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.     

LINE1 family member is negative regulator of HLA-G expression

Class Ia molecules of human leucocyte antigen (HLA-A, -B and -C) are widely expressed and play a central role in the immune system by presenting peptides derived from the lumen of the endoplasmic reticulum. In contrast, class Ib molecules such as HLA-G serve novel functions. The distribution of HLA-G is mostly limited to foetal trophoblastic tissues and some tumour tissues. The mechanism required for the tissue-specific regulation of the HLA-G gene has not been well understood. Here, we investigated the genomic regulation of HLA-G by manipulating one copy of a genomic DNA fragment on a human artificial chromosome. We identified a potential negative regulator of gene expression in a sequence upstream of HLA-G that overlapped with the long interspersed element (LINE1); silencing of HLA-G involved a DNA secondary structure generated in LINE1. The presence of a LINE1 gene silencer may explain the limited expression of HLA-G compared with other class I genes.     

Linking two immuno-suppressive molecules: indoleamine 2,3 dioxygenase can modify HLA-G cell-surface expression

Nonclassical human leukocyte antigen (HLA) class I molecule HLA-G and indoleamine 2,3 dioxygenase (INDO) in humans and mice, respectively, have been shown to play crucial immunosuppressive roles in fetal-maternal tolerance. HLA-G inhibits natural killer and T cell function by high-affinity interaction with inhibitory receptors, and INDO acts by depleting the surrounding microenvironment of the essential amino acid tryptophan, thus inhibiting T cell proliferation. We investigated whether HLA-G expression and INDO function were linked. Working with antigen-presenting cell (APC) lines and monocytes, we found that functional inhibition of INDO by 1-methyl-tryptophan induced cell surface expression of HLA-G1 by HLA-G1-negative APCs that were originally cell-surface negative, and that in reverse, the functional boost of INDO by high concentrations of tryptophan induced a complete loss of HLA-G1 cell surface expression by APCs that were originally cell-surface HLA-G1-positive. This mechanism was shown to be posttranslational because HLA-G protein cell contents remained unaffected by the treatments used. Furthermore, HLA-G cell surface expression regulation by INDO seems to relate to INDO function, but not to tryptophan catabolism itself. Potential implications in fetal-maternal tolerance are discussed.     

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.     

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。     

HLA-G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells.

Human NK cells adhere to and lyse porcine endothelial cells (pEC) and therefore may contribute to the cell-mediated rejection of vascularized pig-to-human xenografts. Since MHC class I molecules inhibit the cytotoxic activity of NK cells, the expression of HLA genes in pEC has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity. HLA-G, a minimally polymorphic HLA class I molecule that can inhibit a wide range of NK cells, is an especially attractive candidate for this purpose. In this study we tested whether the expression of HLA-G on pEC inhibits the molecular mechanisms that lead to adhesion of human NK cells to pEC and subsequent xenogeneic NK cytotoxicity. To this end two immortalized pEC lines (2A2 and PED) were stably transfected with HLA-G1. Rolling adhesion of activated human NK cells to pEC monolayers and xenogeneic cytotoxicity against pEC mediated by polyclonal human NK lines as well as NK clones were inhibited by the expression of HLA-G. The adhesion was partially reversed by masking HLA-G on pEC with anti-HLA mAbs or by masking the HLA-G-specific inhibitory receptor ILT-2 on NK cells with the mAb HP-F1. The inhibition of NK cytotoxicity by HLA-G was only partially mediated by ILT-2, indicating a role for other unknown NK receptors. In conclusion, transgenic expression of HLA-G may be useful to prevent human NK cell responses to porcine xenografts, but is probably not sufficient on its own. Moreover, the blocking of rolling adhesion by HLA-G provides evidence for a novel biological function of HLA molecules.     

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.     

Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E,F, and G in maternal-placental immune recognition

In this study we focused on the structure and expression of the HLA-E, F, and G class I complexes in placental tissue. Structural analysis included an examination of the peptides bound to soluble and membrane forms of the HLA-G complex isolated directly from placenta. An important distinction was observed from HLA-G bound peptides previously isolated from transfectant cells. Thus, the number of distinct moieties bound to placental-derived proteins was substantially lower than that bound to transfectant-derived HLA-G. Indeed, a single peptide species derived from a cytokine-related protein alone accounted for 15% of the molar ratio of HLA-G bound peptide. To further examine HLA-E and its potential to bind peptide, notably that derived from HLA-G, we combined new Abs to examine expression in placental tissues for all the known forms of the nonclassical class I molecules. Whereas membrane HLA-G was found in extravillous trophoblasts, soluble HLA-G was found in all placental trophoblasts, including villous cytotrophoblasts and syncitiotrophoblasts. Further, HLA-E was found in all cells that expressed either form of HLA-G, consistent with HLA-E being complexed with the HLA-G signal sequence-derived nonamer in these cells. Finally, using new reagents specific for HLA-F, a restricted pattern of expression was observed, primarily on extravillous trophoblasts that had invaded the maternal decidua. Comparative staining indicated that HLA-F was on the surface of these cells, defining them as the first to demonstrate surface expression of this Ag and the first cell type identified to express all three nonclassical HLA class I Ags simultaneously.     

β-defensin-3 negatively regulates TLR4–HMGB1 axis mediated HLA-G expression in IL-1β treated glioma cells

The non-classical HLA class I antigen HLA-G contributes to immune escape mechanisms in glioblastoma multiforme (GBM). We have previously shown that IL-1β–HIF-1α axis connects inflammatory and oncogenic pathways in GBM. In this study, we investigated the role of IL-1β induced inflammation in regulating HLA-G expression. IL-1β increased HLA-G and Toll like receptor 4 (TLR4) expression in a HIF-1α dependent manner. Inhibition of TLR4 signaling abrogated IL-1β induced HLA-G. IL-1β increased HMGB1 expression and its interaction with TLR4. Inhibition of HMGB1 inhibited TLR4 and vice versa suggesting the existence of HMGB1–TLR4 axis in glioma cells. Interestingly, HMGB1 inhibition prevented IL-1β induced HLA-G expression. Elevated levels of HMGB1 and β-defensin 3 were observed in GBM tumors. Importantly, β-defensin-3 prevented IL-1β induced HLA-G, TLR4, HMGB1 expression and release of pro-inflammatory mediators. Our studies indicate that β-defensin-3 abrogates IL-1β induced HLA-G expression by negatively affecting key molecules associated with its regulation.