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HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8 + T cell cytotoxic functions. Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8 + T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition. This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2 + CD8 + T cell and ILT2 + NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The HLA-G molecule plays a crucial role in the protection of the fetus against aggression by the mother's immune system. Recently, it was shown that HLA-G was involved in the protection of the transplanted tissues, via the inhibition of all immune effectors that mediate graft rejection. The inhibitory functions of HLA-G were studied in vitro using allo- and xeno-geneic models, ex vivo on transplanted tissues biopsies, and in an in vivo animal model. In this review, we will summarize recent results which show that HLA-G acts as a regulator of immune function, seems to be directly involved in transplant acceptation, and should be taken into consideration when monitoring transplanted patients' status.

The Human Leucocyte Antigen-G (HLA-G) is a non-classical MHC class I molecule of low polymorphism, restricted tissue distribution and tolerogeneic functions. It is clearly demonstrated that HLA-G contributes to fetal graft tolerance by the maternal immune system. The tolerogeneic properties of HLA-G act via specific inhibitory receptors present on immunocompetents cells: HLA-G inhibits natural killer cells (NK) and CD8+ T cell cytotoxicity, suppresses CD4+ T cell proliferation in response to allogeneic stimulation and promotes T helper 2 (Th2) type responses. The soluble HLA-G protein is spontaneously secreted by allo-sensitized CD4+ T cells during mixed lymphocyte reactions (MLR), and inhibits their proliferative response. Finally, inhibition of dendritic cell maturation has been observed in HLA-G transgenic mice. In human organ transplantation, our group has reported in cardiac and liver-kidney transplanted patients, a positive correlation between the de novo ectopic expression of HLA-G in both patient's serum and graft biopsies, and a lower rate of acute rejection episodes of the grafts. Moreover no chronic graft rejection has been detected in those populations. These results support the involvement of HLA-G in regulatory mechanisms that may occur during human allotransplantation.

We recently reported that the nonclassical HLA class I molecule HLA-G was expressed in the endomyocardial biopsies and sera of 16% of heart transplant patients studied. The aim of the present report is to identify cells that may be responsible for HLA-G protein expression during the allogeneic reaction. Carrying out mixed lymphocyte cultures in which the responder cell population was depleted either in CD4(+) or CD8(+) T cells, we found that soluble HLA-G5 protein but not the membrane-bound HLA-G isoform was secreted by allo-specific CD4(+) T cells from the responder population, which suppressed the allogeneic proliferative T cell response. This inhibition may be reversed by adding the anti-HLA-G 87G antibody to a mixed lymphocyte culture. That may indicate a previously uncharacterized regulatory mechanism of CD4(+) T cell proliferative response.

Recent developments in the field of HLA-G research have revealed that, besides its involvement during pregnancy, HLA-G is expressed in peripheral tissues during pathological processes, such as viral infections, malignancies and organ transplantation. Here, we discuss recent findings regarding the influence of HLA-G on the T helper (Th) cytokine balance (favoring Th2-type responses), and the expression of HLA-G during chronic, cutaneous inflammatory diseases, such as psoriasis and atopic dermatitis. We propose a novel role for HLA-G as a tissue-protective molecule in inflammatory responses.

HLA-G is a nonclassical class I antigen mainly expressed at the maternofetal interface during pregnancy where it is thought to down-modulate maternal immune response against the semiallogeneic fetus. Recent studies indicate that ectopic up-regulation of HLA-G expression on melanoma cells may also favor their escape from antitumor immune response. HLA-G expression was here investigated on paraffin-embedded tumor and adjacent normal renal tissues of 18 renal cell carcinoma (RCC) patients. We provide evidence that HLA-G antigen is differentially expressed in carcinoma and normal renal cells and that up-regulation of this antigen in the tumor cells is more frequent than alterations of other MHC class I or class II antigens. We also demonstrated that HLA-G cell surface expression and secretion is maintained in a tumor cell line (DM) established from an HLA-G-positive RCC lesion. Furthermore, we show that type I (alpha and beta) and, in particular, type II (gamma) IFN treatment enhances steady-state mRNA levels and cell surface expression of HLA-G in the DM cell line. As several studies suggest that HLA-G displays various functional features that allow down-modulation of immune response in vitro, we propose that selective in vivo expression of HLA-G may participate in the impairment of antitumor immunity in RCC.

Recent data have suggested that in psoriasis, the T-infiltrating cells could be submitted to regulatory pathways, possibly through natural killer receptors. HLA-G binds to different natural killer receptors and is able to inhibit T-cell functions. Because this molecule is induced by interferon-gamma, a major cytokine in psoriasis, we asked whether HLA-G and its receptor might be expressed in this disease. Specific RNAs for HLA-G1 and HLA-G5 were consistently found in lesional skin specimens, soluble HLA-G5 transcripts being found only in psoriasis. HLA-G protein was found in all psoriatic sections, but never in normal skin controls. Double labeling demonstrated that HLA-G-positive cells were CD68(+), CD11c(+) macrophages. The NKR ILT2 was also present in psoriatic skin, the T CD4(+)-infiltrating cells expressing indeed ILT2. The demonstration of HLA-G and ILT2 expression in psoriatic skin suggests that this pathway may act as an inhibitory feed back aimed to down-regulate the deleterious effects of T-cell infiltrate in this disease.

HLA-G is a nonclassical MHC class I molecule that plays a major role in maternal-fetal tolerance. Four membrane-bound (HLA-G1 to -G4) and two soluble (HLA-G5, and -G6) proteins are generated by alternative splicing. Only HLA-G1 has been extensively studied in terms of both expression and function. We provide evidence here that HLA-G2, -G3, and -G4 truncated isoforms reach the cell surface of transfected cells, as endoglycosidase H-sensitive glycoproteins, after a 2-h chase period. Moreover, cytotoxicity experiments show that these transfected cells are protected from the lytic activity of both innate (NK cells) and acquired (CTL) effectors. These findings highlight the immunomodulatory role that HLA-G2, -G3, and -G4 proteins will assume during physiologic or pathologic processes in which HLA-G1 expression is altered.

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.

Preeclampsia (PE) is a multisystem disorder of human pregnancy, occurring in 5%-10% of all population births and represents the leading cause of both fetal and maternal morbidity and mortality in pregnancy. Although the disorder only becomes clinically apparent late in pregnancy, the underlying pathology indicates that invasion of fetal trophoblasts into maternal spiral arteries during early pregnancy is shallow or absent in PE. A large number of epidemiologic studies have been carried out and they demonstrate that the disorder is highly heritable and occurs with a high incidence in all populations. Studies have shown that PE is largely under genetic control, but the mode of its inheritance remains unclear. Genetic studies have been carried out using both large scale linkage analysis and candidate gene approaches; however, the genetic mechanisms underlying the disorder have yet to be determined. We focus on the potential role of HLA-G, a nonclassical class I HLA located on chromosome 6, which appears to be a key component in trophoblast invasion. We examine the hypothesis that HLA-G may have a key role in both genetic susceptibility to, and pathogenesis of, PE.

The CDK10/PISSLRE gene has been shown to encode two different CDK-like putative kinases. The function(s) of the gene products are unknown, although a role at the G2/M transition has been suggested. We characterised two novel cDNAs. CDK10 mRNA quantity was not found to be correlated with cell proliferation status in HeLa or WI38 cell cultures or in human tissues. Relative levels of the four CDK10 isoforms were studied by RT-PCR, of which three were principally expressed. The two initially cloned isoforms predominated in human tissues, except in brain and muscle. Relative isoform levels did not vary during the cell cycle in culture, except when cells entered into the cell cycle. Finally, the predominant isoforms were shown to have different translation initiation sites and to have different subcellular distribution, due to an alternatively spliced nuclear localisation signal., (Copyright 2000 Academic Press.)

HLA-G is a non-classical major histocompatibility complex class I molecule selectively expressed on cytotrophoblasts. We have demonstrated ex vivo (from voluntary pregnancy interruption samples) the protector role of the HLA-G molecule present on the surface of cytotrophoblast cells versus the lysis carried out by the decidual uterine NK cells. This occurs under semi-allogenic conditions (maternal uterine NK cells and their trophoblast counterparts), as well as in allogenic conditions (maternal uterine NK cells and trophoblast cells from different mothers), thus defining the absence of maternal rejection at the moment of the implantation of the fertilized egg during pregnancy. Moreover, the expression of HLA-G on the cytotrophoblasts permits migration in maternal circulation and infiltration of maternal tissue (particularly in the skin), thereby probably creating a general state of tolerance. In the context of heart transplantation, in preliminary studies, we show that the presence of HLA-G in cardiac biopsy tissue prelevated from grafted patients significantly reduces acute rejects and shows an absence of chronic rejects. In the tumour context, the expression of HLA-G protein at the surface of primitive melanoma and metastatic cells confers protection from NK and CTL lytic activity. This suggests that HLA-G expression may impede the elimination of malignant cells by anti-tumour immune effector cells, constituting a newly described mechanism by which tumour cells may evade immunosurveillance. From there on E.D. Carosella introduced the breakthrough concept of 'HLA a tolerance molecule' in the heart of histocompatibility antigens, which had been described up till then as antigenes of defence and rejection, and the dramatic role of HLA-G in immunotolerance.

HLA-G is a nonclassical major histocompatibility complex class I molecule selectively expressed on cytotrophoblasts at the fetal-maternal interface, where it plays a role in maternofetal tolerance. In this review, attempts were made to summarize the current state of knowledge of the effects of HLA-G on both natural killer cell and T cell functions and their implications in transplantation.

The nonclassical histocompatibility class I gene HLA-G has a tissue-restricted expression. To explore mechanisms involved in HLA-G transcriptional regulation, we have investigated the effect of stress, including heat shock and arsenite treatment, on HLA-G expression in tumor cell lines. We show that stress induces an increase of the level of the different HLA-G alternative transcripts without affecting other MHC class I HLA-A, -B, -E, and -F transcripts. A heat shock element (HSE) that binds to heat shock factor 1 (HSF1) on stress conditions was further identified within the HLA-G promoter. Considering the ability of HLA-G to modulate the function of immunocompetent cells, we hypothesize a new feature of HLA-G as a signal regulating the immune response to stress.

HLA-G is a non-classical major histocompatibility complex class I molecule that differs from the classical HLA-A, -B and -C molecules by (i) alternative splicing of mRNAs encoding for at least four membrane-bound and two soluble HLA-G isoforms, (ii) a limited polymorphism, and (iii) a tissue-restricted distribution. Studies over the past few years have elucidated the function of HLA-G demonstrating inhibition of both NK cell- and T cell-mediated cytolysis. Furthermore, aside from its expression during pregnancy, we have shown that HLA-G is also expressed in solid tumor cells (i.e. human melanoma cell lines and ex vivo melanoma biopsies). Here we present a review of the current state of knowledge of the immunotolerant functions of HLA-G and their implications in materno-fetal tolerance and tumor immunosurveillance.

HLA-G molecule is thought to play a major role in down-regulating the maternal immune response by inhibiting NK and T cell cytolytic activities. We examined the molecular regulatory mechanisms that may control the restricted expression pattern of the HLA-G gene. We first analyzed protein interactions between nuclear extracts from the HLA-G-positive JEG-3 choriocarcinoma and the HLA-G-negative NK-like YT2C2 cell lines to a 244 bp regulatory element located 1.2 kb from the HLA-G gene, previously shown to direct HLA-G expression in transgenic mouse placenta. This allowed characterization of cell-specific DNA-protein interactions that could account for differential cell-specific expression of the HLA-G gene. In particular two DNA-protein complexes were exclusively observed in YT2C2, suggesting that this HLA-G regulatory element is a target for putative cell-specific repressor factors. We further mapped nuclear factor binding sites to a 70 bp fragment in the upstream region of the regulatory element. We then investigated the effect of IFN-gamma on HLA-G gene expression. HLA-G cell surface expression was enhanced by IFN-gamma treatment in JEG-3 and U937 cell lines and peripheral blood monocytes while no effect was observed in tera-2 teratocarcinoma cell line. HLA-G transcriptional activity was increased only in JEG-3 and U937 cell lines. Activity of the 1.4-kb HLA-G promoter region was unchanged after IFN-gamma treatment in JEG-3 and Tera-2. These results suggest that both post-transcriptional and transcriptional mechanisms implicating IFN-responsive regulatory sequences outside the 1.4 kb-region are involved in IFN-gamma gene activation of the HLA-G gene.

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.

Expression of the non-classical HLA-G class I antigen is physiologically restricted to a limited number of tissues including trophoblasts, and is thought to play a role in establishing tolerance of the fetus by the maternal immune system. We investigated whether ectopic expression of HLA-G could also be detected in tumor cells and confer them the ability to escape immune cytotoxic responses. High levels of all alternatively spliced HLA-G transcripts could be detected in melanoma cells by RT-PCR. Analysis of biopsies from a melanoma patient revealed a higher HLA-G transcription level in skin metastasis as compared to healthy skin, while specific amplification of the HLA-G5 transcript was only observable in the tumor. HLA-G protein expression could also be detected in two melanoma cell lines. HLA-G-positive tumors inhibit cytotoxic lysis by the NK cell line YT2C2-PR. This inhibition is not observed with B-EBV cell lines bearing matched class I specificities, and is thought to occur through interaction of HLA-G with inhibitory receptors that are distinct from known KIRs interacting with HLA-E or classical class I molecules. Together, these results confirm that HLA-G expression at the surface of tumor cells can participate in the evasion of antitumoral immune responses and favor tumor progression.

The expression pattern of MHC class I genes in trophoblast cells at the feto-maternal interface is thought to be the basis of the maintenance of pregnancy by protecting the fetus from maternal immune rejection. Transcription of classical HLA class I genes is low or undetectable in most trophoblast cells as well as in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells. The aim of this study was to characterize the regulatory mechanisms that repress HLA-A transcription in these cell types. We show that the -335 to ATG region of the HLA-A11 gene promoter is inactive in JEG-3 and BeWo cells while able to control efficient reporter gene transcription in other cell types, indicating that this region is the target for downregulation of HLA-A expression in choriocarcinoma cell lines. The regulatory sequence involved in HLA-A gene repression was further mapped to a proximal regulatory element within the -107 to +2 ATG region of the promoter. We show that the HLA-A promoter activity cannot be induced by interferon-gamma (IFN-gamma) and that exogenous MHC class II transactivator CIITA is able to induce HLA class I promoter activity in these cells. Stringent transcriptional regulatory mechanisms, implicating the lack of basal and IFN-gamma-inducible class I promoter activity, are thus involved in the downregulation of HLA-A expression in JEG-3 and BeWo trophoblast-derived choriocarcinoma cells.

Nonclassical MHC class I HLA-G antigen expression is tissue specific and is thought to play a role in tolerance of the semiallogeneic fetus by the maternal immune system. Ectopic expression of HLA-G by tumor cells provides them with an additional mechanism of escape from immunosurveillance by host cytotoxic effector mechanisms. The aim of this study was to assess the expression of nonclassical HLA-G antigens in ex vivo human melanoma biopsies. HLA-G mRNA levels corresponding to both membrane-bound and soluble protein isoforms were analyzed in tumor specimens obtained from primary or metastatic melanomas of 23 patients. High levels of HLA-G transcription were detected in tumor specimens in 5 of 23 patients and found to be comparable in both lymph node and skin metastases. HLA-G mRNA transcript levels at tumor sites in 18 of these patients were compared with those in samples of their own healthy skin and were higher in the tumor tissue in 12 patients. Differential expression of mRNA transcripts corresponding to soluble and membrane-bound HLA-G was also observed in some tumor biopsies. HLA-G protein expression was detected in tumors that exhibited high levels of HLA-G transcription by immunofluorescence of frozen sections and Western blot analysis of both tumor and healthy skin biopsies, using anti-HLA-G-specific monoclonal antibodies. This work provides evidence that HLA-G gene transcription and protein expression can be up-regulated ex vivo in melanoma. Our finding that several of the tumors studied expressed high levels of HLA-G provides additional clues as to how a tumor can be selected in vivo to escape from cytotoxic antitumor responses, constituting a new parameter to be considered in the design of therapeutic approaches aimed at enhancing antitumor immune responses.

HLA-G is a non-classical MHC class I molecule involved in immune tolerance. We present our results concerning the effects of HLA-G on the cellular immune response, where it impairs both NK and T cell functions. We describe the NK inhibitory properties of HLA-G ex vivo, demonstrating its role in materno-fetal tolerance, which is supported by our in vitro studies using membrane-bound HLA-G1- and HLA-G2-transfected cells and a full-length soluble HLA-G molecule. We also report how HLA-G interacts at the T cell level, here exemplified by its inhibitory effect on both T cell allogeneic proliferative and Ag-specific CTL responses., (Copyright 1999 Academic Press.)

The non-classical class I antigen HLA-G has been shown to play a role in foeto-maternal tolerance. It interacts with inhibitory receptors to down-regulate natural killer and T cell cytotoxic functions. We here report our investigations on HLA-G expression in melanoma cells and its implication in the induction of immune tolerance to tumours. We provide the first evidence that both malignant human melanoma cell lines and ex vivo biopsies can exhibit high levels of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns. We further demonstrated that melanoma cells that express HLA-G inhibit NK cytolysis. We thus propose that HLA-G expression may impede the elimination of malignant cells by anti-tumour immune effector cells, constituting a newly described mechanism by which tumour cells may evade immunosurveillance., (Copyright 1999 Academic Press.)

Background: Polymorphic eruptions of pregnancy (PEP) are common cutaneous disorders of unknown origin that occur usually after week 34 of gestation. Since pregnancy is associated with peripheral-blood chimerism, particularly during the third trimester, we studied the role of fetal cells in the development of the skin lesions., Methods: We studied samples of skin from ten women with PEP who were carrying male fetuses and 26 women with normal skin or non-PEP skin disorders (13 carrying male and 13 carrying female fetuses). Epidermis and dermis were dissected from the samples, and the DNA was extracted. PCR with primers specific for the SRY gene was used to detect male DNA., Findings: Male DNA was detected in dermis or epidermis from skin lesions of six of the ten women with PEP. No male DNA was detected in any of the 26 women without PEP., Interpretation: Fetal cells can migrate to skin during gestation, where they seem to be associated with the development of cutaneous disorders of pregnancy.

Problem: Human leukocyte antigen (HLA)-G is a major histocompatibility complex class I antigen, which is referred to as nonclassical because it displays a tissue-restricted distribution in the placenta, a reduced cytoplasmic domain, a limited polymorphism, and several isoforms. The HLA-G antigen is thought to play an essential role during pregnancy by protecting the semi-allogeneic fetus from recognition and destruction by maternal immune cells., Method of Study: Alternative splicing of HLA-G mRNA was analyzed by Southern blot of reverse transcriptase-polymerase chain reaction products from trophoblasts of the first trimester of gestation and term placenta. The regulation of HLA-G gene expression was investigated by electrophoretic mobility shift assays using nuclear extracts from cells expressing different levels of HLA-G gene activity. Using polymerase chain reaction-single strand conformational polymorphism and sequencing, we studied HLA-G gene polymorphism in families from the Centre d'Etude du Polymorphisme Humain in Paris. To understand the function of the HLA-G molecule, cytotoxicity assays were carried out with peripheral blood mononuclear cells or polyclonal natural killer effectors cells from 30 different donors against HLA-G1 and HLA-G2 transfectants., Results: Four main aspects have been elucidated: 1) The primary transcript of the HLA-G gene is alternatively spliced into five main mRNA forms: HLA-G1 (full length), HLA-G2 (minus exon 3), which encodes a membrane-bound isoform associated with beta-2 microglobulin, HLA-G3 (minus exons 3 and 4), HLA-G4 (minus exon 4), and HLA-G5 (plus intron 4), which encodes a soluble form of the HLA-G antigen; 2) specific nuclear factors bind to an important regulatory element located more than 1.2 kb from the HLA-G gene. Three specific complexes are observed in cells that show HLA-G transcriptional activity and an additional factor that could correlate with the repression of HLA-G gene expression that is detected in natural killer cells; 3) we observed an important genomic polymorphism in exon 3 but a very low polymorphism at the protein level; 4) HLA-G1 and HLA-G2 transfectants clearly demonstrated that both HLA-G isoforms are capable of inhibiting natural killer lytic activity., Conclusion: These results suggest that HLA-G acts as the public ligand for natural killer inhibitory receptors, thus protecting the fetus against maternal rejection.