Expression of HLA-G in the human placenta

The unique structure of the human placenta provides a separation between maternal and fetal tissues and circulations. Although the maternal–fetal proximity is very close, the placenta and the fetus are not rejected by the maternal immune system. Among the various factors implicated in the mother's tolerance of the fetus, a key factor has been attributed to HLA-G molecules, which are expressed by specialized trophoblast cells. By the alternative splicing of its primary mRNA, HLA-G, belonging to Class I MHC molecules, results in four membrane bound (HLA-G1, -G2, -G3, -G4) and three soluble proteins (HLA-G5, -G6, -G7). Which of these HLA-G isoforms are expressed on the cell surface and which are secreted was the aim of our study.
Whereas the generally agreed upon opinion is that the full length HLA-G1 isoform is expressed by extravillous cytotrophoblast cells, the source of soluble HLA-G5 isoform and the expression of all other isoforms in the human placenta is still under debate. However, soluble HLA-G products, which might be present in body fluids, could also come from membrane bound HLA-G1 by shedding; a process, that has been described for classical HLA molecules. HLA-G expression in organs, other than placenta and certain tumors, has not yet been explained convincingly.
Since immunological detection methods crucially depend on the antibodies and preparation techniques used, we investigated HLA-G specific antibodies for their specificity and binding properties. We compared the binding ability of some antibodies on HLA-G1, G2, -G5 transfected cell lines with naturally expressed placental HLA-G molecules. A broad range of methods, from immunolocalization to protein-biochemical and Elisa techniques, were used to clarify which HLA-G isoform is actually expressed in the normal placenta.     

膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响

目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.     

Expression of Epstein-Barr Virus-Induced Gene 3, an Interleukin-12 p40-Related Molecule, throughout Human Pregnancy : Involvement of Syncytiotrophoblasts and Extravillous Trophoblasts

In human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation.     

HLA-G1 and HLA-G5 isoforms have an additive effect on NK cytolysis

The immunotolerant HLA-G could generate seven isoforms including HLA-G1–-G7. The suppressive function of either HLA-G1 or HLA-G5 isoform to NK cell cytolysis has been well established. Whether HLA-G1 and HLA-G5 isoform have an additive effect on the cytolysis of NK cells remain to be explored. In this study, effects of expression of HLA-G1 and HLA-G5 isoforms and their combination on NK cytolysis was investigated. NK cell cytolysis was analyzed by detecting the NK cell surface CD107a expression. In this study, data showed that the inhibition capacity is dependent on the level of both HLA-G1 and HLA-G5 expression, but the HLA-G5 isoform has a more potent inhibition effect on the NK cytolysis (p < 0.01). Furthermore, HLA-G1 and HLA-G5 have an additive effect on the suppression of NK cell cytolysis. Our study provided further understanding for the roles of HLA-G1 and HLA-G5 isoform expression in target cells immune escaping from NK cells.     

HLA-G mRNA差异表达对细胞膜表面HLA-G异构体分子表达的影响

目的 探讨HLA-G异构体(HLA-G1～6)mRNA差异表达对HLA-G分子在细胞表面表达的影响.方法 通过RT-PCR方法分析卵巢癌细胞株HO-8910、H0-8910PM、OVCAR-3,白血病细胞株Jurkat、K562、HL60、MUTZ-1,绒癌细胞株JEG-3、JAR内HLA-G异构体mRNA的表达种类,采用流式细胞术分析上述细胞株细胞表面及细胞内HLA-G分子的分布及表达水平.结果 阳性对照JEG-3细胞内表达HLA-G1～6 mRNA,阴性对照JAR不表达HLA-G1～6 mRNA.HLA-G1 mRNA在HO-8910、HO-8910PM、OVCAR-3、MUTZ-1、Jurkat细胞内表达.除阳性对照JEG-3外,其他细胞均不表达HLA-G2 mRNA;表达HLA-G3 mRNA的细胞有HO-8910、HO-8910PM、K562、HL60、MUTZ-1、OVCAR-3、Jurkat;表达HLA-G4 mRNA的细胞有HO-8910、HO-8910PM、HL60、Jurkat;表达HLA-G5mRNA的细胞为Jurkat.FACS分析显示在JEG-3、HO-8910PM、Jurkat细胞膜表面表达HLA-G分子,而除JAR细胞外,其他细胞内均表达HLA-G分子.结论 HLA-G2、-G3、-G4、-G5、-G6均不能在细胞表面表达,而HIJA-G1分子则能在特定的细胞表面表达.     

HCMV感染对人THP-1细胞HLA-G及其受体表达的影响

目的:研究人巨细胞病毒(HCMV)感染对人THP-1细胞人类白细胞抗原G(HLA-G)异构体及其受体表达的影响探讨HLA-G在HCMV逃逸宿主免疫应答中的作用。方法:HCMV Towne株感染THP-1细胞后,采用RT-PCR和Western blot检测HLA-G异构体mRNA和蛋白水平,流式细胞术检测THP-1细胞HLA-G及其表面受体ILT2、ILT4的表达,ELISA检测细胞培养上清中IL-10及可溶性HLA-G(sHLA-G)水平,同时检测细胞存活率。结果:HCMV感染后细胞未出现明显凋亡,细胞存活率高。HCMV感染THP-1细胞1 d后HLA-G1、-G3、-G4和-G5的mRNA表达明显上调,HLA-G1和HLA-G5的蛋白表达明显上调。THP-1细胞HLA-G、ILT2和ILT4的表达在感染1 d后明显上调。sHLA-G水平在感染1 d后显著升高,与对照组比较差异有统计学意义(P0.01)。THP-1细胞培养上清液IL-10水平在感染1 d后明显上调,与对照组比较,差异有统计学意义(P0.05)。结论:HCMV感染THP-1细胞能诱导HLA-G异构体的差异表达,以HLA-G1和HLA-G5为主,且上调其表面受体ILT2/ILT4的表达。同时,HCMV感染能诱导THP-1细胞分泌IL-10。该研究为进一步探讨HCMV逃避机体免疫应答的机制提供实验依据。     

sHLA-G1 and HLA-G5 levels are decreased in Tunisian women with multiple abortion

Pregnancy is associated with increased levels of soluble (s) human leukocyte antigen (HLA)-G molecules, while during abortion these molecules are decreased. To date, little is known about the role of sHLA-G isoforms during abortion. In this study, we investigated the levels of total sHLA-G and its isoforms: HLA-G1 (membrane shedded isoform) and alternative spliced HLA-G5 in plasma samples obtained from 55 women who had experienced spontaneous abortion, 108 pregnant healthy women and 56 non pregnant healthy women.We found that pregnant women exhibited higher amounts of sHLA-G compared to either non pregnant women or women with abortion. Among women who had experienced spontaneous abortion, women with recurrent abortions (RSA) had lower sHLA-G than women with only one abortion. In particular, RSA women were characterized by the absence of sHLA-G1 isoform, suggesting a possible implication in abortion event.     

First trimester human endovascular trophoblast cells express both HLA-C and HLA-G.

PROBLEM: In human pregnancies, trophoblasts, in contrast to placental connective tissue and the fetus itself, come into direct contact with the maternal allorecognizing system at special sites. Villous syncytiotrophoblasts washed around by maternal blood lack HLA class I proteins, whereas extravillous trophoblasts, which deeply invade maternal uterine tissues, express high amounts of HLA-G and also HLA-C, the latter to a lesser degree, however. A subpopulation of extravillous trophoblasts, the endovascular trophoblast, enters maternal spiral artery lumen and, like syncytiotrophoblast, comes into direct contact with maternal blood. Less is known about HLA class I distribution on this endovascular trophoblast subpopulation. METHOD OF STUDY: A comparative immununohistochemical analysis was done on decidual cryo-sections containing trophoblast-invaded spiral arteries using different anti-HLA class I monoclonal antibodies (mAbs) and a peroxidase-labeled streptavidinbiotin detection system. RESULTS: MAbs W6/32 (anti-HLA-A, -B, -C, -G), HCA2 (anti-HLA-A, -G) G233 and 87G (both anti-HLA-G) resulted in strong positivity on endovascular trophoblasts. L31 (anti-HLA-C) and HC10 (anti-HLA-B, -C) revealed clear positivity, whereas TU149 (anti-HLA-B, -C, some -A) produced a heterogeneous staining pattern, faintly positive on some endovascular trophoblastic cells and negative on others. MAb LA45 (anti-HLA-A, -B) did not bind to any endovascular trophoblast, neither did BFL.1 (anti-HLA-G) nor 16G1 (anti-HLA-G, soluble). CONCLUSION: This study shows that trophoblastic cells belonging to the endovascular subpopulation express considerable amounts of HLA-G and slightly less HLA-C.     

Exosomes bearing HLA-G are released by melanoma cells

Tumor cells release membrane vesicles, named exosomes, capable of specific cytotoxic T-lymphocyte activation by transferring tumor antigens to dendritic cells. By contrast, the nonclassical human leucocyte antigen (HLA)-G class I molecule displays immunotolerant properties and can be ectopically expressed by tumor cells, thereby allowing their escape from immunosurveillance. We describe here that a melanoma cell line, named Fon, established from an HLA-G-positive melanoma biopsy, spontaneously expressed high levels of the HLA-G1 membrane-bound isoform. Exosomes released by Fon cells were purified and analyzed both for their density on sucrose gradient and their protein composition by Western blotting and flow cytometry. Besides the expression of well-described proteins such as Lamp-2, notably, these melanoma-derived exosomes bore HLA-G1. In addition, exosomes harboring HLA-G1 were secreted by the HLA-G-negative M8 melanoma cells transfected with the HLA-G1 cDNA. Thus, the presence of tolerogenic HLA-G molecules on melanoma-derived exosomes may provide a novel way for tumors to modulate host's immune response.     

HLA-G in reproduction: studies on the maternal-fetal interface

For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.