Role of HLA-G in innate immunity through direct activation of NF-kappaB in natural killer cells.

HLA-G is a non-classical HLA class I molecule involved in immunotolerance. HLA-G protects the fetus from maternal immune recognition and promotes allograft acceptance and tumor escape. Its low polymorphism and primary function, which is not peptide presentation to T lymphocytes, led us to compare the signal transduced after interaction between HLA-G and its receptor to those of innate immunity receptors with their ligands. We investigated the role of HLA-G in the classical NF-kappaB pathway in natural killer (NK) cells, which is the major pathway activated by innate immunity receptors. In NK cells stimulated with HLA-G1-expressed cells, we demonstrate that HLA-G induces the phosphorylation and the degradation of IkappaBalpha leading to nuclear translocation of NF-kappaB. This effect is independent of the presence of ILT-2 receptors and is still observed using a peptide corresponding to the alpha-1 domain of HLA-G. All these data support an unsuspected role for HLA-G in innate immunity by activating classical NF-kappaB pathway in NK cells.

Cellular co-localization of intron-4 containing mRNA and HLA-G soluble protein in melanoma analyzed by fluorescence in situ hybridization.

HLA-G5, -G6, and -G7 soluble isoforms of the immunosuppressive HLA-G molecule are produced from the splice variants of the primary HLA-G mRNA transcript containing intron-4 that encodes a specific 21 amino acids tail. In particular, HLA-G5 interacts with the inhibitory ILT2/4 and KIR2DL4 receptors that are expressed on immune cells. Acquisition of soluble HLA-G in the microenvironment may turn a HLA-G non-expressing cell into a HLA-G-bearing one. To address the question of how to distinguish cells that express soluble HLA-G generated by alternative splicing from those that have acquired HLA-G, we have developed a method capable of detecting intron-4 containing mRNA and protein in situ simultaneously. M8 melanoma cell line either transfected or not with HLA-G5 cDNA was analyzed by indirect immunofluorescence confocal microscopy using double staining with a HLA-G intron-4 digoxygenin labeled probe along with a monoclonal antibody directed against the 21 amino acid tail. The combined fluorescence in situ hybridization was also used on the HLA-G-positive choricarcinoma cell line JEG-3. This method would be helpful to follow-up bona fide HLA-G expression in a heterogeneous cell population and to elucidate the mechanisms underlying soluble HLA-G mediated immune modulation in physiological conditions such as pregnancy and pathophysiological situations such as cancer.

Hypoxia modulates HLA-G gene expression in tumor cells.

Human leukocyte antigen G (HLA-G) molecules are expressed in cytotrophoblasts and play a key role in maintaining immune tolerance at the maternal-fetal interface. HLA-G expression was also reported in inflammatory diseases, organ transplantation, and malignant tumors. The regulatory mechanisms of HLA-G gene expression differ from those of classical HLA class I genes and are still only partially elucidated. Focusing on tumor cells, we previously demonstrated a tight control of HLA-G gene expression by cis-acting epigenetic mechanisms. In the present study, we hypothesized that these processes are dependent of microenvironment conditions, and more particularly, stress conditions like hypoxia. Cellular response to hypoxia is mainly driven by a key transcription factor, hypoxia-inducible factor 1 (HIF-1), and other factors, such as NF-kappaB, involved in angiogenesis and cell survival. Here we confirmed the influence of hypoxia on HLA-G gene induction in the HLA-G-negative M8 melanoma cell line. Moreover, upon treatment with the hypoxia-mimicking desferrioxamine, we demonstrated a decrease in HLA-G gene expression in melanoma FON and choriocarcinoma JEG-3 cell lines, both expressing constitutively HLA-G. Finally, we demonstrated for the first time that the modulation of HLA-G gene expression is dependent of HIF-1 stabilization and thus might be relevant for the control of HLA-G gene expression in hypoxic tumors.

The 14 bp deletion-insertion polymorphism in the 3' UT region of the HLA-G gene influences HLA-G mRNA stability.

Human leukocyte antigen (HLA)-G molecules are generated by an alternative splicing of the primary transcript of the gene and display specialized function in regulating the immune response. Although HLA-G gene polymorphism is low, it may influence levels of protein expression and, in some cases, has been associated with pregnancy diseases. The HLA-G gene exhibits 14 alleles generating six proteins with minor variations and a null allele. HLA-G allelic variants may be also characterized by a 14 bp deletion-insertion polymorphism located in the 3' UT region of the HLA-G gene. The presence of the 14 bp insertion is known to generate an additional splice whereby 92 bases are removed from the start of exon 8. To analyze the effect of this deletion on the stability of HLA-G mRNAs, we performed actinomycin D treatments on both JEG-3 choriocarcinoma cell line and M8 melanoma cell line transfected with HLA-G*010102 allele. We observed that HLA-G mRNAs having the 92-base deletion are more stable than the complete mRNA forms, suggesting that this region may be involved in the mechanisms controlling post-transcriptional regulation of HLA-G molecule associated with allelic variants.

Switch of HLA-G alternative splicing in a melanoma cell line causes loss of HLA-G1 expression and sensitivity to NK lysis.

Considerable information has been accumulated on HLA-G expression in tumor lesions in which HLA-G is viewed as a way to turn off anti-tumoral immunity. Nevertheless, there is little data concerning the mechanisms by which expression and function of HLA-G are regulated in malignant cells. Here, we have addressed these points by studying a melanoma cell line derived from a surgically-removed HLA-G-positive melanoma lesion. We show that HLA-G expression in melanoma cells can be regulated at the mRNA splicing level. Indeed, melanoma cells rapidly switched from cell-surface HLA-G1 to intra-cellular HLA-G2 expression. This mechanism restored tumor sensitivity to NK lysis. Moreover, switch from HLA-G1 to HLA-G2 was strong enough to prevent re-expression of immunoprotective HLA-G1 even following treatments with cytokines and DNA demethylating agent. Modulating HLA-G at the mRNA splicing level would be an efficient way of lifting in vivo HLA-G-mediated tumor immune escape.

HLA-G gene activation in tumor cells involves cis-acting epigenetic changes.

The tissue distribution of HLA-G molecules is broader than originally reported in trophoblastic cells. On the basis of numerous studies, HLA-G is also expressed in malignant tumors and involved in tumor immune escape. The mechanisms of HLA-G gene regulation differ from those of classical HLA class I genes and involve epigenetic processes. Here, we provide additional evidence on the influence of DNA demethylation on HLA-G activation. We also analyze the 5' regulatory region of HLA-G in 2 cellular models, melanoma (FON, M8) and choriocarcinoma (JEG-3, JAR), either expressing HLA-G transcripts or not. The data strongly suggest that HLA-G is silenced as a result of CpG site hypermethylation within a 5' regulatory region encompassing 450 bp upstream of the start codon, whereas it is activated upon demethylation. This result correlates with the acetylation status of histones within this region and the putative locus control region located at -1.2 kb. cis-acting epigenetic changes and the fact that demethylating agents activate HLA-G expression at least 5 days following treatment should be taken into account in epigenetic cancer therapies.

HLA-G gene repression is reversed by demethylation.

The HLA-G molecule plays an important role in immune tolerance, protecting the fetus from maternal immune attack, and probably contributes to graft tolerance and tumor escape from the host immune system. HLA-G expression is tightly regulated and involves mechanisms acting in part at the transcriptional level. Nevertheless, almost all regulatory sequences that govern constitutive and inducible HLA class I gene transcription are disrupted in the HLA-G gene promoter, suggesting an unusual regulatory process. In further investigating the molecular mechanisms of HLA-G gene activation, we evaluated the influence of epigenetic mechanisms on seven HLA-G-negative cell lines that exhibit various phenotypes. Exposure of cells to histone deacetylase inhibitors, or to the demethylating agent 5-aza-2'-deoxycytidine, revealed that HLA-G gene transcription is inhibited by DNA methylation. Reversal of methylation-mediated repression may directly induce HLA-G cell-surface expression, supporting the idea that HLA-G might be activated by such a mechanism during malignancy, inflammation, and allogenic reactions.