roles of Mst1/2 in mouse placental development.

PMID 24595170

2013

Molecular pathways: human leukocyte antigen G (HLA-G)

Clin Cancer Res. 2013 Oct 15;19(20):5564-71. doi: 10.1158/1078-0432.CCR-12-3697. Epub 2013 Jul 29.

Curigliano G1, Criscitiello C, Gelao L, Goldhirsch A. Author information

Abstract Human leukocyte antigen G (HLA-G) is a nonclassical MHC class I molecule that exerts important tolerogenic functions. Its main physiologic expression occurs in the placenta, where it participates in the maternal tolerance toward the fetus. HLA-G expression was found in embryonic tissues, in adult immune privileged organs, and in cells of the hematopoietic lineage. It is expressed in various types of primary solid (melanoma, head and neck, lung, urogenital, gastrointestinal, and breast cancers) and hematologic malignancies (acute leukemia, lymphomas) and metastases. HLA-G ectopic expression is observed in cancer, suggesting that its expression is one strategy used by tumor cells to escape immune surveillance. In this review, we will focus on HLA-G expression in cancers and its association with the prognosis. We will highlight the underlying molecular mechanisms of impaired HLA-G expression, the immune tolerant function of HLA-G in tumors, and the potential diagnostic use of membrane-bound and soluble HLA-G as a biomarker to identify tumors and to monitor disease stage. As HLA-G is a potent immunoinhibitory molecule, its blockade remains an attractive therapeutic strategy against cancer. Elimination of HLA-G-expressing cancer cells would be important in the efficacy of anticancer therapies. ©2013 AACR.

PMID 23897901

2012

MiRNA-mediated control of HLA-G expression and function

PLoS One. 2012;7(3):e33395. doi: 10.1371/journal.pone.0033395. Epub 2012 Mar 16.

Manaster I1, Goldman-Wohl D, Greenfield C, Nachmani D, Tsukerman P, Hamani Y, Yagel S, Mandelboim O. Author information

Abstract

HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT) of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3'UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3'UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G.

PMID 22438923

HLA-G 2012 conference: the 15-year milestone update

Tissue Antigens. 2013 Mar;81(3):127-36. doi: 10.1111/tan.12053. Epub 2013 Jan 24.

Loustau M1, Wiendl H, Ferrone S, Carosella ED. Author information

Abstract The non-classical human leukocyte antigen (HLA) Class I molecule HLA-G is best known for its tolerogenic function at the maternal-fetal interface, where it protects the fetus from destruction by the immune system of its mother. Yet, HLA-G has been the topic of intense investigations and its functions reach much further than originally believed. International conferences on HLA-G have taken place every 3 years since 1998, and the Sixth International Conference on HLA-G, that took place in Paris in July 2012. It counted 180 attendees from 28 countries, 35 speakers in plenary sessions, and 63 presentations of research in symposia and poster sessions, bringing new insight in HLA-G research. Here we summarize the major advances on the function and nature of HLA-G molecule that were reported, with particular interest on the findings in new mechanisms of action through regulatory cells, its relevance in cancer as well as in the molecular structure and functions of HLA-G, which are key for its clinical application. © 2013 John Wiley & Sons A/S.

PMID 23347068

Multimeric structures of HLA-G isoforms function through differential binding to LILRB receptors

Cell Mol Life Sci. 2012 Jul 17. [Epub ahead of print]

Howangyin KY1, Loustau M, Wu J, Alegre E, Daouya M, Caumartin J, Sousa S, Horuzsko A, Carosella ED, Lemaoult J. Author information

Abstract The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α(1)-α(2)-α(3) non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α(1)-α(3) structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α(1)-α(3)-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.

PMID 22802125

Figure 1 HLA-G isoforms - Alternative splicing of HLA-G primary transcript yields 7 isoforms. Excision of one or two exons encoding globular domain generates truncated isoforms, and translation of intron 4 or intron 2 yield secreted isoforms that lack the transmembrane domain.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884069/figure/F1

"Taken together, these data clearly show that HLA-G isoforms present important structural variations which may correspond to different biological functions. As mentioned before, HLA-G1 and HLA-G5 (α1–α2–α3 associated to B2M) are the most extensively studied isoforms, probably because of their alleged higher abundance, their availability for in vitro experiments, and the existence of specific antibodies directed against them. Thus, these isoforms are believed to be the main isoforms responsible immune regulation in vivo. However, we do not know to which extent this is true. Isoforms and B2M-free HLA-G structures may also play a specific role. Not knowing the precise structure of HLA-G truncated isoforms implies that we do not know which receptors they bind to, either. For instance, in this work, we studied the HLA-G2 or -G6 interaction with LILRB2 because this isoform contains the α3 domain and could potentially bind to LILRB2. However, we do not know what the HLA-G3 receptor could be, or that of HLA-G4, despite our data indicating that this isoform may bind LILRB2. A recent work [47] describes binding of B2M-associated and B2M-free forms of MHC to members of the LILR family, and demonstrates that in addition to LILRB2, B2M-free forms are recognized by several members of the LILR family. In fact, “activating” members of the LILR family show a preference for these forms. It is therefore possible that it is also the case for HLA-G. This would support the notion that HLA-G structural variations may be relevant in biological function modulations. It is also intriguing to consider that, similarly to classical HLA Class I molecules, HLAG has activating receptors. Indeed, it is possible that other HLA-G receptors exist, isoform-specific or not, and studying HLA-G structures other than that of B2M-associated HLA-G1 and -G5 might allow us to identify them. Finally, our data highlight the fact that even though we do know the physiological importance of HLA-G, we do not know which of its structures are relevant in vivo."

2006

HLA-G in human reproduction: aspects of genetics, function and pregnancy complications

Hum Reprod Update. 2006 May-Jun;12(3):209-32. Epub 2005 Nov 9.

Hviid TV. Author information

Abstract

The non-classical human leukocyte antigen (HLA) class Ib genes, HLA-E, -G and -F, are located on chromosome 6 in the human major histocompatibility complex (MHC). HLA class Ib antigens resemble the HLA class Ia antigens in many ways, but several major differences have been described. This review will, in particular, discuss HLA-G and its role in human reproduction and in the human MHC. HLA-G seems to be important in the modulation of the maternal immune system during pregnancy and thereby the maternal acceptance of the semiallogenic fetus. Recent findings regarding aspects of HLA-G polymorphism, the possible significance of this polymorphism in respect to HLA-G function and certain complications of pregnancy (such as pre-eclampsia and recurrent spontaneous abortions (RSA)) are discussed together with possible importance to IVF. Finally, aspects of a possible role of HLA-G in organ transplantation and in inflammatory or autoimmune disease, and of HLA-G in an evolutionary context, are also briefly examined.

PMID 16280356

The non-classical Human Leukocyte Antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions.

trophoblast cells, which originate from the fetus, do not express classical HLA class Ia and II antigens, except for a possible weak expression of HLA-C

Major histocompatibility complex (MHC)

short arm of chromosome 6, ∼4 Mb and encodes at least ∼130 functional genes.
classical HLA class Ia and II genes (HLA-A, -B, -C, -DR, -DQ and -DP)
non-classical HLA class Ib genes
HLA class Ib antigens, HLA-E, -F and -G

fetus inherits one HLA haplotype from the mother and one from the father and is thereby semiallogenic for the mother.

HLA-G

gene is located on chromosome 6 close to HLA-A
inhibit cytotoxic T-lymphocyte (CTL) response
inhibit natural killer (NK) functions
in both cases by direct interaction with the receptors ILT2 and ILT4 and with the killer Ig-like receptor 2 DL4 (KIR2DL4 receptor)

short cytoplasmic tail important for reduced spontaneous endocytosis of HLA-G
seven HLA-G mRNA and protein isoforms are generated by alternative splicing of mRNA.
- four membrane-bound isoforms (HLA-G1 to -G4)
- three soluble isoforms (HLA-G5, -G6, -G7)
HLA-G mRNA has been detected in many different tissues, HLA-G protein expression has been described repeatedly only on and by the trophoblast cells in placenta on and by certain immune cells (in most cases monocytes) and in the thymus

Figure 2. human leukocyte antigen-G (HLA-G) gene and expression. http://humupd.oxfordjournals.org/content/12/3/209/F2.large.jpg

Figure 4. Expression of human leukocyte antigen (HLA) molecules during pregnancy http://humupd.oxfordjournals.org/content/12/3/209/F4.large.jpg

Crystal structure of HLA-G: a nonclassical MHC class I molecule expressed at the feral-maternal interface

Proc Natl Acad Sci U S A. 2005 Mar 1;102(9):3360-5. Epub 2005 Feb 17.

Clements CS1, Kjer-Nielsen L, Kostenko L, Hoare HL, Dunstone MA, Moses E, Freed K, Brooks AG, Rossjohn J, McCluskey J. Author information

Abstract HLA-G is a nonclassical major histocompatibility complex class I (MHC-I) molecule that is primarily expressed at the fetal-maternal interface, where it is thought to play a role in protecting the fetus from the maternal immune response. HLA-G binds a limited repertoire of peptides and interacts with the inhibitory leukocyte Ig-like receptors LIR-1 and LIR-2 and possibly with certain natural killer cell receptors. To gain further insights into HLA-G function, we determined the 1.9-A structure of a monomeric HLA-G complexed to a natural endogenous peptide ligand from histone H2A (RIIPRHLQL). An extensive network of contacts between the peptide and the antigen-binding cleft reveal a constrained mode of binding reminiscent of the nonclassical HLA-E molecule, thereby providing a structural basis for the limited peptide repertoire of HLA-G. The alpha3 domain of HLA-G, a candidate binding site for the LIR-1 and -2 inhibitory receptors, is structurally distinct from the alpha3 domains of classical MHC-I molecules, providing a rationale for the observed affinity differences for these ligands. The structural data suggest a head-to-tail mode of dimerization, mediated by an intermolecular disulfide bond, that is consistent with the observation of HLA-G dimers on the cell surface.

PMID 15718280

http://www.pnas.org/content/102/9/3360.long

2003

CXCL12 expression by invasive trophoblasts induces the specific migration of CD16- human natural killer cells

Blood. 2003 Sep 1;102(5):1569-77. Epub 2003 May 1.

Hanna J1, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, Katz G, Haimov-Kochman R, Fujii N, Yagel S, Peled A, Mandelboim O. Author information

Abstract

In the maternal decidua, natural killer (NK) cells, characterized by lack of CD16, are found in direct contact with the fetal extravillous trophoblasts (EVTs). It is yet unknown which factors contribute to the specific homing of this unique NK subset to the decidua. In this study we analyze the chemokine receptor repertoire on various NK populations derived from the peripheral blood and decidua. We show that CXCR4 and CXCR3 receptors are preferentially expressed on CD16- NK subsets derived either from the peripheral blood or the decidua and that these receptors are involved in migration of all NK subsets to their ligands. We further demonstrate in vivo that invading EVTs that eventually perform endovascular invasion express CXCL12, the ligand for CXCR4, but not ligands for CXCR3. Indeed, specific accumulation of the CD16- NK cells at the expense of CD16+ cells was observed only when in vitro migration was performed with ligands for CXCR4. Finally, incubation of the peripheral blood CD16- NK cells with cytokines present in the decidua, especially interleukin 15 (IL-15), resulted in the expression of chemokine receptor repertoire similar to that observed on decidual NK cells, suggesting an additional important regulatory effect of local decidual cytokines.

PMID 12730110