A specific interferon (IFN)-stimulated response element of the distal HLA-G promoter binds IFN-regulatory factor 1 and mediates enhancement of this nonclassical class I gene by IFN-beta.

Type I interferons display a broad range of immunomodulatory functions. Interferon beta increases gene expression at the transcriptional level through binding of factors to the interferon-stimulated response element (ISRE) within the promoters of interferon-inducible genes, such as HLA class I. Despite mutation of the class I ISRE sequence within the nonclassical HLA-G class I gene promoter, we show that interferon beta enhances both transcription and cell surface expression of HLA-G in trophoblasts and amniotic and thymic epithelial cells that selectively express it in vivo. Deletion and mutagenesis analysis of a putative interferon-regulatory factor (IRF)-1 binding site within the HLA-G promoter show that HLA-G transactivation is mediated through an ISRE sequence 746 base pairs upstream from ATG, which is distinct from the interferon-responsive element described within proximal classical class I gene promoters. Electrophoretic mobility shift analysis and supershift analysis further demonstrate that interferon-responsive transcription factors, including IRF-1, specifically bind to the HLA-G ISRE. Our results provide evidence that IRF-1 binding to a functional ISRE within the HLA-G promoter mediates interferon beta-induced expression of the HLA-G gene. These observations are of general interest considering the implication of HLA-G in mechanisms of immune escape involved in fetal-maternal tolerance and other immune privilege situations.

Modulation of HLA-G expression in human thymic and amniotic epithelial cells.

Expression of the nonclassical HLA class I antigen, HLA-G, is tightly regulated. HLA-G physiologic expression is mostly restricted to some placental and thymic cell types. Only few established cell lines express HLA-G in vitro. Cytokine-induced expression of HLA-G is hardly observed and also depends on the cell lineage. We assessed expression and cytokine regulation of HLA-G in primary cultures derived from human thymus and amnion epithelial cells, which also express HLA-G in vivo. We show that HLA-G cell surface expression is maintained, but decreases gradually, in primary cultures derived from human thymus and amnion epithelial cells. We also show that IFN-gamma re-induces HLA-G cell surface expression and upregulates classical class I gene expression in both primary cultures and in a thymus derived cell line. We further show that IFN-gamma also upregulates levels of HLA-G transcripts in TEC primary cultures. This study provides evidence that IFN-gamma induction of HLA-G expression occurs in the human amnion and the thymus, and is mediated at the transcriptional level in these tissues. These results also suggest a role for the microenvironment in regulating HLA-G in vivo gene expression in the thymus and amnion membrane.

IL-10 selectively induces HLA-G expression in human trophoblasts and monocytes.

HLA-G plays an essential role in feto-maternal tolerance by inhibiting lysis by maternal NK cells. The factors that allow tissue-specific activation of HLA-G gene expression in trophoblasts remain to be characterized. We investigated the potential effect of IL-10, a cytokine which is secreted in placenta, on HLA-G gene transcription in trophoblasts. Using Northern blot, RNase protection assay and RT-PCR analysis, we demonstrated that IL-10 enhances steady-state levels of HLA-G transcription in cultured trophoblast cells. We further tested the effect of IL-10 on HLA-G gene transcription and protein expression in peripheral blood monocytes, showing that IL-10 can up-regulate HLA-G cell surface expression in this cell type. This effect of IL-10 is selective, since classical MHC class I products and MHC class II are down-regulated in monocytes following IL-10 treatment. Induction of HLA-G expression by IL-10 on monocytes may thus play a role in down-regulation of the immune response. We propose that IL-10 secretion by trophoblasts during pregnancy may also influence the HLA class I expression pattern at the feto-maternal barrier, thus protecting the fetus from rejection. This should be taken into consideration in the design of treatment for pathologies of pregnancy.

Specific binding of nuclear factors to the HLA-G gene promoter correlates with a lack of HLA-G transcripts in first trimester human fetal liver.

The nonclassical MHC class I HLA-G antigen is expressed in cytotrophoblasts during pregnancy and may play a role in inhibiting lysis by maternal natural killer cells. HLA-G gene transcription was analyzed in human fetal liver of 6-8 wk of gestation, a development stage where classical HLA class I expression is very reduced. We demonstrated that HLA-G transcription is undetectable in these cells and we investigated the molecular mechanisms that control the lack of HLA-G gene transcription. We compared protein interactions of nuclear extracts from first trimester fetal livers, YT2C2-PR (HLA-G negative) and JEG-3 (HLA-G positive) cell lines to a 244-bp EcoR I/Hind III DNA region located 1.2 kb from the HLA-G gene, previously shown to direct HLA-G expression in transgenic mouse placenta. A strong specific C7-factor was specifically detected in first trimester fetal liver that could account for the inhibition of HLA-G transcription. Interaction of C7-factor and cell-specific factors previously detected in YT2C2 cell line (C5, C6) with two distinct regulatory regions identify this 244-bp EcoR I/Hind III fragment as a putative target for inhibition of HLA-G transcription.

HLA-G gene transcriptional regulation in trophoblasts and blood cells: differential binding of nuclear factors to a regulatory element located 1.1 kb from exon 1.

The HLA-G antigen is specifically expressed on trophoblasts at the maternal-fetal interface, while expression of classical class I HLA-A, -B, -C products is repressed in this tissue. The transcriptional level of the HLA-G gene is high in trophoblast cells and in JEG-3 choriocarcinoma cells, is markedly reduced in blood cells, and is shown here to be undetectable in the YT2C2 NK cell line. In an attempt to understand molecular mechanisms controlling cell-specific transcriptional regulation of the HLA-G gene in these cells, we focused our study on protein interaction with a 244-bp region located over 1.1 kb from exon 1, which has been shown to direct HLA-G expression in transgenic mouse trophoblast. Three specific complexes were detected, two of which are found exclusively in cells showing HLA-G transcriptional activity. The YT2C2 nuclear extracts contain restricted DNA-binding activity of an additional factor which could correlate with repression of HLA-G transcription in these cells.

[Alternative transcripts of the MHC of the non-classical class I HLA-G gene in the in trophoblast during the first pregnancy trimester and in the placenta at term]. / Transcrits différentiels du gène du CMH de classe I non classique HLA-G dans le trophoblaste de 1er trimestre de gestation et le placenta à terme.

HLA-G, a non-classical class I gene, is located within the human major histocompatibility complex locus. It has a tissue-specific expression in trophoblast, where the products of HLA-A, -B and -C classical genes are absent. Therefore, the HLA-G gene may have a role during pregnancy in inducing protection of the semi-allogeneic fetus from recognition and destruction by maternal immune cells. The primary transcript of the HLA-G gene is alternatively spliced into 6 mRNA forms (HLA-G1 to HLA-G6), 2 of them may encode soluble forms of the HLA-G antigen (HLA-G5 and HLA-G6). In this work, by using reverse transcription polymerase chain reaction technique, we demonstrate that all transcripts are detected in similar amounts, both in the first and third trimester of gestation. These results are discussed in context of putative function of HLA-G antigen isoforms.

HLA-G mRNA forms in human trophoblasts and peripheral blood lymphocytes: potential use in prenatal diagnosis.

HLA-G limited polymorphic gene maps to the human major histocompatibility complex (MHC) class I subregion and encodes the molecule which is the only MHC class I antigen expressed on cytotrophoblast cells at the maternal-fetal interface. In this tissue, HLA-G primary mRNA is differentially spliced. We have used a sensitive hot start reverse transcriptase-polymerase chain reaction (RT-PCR) technique to investigate the expression of HLA-G gene in first trimester trophoblasts and adult peripheral blood cells. PCR amplification with HLA-G primers specific of exon 3 has enabled us to demonstrate a novel alternatively spliced form of HLA-G mRNA present in fetal first trimester trophoblasts and lacking exon 4 (HLA-G4). Cloning the whole PCR product and hybridizing recombinant bacterial colonies with specific probes has permitted evaluation of HLA-G4 vs. full length mRNA frequency at approximately 1:200. Moreover, the presence of HLA-G transcripts was found at a very weak level in adult peripheral blood lymphocytes and equally in B- and T-cell populations. These results are relevant in the context of immune tolerance and in the potential use of HLA-G transcripts as a marker for RT-PCR detection of the fetal cells in maternal as a marker for RT-PCR detection of the fetal cells in maternal blood.