Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2＇-deoxycytidine

The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.     

HLA-G: fetomaternal tolerance

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*0105N null allele encodes functional HLA-G isoforms

Expression of the nonclassical HLA class I antigen, HLA-G, is associated with immune tolerance in view of its role in maintaining the fetus in utero, allowing tumor escape, and favoring graft acceptance. Expressed on invasive trophoblast cells, HLA-G molecules bind inhibitory receptors on maternal T lymphocytes and NK cells, thereby blocking their cytolytic activities and protecting the fetus from maternal immune system attack. The HLA-G gene consists of 15 alleles, including a null allele, HLA-G*0105N. HLA-G*0105N presents a single base deletion, preventing translation of both membrane-bound (HLA-G1) and full-length soluble isoforms (HLA-G5) as well as of the spliced HLA-G4 isoform. The identification of healthy subjects homozygous for this HLA-G null allele suggests that the HLA-G*0105N allele may generate other HLA-G isoforms, such as membrane-bound HLA-G2 and -G3 and the soluble HLA-G6 and -G7 proteins, which may substitute for HLA-G1 and -G5, thus assuming the immune tolerogeneic function of HLA-G. To investigate this point, we cloned genomic HLA-G*0105N DNA and transfected it into an HLA-class I-positive human cell line. The results obtained indicated that HLA-G proteins were indeed present in HLA-G*0105N-transfected cells and were able to protect against NK cell lysis. These findings emphasize the role of the other HLA-G isoforms as immune tolerogeneic molecules that may also contribute to maternal tolerance of the semiallogenic fetus as well as tumor escape and other types of allogeneic tissue acceptance.     

Human leukocyte antigen-G (HLA-G) as a marker for diagnosis, prognosis and tumor immune escape in human malignancies

Human leukocyte antigen-G (HLA-G) is a non-classical HLA-class Ib molecule with multiple immunoregulatory properties. Its main functions in physiological conditions are to abolish maternal immune cell activity against fetus and to establish immune tolerance at the maternal-fetal interface. In oncology, HLA-G molecules are aberrantly expressed in a variety of human neoplastic diseases and play an important role in the escape of tumor cells from immune surveillance. In the past few years, making use of HLA-G protein expression in tissues and circulating levels in body fluids as a tumor marker have been the focus of extensive research in the diagnosis and prognosis of several human malignancies. In addition, this molecule might be a promising target for future immune therapeutic approaches based on its immune tolerant functions and its highly specific expression for malignant transformation. In this review, we will summarize available literature data as well as our own works on HLA-G in cancer, and address some of the issues concerning its application in human neoplasia.     

人类白细胞抗原-G分子结构与功能

人类白细胞抗原G（humanleukocyteantigen．G,HLA—G）属于非经典的HLAI类分子,是机体内一个重要的免疫耐受分子。HLA—G分子可通过与受体结合直接抑制多种免疫活性细胞的生物学功能,或通过诱导产生免疫调节细胞间接抑制机体的免疫应答。研究显示,HLA-G基因多态性及分子表达在母胎免疫、器官移植、肿瘤的发生发展、感染和自身免疫中均有重要意义。     

HLA-E and HLA-G expression in classical HLA class I-negative tumors is of prognostic value for clinical outcome of early breast cancer patients

Nonclassical HLAs, HLA-E and HLA-G, are known to affect clinical outcome in various tumor types. We examined the clinical impact of HLA-E and HLA-G expression in early breast cancer patients, and related the results to tumor expression of classical HLA class I. Our study population (n = 677) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1995. Tissue microarray sections of arrayed tumor and normal control material were immunohistochemically stained for HLA-E and HLA-G. For evaluation of HLA-E and HLA-G and the combined variable, HLA-EG, a binary score was used. Expression of classical HLA class I molecules was determined previously. HLA-E, HLA-G, and HLA-EG on breast tumors were classified as expression in 50, 60, and 23% of patients, respectively. Remarkably, only in patients with loss of classical HLA class I tumor expression, expression of HLA-E (p = 0.027), HLA-G (p = 0.035), or HLA-EG (p = 0.001) resulted in a worse relapse-free period. An interaction was found between classical and nonclassical HLA class I expression (p = 0.002), suggestive for a biological connection. We have demonstrated that, next to expression of classical HLA class I, expression of HLA-E and HLA-G is an important factor in the prediction of outcome of breast cancer patients. These results provide further evidence that breast cancer is immunogenic, but also capable of evading tumor eradication by the host's immune system, by up- or downregulation of HLA class Ia and class Ib loci.     

HLA-E and HLA-G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2-dependent and -independent pathways

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab')2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.     

Modulation of HLA-G expression in human neural cells after neurotropic viral infections

HLA-G is a nonclassical human major histocompatibility complex class I molecule. It may promote tolerance, leading to acceptance of the semiallogeneic fetus and tumor immune escape. We show here that two viruses-herpes simplex virus type 1 (HSV-1), a neuronotropic virus inducing acute infection and neuron latency; and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection-upregulate the neuronal expression of several HLA-G isoforms, including HLA-G1 and HLA-G5, the two main biologically active isoforms. RABV induces mostly HLA-G1, and HSV-1 induces mostly HLA-G3 and HLA-G5. HLA-G expression is upregulated in infected cells and neighboring uninfected cells. Soluble mediators, such as beta interferon (IFN-beta) and IFN-gamma, upregulate HLA-G expression in uninfected cells. The membrane-bound HLA-G1 isoform was detected on the surface of cultured RABV-infected neurons but not on the surface of HSV-1-infected cells. Thus, neuronotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially. HLA-G may therefore be involved in the escape of certain viruses from the immune response in the nervous system.     

Melanoma: Where we are and where we go

Melanoma is known as an aggressive tumor which shows an increasing incidence and poor prognosis in the metastatic phase. Hence, it seems that diagnosis and effective management (including early diagnosis, choosing of the effective therapeutic platform, caring, and training of patients for early detection) are major aspects of melanoma therapy. Early detection of melanoma is a key point for melanoma therapy. There are various diagnosis options such as assessing of biopsy, imaging techniques, and biomarkers (i.e., several proteins, polymorphism, and liquid biopsy). Among the various biomarkers, assessing circulating tumor cells, cell-free DNAs, cell-free RNAs, and microRNAs (miRNAs) have emerged as powerful diagnosis tools for melanoma patients. Deregulations of these molecules are associated with melanoma pathogenesis. After detection of melanoma, choosing of effective therapeutic regimen is a key step for recovery of melanoma patients. Several studies indicated that various therapeutic approaches including surgery, immunotherapy, systematic therapy, radiation therapy and antibodies therapy could be used as potential therapeutic candidates for melanoma therapy. Caring for melanoma patients is one of the important components of melanoma therapy. Caring and training for melanoma patients could contribute to better monitoring of patients in response to various therapeutic options. Here, we summarized various diagnosis approaches such as assessing biopsy, imaging techniques, and utilization of various biomarkers (i.e., proteins, CTCs, cfDNAs, and miRNAs) as a diagnostic biomarker for detection and monitoring patients with melanoma. Moreover, we highlighted various therapeutic options and caring aspects in patients with melanoma.     

Low Expression of Human Histocompatibility Soluble Leukocyte Antigen-G (HLA-G5) in Invasive Cervical Cancer With and Without Metastasis, Associated With Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICC^W)] and absence [ICC without metastasis (ICC^WT)] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICC^WT (n=52 patients) and ICC^W (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies. (J Histochem Cytochem 58:405–411, 2010)     

RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line

The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves the regulation of multiple genes. We have previously shown that the receptor for advanced glycation end products (RAGE) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized that up-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switch of these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line that was established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE in these cells is associated with mesenchymal-like morphologies of the cells. These cells demonstrate higher migration abilities and reduced proliferation properties, suggesting that the cells have switched to a metastatic phenotype. At the molecular level, we show that RAGE overexpression is associated with the up-regulation of the RAGE ligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE in the metastatic switch of melanoma cells.     

HLA-G与自身免疫病相关性研究进展

非经典人类白细胞抗原G（human leukocyte antigen-G,HLA-G）是机体内一类重要的免疫调节分子,包括膜结合型HLA—G（mHLA—G）及可溶性HLA—G（sHLA—G）两种分子表达形式。HLA-G分子可通过与受体结合直接抑制多种免疫活性细胞的生物学功能,或通过诱导产生免疫调节细胞间接抑制机体的免疫应答。研究显示,HLA—G基因多态性及分子表达在母胎免疫、感染、自身免疫病及肿瘤的发生发展中均有重要意义。对HLA—G在自身免疫病中的作用作一综述。     

Differential expression of alternatively spliced transcripts of HLA-G in human preimplantation embryos and inner cell masses

It has been reported that preimplantation human embryos secrete HLA-G, and the levels may be predictive of their ability to implant. However, it is not known which of the membrane-bound (HLA-G 1-4) and soluble (HLA-G 5-6) alternatively spliced forms are present, nor the developmental stage at which they appear. Therefore, we have investigated HLA-G mRNA isoform expression on single embryos at the two-, four-, six-, and eight-cell, morula, and blastocyst stages. The percentage of embryos expressing each HLA-G isoform mRNA increased with developmental stage, but contrary to expectation, HLA-G5 mRNA was not detected in single two- to eight-cell embryos and was only expressed by 20% of morulae and blastocysts. Similarly, soluble HLA-G6 mRNA was not detected until the blastocyst stage and then in only one-third of embryos. In contrast, labeling with MEM G/9 Ab (specific for HLA-G1 and -G5) was observed in 15 of 20 two- to eight-cell embryos and 5 of 5 blastocysts. This disparity between mRNA and protein may be due to HLA-G protein remaining from maternal oocyte stores produced before embryonic genome activation and brings into question the measurement of soluble HLA-G for clinical evaluation of embryo quality. Although HLA-G is expressed in the preimplantation embryo, later it is primarily expressed in the invasive trophoblast of the placenta rather than the fetus. Therefore, we have investigated whether down-regulation of HLA-G first occurs in the inner cell mass (precursor fetal cells) of the blastocyst and, in support of this concept, have shown the absence HLA-G1 and -G5 protein and mRNA.     

Tolérance fœtomaternelle : rôle de la molécule HLA-G dans la protection du fœtus contre l'activité natural killer maternelle

HLA-G is a non-classical major histocompatibility complex class I molecule selectively expressed on extravillous trophoblast cells at the fetal—maternal interface. HLA-G may play an important role in maintaining maternal immune tolerance of the semi-allogenic fetus. In this study, we demonstrate for the first time the protective role of HLA-G during pregnancy. Indeed, cytotrophoblast cells of the fetus are resistant to lytic activity by maternal decidual natural killer cells. In order to precisely characterize the immunological functions of HLA-G products, we have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against NK cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA-class I negative human K562 cell line. We demonstrate that both HLA-G1 and HLA-G2 transfectants inhibit NK cytolysis observed in peripheral blood from 25 donors (males and females). This led us to the conjecture that HLA-G is the public ligand for natural killer inhibitory receptors present in all individuals.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

HLA-G在肿瘤组织中表达情况研究进展

人类白细胞抗原G(human leukocyte antigen,HLA-G)属于非经典HLA-I类分子,在多种肿瘤细胞上均有表达。从结构上可以将HLA-G分为7种亚型:膜结合型HLA-G1-HLA-G4和可溶型HLA-G5-HLA-G7。研究表明,HLA-G1和HLA-G5具有明确的生物学活性也是研究较为深入的两种亚型,他们可以与T淋巴细胞、B淋巴细胞和NK细胞表面的ILT2/CD85j/LILRB1,ILT4/CD85d/LILRB2,KIR2DL4/CD158d受体结合而发挥免疫抑制功能。目前,HLA-G分子可以在肝癌、肾癌、肺癌、胃癌、食道癌、鼻咽癌、卵巢癌、乳腺癌、宫颈癌、直肠癌和血液肿瘤中表达。本文从HLA-G分子的结构和功能出发,综述了HLA-G分子在上述肿瘤中表达的情况,旨在分析HLA-G在各种肿瘤组织中表达的特点以及临床意义,为临床早期诊断和治疗肿瘤提供参考。     

Breast cancer suppressor candidate-1 (BCSC-1) is a melanoma tumor suppressor that down regulates MITF

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate-1 (BCSC-1) as a novel tumor suppressor in melanoma. BCSC-1 expression is decreased in human MM, and its ectopic expression in MM-derived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC-1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC-1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.     

HLA-G expression in human embryonic stem cells and preimplantation embryos

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.     

Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.