HLA-G Protein Expression Research Articles

HLA-G in melanoma: can the current controversies be solved?

The potential role of HLA-G in tumor immune escape has stimulated interest in the analysis of HLA-G antigens in malignant cells. Malignant melanoma is the tumor which has been mostly analyzed for HLA-G expression. Results obtained by seven groups of investigators about HLA-G expression in 108 melanoma cell lines have been concordant. HLA-G mRNA has been found in about 50% of the cell lines tested, whereas HLA-G protein has been found in less than 1% of the cell lines analyzed. In contrast, results obtained from six groups of investigators about HLA-G protein expression in 133 melanoma lesions have been conflicting. The possible causes of these conflicting results as well as the reasons for the discrepancy in HLA-G expression between cultured melanoma cell lines and surgically removed lesions have been discussed. Lastly, data about the potential clinical relevance of HLA-G expression in melanoma has been reviewed. The available data in the literature strongly suggest that progress in this exciting research area would greatly benefit from experiments to solve the current controversies in the field.

Expression of membrane-bound HLA-G at the maternal-fetal interface is not associated with pregnancy maintenance among patients with idiopathic recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) has many known aetiologies. However, using current diagnostic testing, a large fraction of recurrent pregnancy losses remain unexplained. Many of these may have immune underpinnings. HLA-G is a non-classical MHC class I product whose fairly restricted expression at the maternal-fetal interface suggests a role in successful embryonic implantation and/or subsequent pregnancy maintenance. The study of immune-mediated RPL should be enhanced by comparing groups of idiopathic RPL patients with normal fetal chromosomes to RPL patients with known chromosomal abnormalities in their index pregnancies. We hypothesized that if alteration of HLA-G expression at the maternal-fetal interface were associated with immune-mediated RPL, such changes might be detectable using these comparisons. HLA-G protein expression at the maternal-fetal interface in maternal and gestational age-matched women with history of idiopathic RPL and normal male fetuses were compared with expression in RPL patients with known fetal trisomy 16 in their index pregnancy. We detected no significant quantitative differences in the levels of HLA-G between these groups of RPL patients. Within the limitations of this study, we conclude that HLA-G expression is not a major immunological determinant of pregnancy maintenance among patients with idiopathic RPL.

HLA-G protein up-regulation in primary cutaneous lymphomas is associated with interleukin-10 expression in large cell T-cell lymphomas and indolent B-cell lymphomas

AbstractPrimary cutaneous lymphomas (CLs) constitute a spectrum of diseases characterized by a clonal accumulation of lymphocytes in the skin. Most CLs display a Th2 cytokine profile, including expression of interleukin-10 (IL-10). Because the up-regulation of HLA-G, a nonclassical class Ib molecule inducible by IL-10, might account for the immunescape of the malignant clone, HLA-G and IL-10 expression has been investigated in 45 cases of primary CL (10 of B-cell and 35 of T-cell origin) with quantitative polymerase chain reaction (PCR) and immunohistochemistry. HLA-G message was present in all cutaneous B-cell (CBCL) and T-cell (CTCL) lymphomas evaluated. Immunohistochemistry revealed HLA-G protein expression in 23 (51%) of 45 cases (7 of 10 CBCL, 16 of 35 CTCL). While in CBCL mostly indolent types displayed HLA-G positivity, in CTCL HLA-G expression was associated with high-grade histology and advanced stage of the disease. Except for neoplastic and infiltrating lymphocytes, other cells such as macrophages and dendritic cells showed HLA-G immunoreactivity. Furthermore, IL-10 protein expression was demonstrated in 16 (73%) of 22 HLA-G+ cases, which correlated with HLA-G protein presence (P < .001). HLA-G up-regulation together with IL-10 expression in CL might additionally contribute to the evasion of immunosurveillance and facilitate the transition from low- to high-grade lymphomas.

Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: a CD4+ T cell regulatory mechanism.

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.

HLA-G : la tolérance fœto-maternelle

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 antigenes, which had been described up till then as antigenes of defence and rejection, and the dramatic role of HLA-G in immunotolerance.

Modulation of HLA-G antigens expression by human cytomegalovirus: specific induction in activated macrophages harboring human cytomegalovirus infection.

After infection, human CMV (HCMV) establishes a latent and persistent infection in immature myeloid progenitors and peripheral blood monocytes. Completion of the HCMV life cycle is possible upon maturation of monocytes to tissue macrophages and under permissive circumstances, e.g., immunosuppression. We investigated the hypothesis that HLA-G molecules could be induced during HCMV reactivation in activated macrophages to favor virus dissemination. In this study, we provide evidence that HLA-G Ags are produced during viral reactivation in macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. While HLA-G surface expression is up-regulated, classical MHC-I molecules are partially down-regulated by HCMV. In vivo, bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also express HLA-G molecules. The direct correlation between HLA-G Ag induction and HCMV infection was confirmed in U-373 MG astrocytoma cells. Soluble HLA-G expression is stimulated upon HCMV infection, and this modulation depends on the cooperative action of the two immediate-early-1 pp72 and immediate-early-2 pp86 products. Because HLA-G transcription is active in macrophages and U-373 MG astrocytoma cells, it is likely that the modulation of HLA-G protein expression during HCMV replication occurs at a post-transcriptional level. Our data suggest that induction of HLA-G molecules could be an additional mechanism that helps HCMV to subvert host defenses.

HLA-G expression in human melanoma cells: protection from NK cytolysis

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.

Search for a human homologue of the mouse Ped gene.

The Ped gene influences the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. The mouse Ped gene product is a major histocompatibility complex (MHC) class Ib protein called Qa-2. Studies from many human in-vitro fertilization (IVF) clinics suggest that the mouse Ped gene has a human homologue because embryos fertilized at the same time have different cleavage rates, and those embryos that cleave at a faster rate are more likely to result in a viable pregnancy. Candidates for the human homologue of the mouse Ped gene include the MHC class Ib genes HLA-E, HLA-F, and HLA-G. The presence of mRNA for these three genes was tested in 108 spare day 3 human preimplantation embryos from 25 couples by using reverse transcription-polymerase chain reaction (RT-PCR). Of the 86 embryos tested for HLA-E mRNA, 72 were positive (84%), and of the 88 embryos tested for HLA-G mRNA, 39 were positive (44%). None of the 17 embryos tested for HLA-F mRNA were positive (0%). Studies of expression of HLA-G protein were undertaken to ascertain whether HLA-G was attached to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage similar to that found in Qa-2 protein. Treatment of JEG-3 cells, an HLA-G expressing cell line, with phospholipase C did not result in removal of HLA-G showing that HLA-G, unlike Qa-2, is not GPI linked to the cell surface. The pros and cons of HLA-E, HLA-F, and HLA-G as candidates for the human Ped gene are discussed.

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.

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.

HLA‐G protein expression is not induced during malignant transformation

To evaluate the biological relevance of HLA-G expression during tumoral transformation, we analyzed its expression in different malignant cells and immune effector cells infiltrating solid tumors. Our analysis of 33 tumor cell lines and 53 tumoral biopsies demonstrated that: i) six tumor cell lines display HLA-G transcription with differential alternative splicing patterns and only Jeg3 choriocarcinoma and MCF-7 breast adenocarcinoma cell lines express HLA-G translated products; and ii) HLA-G antigens are not expressed in malignantly transformed cells derived from lung (n=18), liver (n=5), colon (n=5), breast (n=10), kidney (n=5), ovary (n=5), and larynx (n=5) tissues ex vivo. The healthy tissues surrounding these tumor tissues do not express HLA-G molecules either. On the other hand, surprisingly, HLA-G products were detected in activated macrophages and dendritic cells localized in tumoral biopsies of 5 out of 18 different lung carcinomas. No HLA-G labelling was observed in resident mononuclear phagocytes of surrounding healthy tissues. Our observations clearly demonstrate that HLA-G is not a marker of malignant cells but appears as a gene expressed in tumor-associated macrophages and dendritic cells, preferentially in those recruited in lung carcinomas. Our findings suggest that specific environmental factors around lung tumors could be involved in the induction of HLA-G protein expression.

HLA-G expression: immune privilege for tumour cells?

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 andex vivobiopsies 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.

Listeria monocytogenes as a probe for trophoblast immune function

HLA class I genes are differentially expressed among subpopulations of cells in first trimester human placentas. In this study, HLA class I protein was detected in extravillous cytotrophoblast cells by immunohistochemistry using the monoclonal antibody W6/32. In the same trophoblast subpopulation, class Ib proteins were identified with two monoclonal antibodies, 87G (anti-HLA-G) and 131 (anti-HLA-AG) and class Ia protein was detected with the monoclonal antibody, 4E (anti-HLA-BC). All of the antibodies also identified antigens on the human trophoblast-derived choriocarcinoma cell line, JEG-3. Therfore, the JEG-3 cells were used as a model system to study cytokine regulation of HLA-G in trophoblast cells. Northern blot hybridization studies showed that interferons (IFN-α, IFN-β, IFN-γ) and tumor necrosis factor-α (TNF-α) modestly enhanced steady state levels of HLA-G mRNA. Yet analysis of HLA-G protein by immunocytochemistry and flow cytometry failed to identify any changes in intracellular or membrane expression of HLA-G protein following cytokine treatment. Resistance to upregulation of HLA class I antigens was not a general feature of JEG-3 cells; IFNs enhanced expression of HLA-BC as well as HLA class I light chain, β2-microglobulin. HLA null Jar choriocarcinoma cells did not contain HLA-G mRNA or antigen and exposure to cytokines had no effect on HLA-G. The results of this study are consistent with the postulate that trophoblast cell expression of HLA-G is stringently regulated and is controlled in part by post-transcriptional mechanisms.

HLA-G expression in melanoma: a way for tumor cells to escape from immunosurveillance.

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Expression of granulocyte-colony stimulating factor receptor during development of the human placenta

The human leukocyte antigen (HLA)-G is expressed by extravillous cytotrophoblast cells in the feto-maternal contact zone. Polymorphisms have been described in the HLA-G gene and have been linked with differences in HLA-G mRNA alternative splicing patterns and protein expression. Differences in the isoform profile or the degree of HLA-G expression may influence cytokine production and, thereby, placental and fetal growth. Associations between a 14 bp deletion polymorphism in the 3′UTR part of the HLA-G gene and birth weight in relation to gestational age and placental weight were studied in 47 pregnancies complicated with preeclampsia and 87 with no preeclampsia. An HLA-G genotype homozygous for the presence of the 14 bp sequence polymorphism was significantly associated with increased birth weight in relation to gestational age (one-way analysis of variance; 2 degrees of freedom: p = 0.02) and with placental weight at birth (>38 weeks of gestation; +14 bp/+14 bp vs others; unpaired t-test: p = 0.03). There was also a slightly higher placental ratio in the offspring with the +14 bp/+14 bp genotype. The implications of these findings are discussed in relation to certain complications of pregnancy and in an evolutionary perspective.

Detection of membrane-bound HLA-G translated products with a specific monoclonal antibody.

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.

Cytokine regulation of HLA-G expression in human trophoblast cell lines

HLA class I genes are differentially expressed among subpopulations of cells in first trimester human placentas. In this study, HLA class I protein was detected in extravillous cytotrophoblast cells by immunohistochemistry using the monoclonal antibody W6/32. In the same trophoblast subpopulation, class Ib proteins were identified with two monoclonal antibodies, 87G (anti-HLA-G) and 131 ( anti-HLA- A G ) and class Ia protein was detected with the monoclonal antibody, 4E ( anti-HLA- B C ). All of the antibodies also identified antigens on the human trophoblast-derived choriocarcinoma cell line, JEG-3. Therfore, the JEG-3 cells were used as a model system to study cytokine regulation of HLA-G in trophoblast cells. Northern blot hybridization studies showed that interferons (IFN-α, IFN-β, IFN-γ) and tumor necrosis factor-α (TNF-α) modestly enhanced steady state levels of HLA-G mRNA. Yet analysis of HLA-G protein by immunocytochemistry and flow cytometry failed to identify any changes in intracellular or membrane expression of HLA-G protein following cytokine treatment. Resistance to upregulation of HLA class I antigens was not a general feature of JEG-3 cells; IFNs enhanced expression of HLA- B C as well as HLA class I light chain, β2-microglobulin. HLA null Jar choriocarcinoma cells did not contain HLA-G mRNA or antigen and exposure to cytokines had no effect on HLA-G. The results of this study are consistent with the postulate that trophoblast cell expression of HLA-G is stringently regulated and is controlled in part by post-transcriptional mechanisms.

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Process of the expression of soluble HLA-G protein

During pregnancy there is an exchange of cells between the fetus and the mother including T lymphocytes that can persist after delivery. Previous studies have described an increased numbers of maternal cells in children with juvenile diabetes as compared to their unaffected siblings. Our objective was to assess the possibility for these chimeric T cells to trigger an anti-beta cell response.We mated OT2 transgenic female mice having T cells specifically targeting ovalbumin to RIP-OVA males expressing ovalbumin in pancreatic β cells. This allowed us to examine RIP-OVA progeny from OT2 mothers to assess the consequences of maternal T cells acquired during gestation or lactation. We quantitatively analyzed the pancreas of RIP-OVA mice from OT2 mothers for islet infiltration and compared them to RIP-OVA mice not exposed to OT2 mothers or to wild-type mice from OT2 mothers.RIP-OVA mice from OT2 mothers had significantly more peri-insulitis (p = 0.0083) compared to wild-type littermates. Similarly RIP-OVA mice from OT2 mothers had more peri-insulitis as compared to RIP-OVA mice from RIP-OVA mothers (p = 0.0073). Presence and specific anti-ovalbumin activity of maternal OT2 cells in the offsprings’ peripheral lymphoid tissues was found in a separate group of mice. In animals presenting islet inflammation, CD3+ infiltrating cells in the pancreas were however derived from the offspring and not from OT2 mothers. In accordance, OT2 and RIP-OVA double transgenic mice with high levels of auto-reactive T cells had more peri-insulitis and sometimes intense insulitis when they were from OT2 mothers as compared to RIP-OVA mothers (p = 0.046).In highly specific fetal/maternal combinations, maternal T cells with activity against the offspring pancreatic beta cells, presumably chimeric in fetal organs, initiate islet inflammation and may therefore predispose to auto-immune diabetes.