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.     

Residues Met^76 and Gin^79 in HLA-G α1 domain involved in KIR2DIA recognition

Human leukocyte antigen-G (HLA-G) has long been speculated as a beneficial factor for a successful pregnancy for its restricted expression on fetal-matemal extravillous cytotrophoblasts and its capability of modulating uterine natural killer cell (uNK) function such as cytotoxicity and cytokine production through NK cell receptors. HLA class I α1 domain is an important killer cell Ig-like receptor (KIR) recognition site and the Met^76 and Gln^79 are unique to HLA-Gin this region. NK cell receptor KIR2DL4 is a specific receptor for HLA-G, yet the recognition site on HLA-G remains unknown. In this study, retroviral transduction was applied to express the wild type HLA-G (HLA-wtG), mutant HLA-G(HLA-mG) on the chronic myelogenous leukemia cell line K562 cells and KIR2DL4 molecule on NK-92 cells,respectively. KIR2DL4-IgG Fc fusion protein was generated to determine the binding specificity between KIR2DL4 and HLA-G. Our results showed that residue Met76, Gln79 mutated to Ala^76.79 in the α1 domain of HLA-G protein could affect the binding affinity between KIR2DL4 and HLA-G, meanwhile, the KIR2DL4 transfected NK-92 cells (NK-92-2DL4) showed a considerably different cytolysis ability against the HLA-wtG and HLA-mG transfected K562 targets.Taken together, our data indicated that residue Met^76 and Gin^79 in HLA-G α1 domain plays a critical role in the recognition of KIR2DL4, which could be an explanation for the isoforms of HLA-G, all containing the α1 domain, with the potential to regulate NK functions.     

Activation of NK cells by an endocytosed receptor for soluble HLA-G

Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4–containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy.     

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.     

The CD85J/leukocyte inhibitory receptor-1 distinguishes between conformed and beta 2-microglobulin-free HLA-G molecules

For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with beta(2)m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.     

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.     

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.     

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.     

Is antigen presentation the primary function of HLA-G?

Human histocompatibility leukocyte antigen (HLA)-G is an antigen-presenting molecule. This review discusses the possibility that this might not be its primary function. HLA-G indeed modulates innate immunity by interacting with immunoglobulin-like receptors and by regulating HLA-E expression and its subsequent interaction with CD94/NKG2 receptors. HLA-G also down-modulates both CD8(+) and CD4(+) T-cell responsiveness.     

Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes

HLA-G is involved in regulating T cell responses. Various mechanisms have been proposed to explain the inhibition of T cell proliferation. In this context, the possible role of HLA-G in cell cycle regulation remains to be explored. Using stably transfected M8 cells expressing the secreted isoform (HLA-G5) of HLA-G, we investigated the role of HLA-G in inducing apoptosis and in controlling the cell cycle of activated T cells. Soluble HLA-G (HLA-G5) inhibited both CD4 and CD8 T cell proliferation. However, HLA-G5 did not induce T cell apoptosis, as determined by 3,3'-diethyloxacarbocyanine and propidium iodine labeling. It induced accumulation of the retinoblastoma protein, but not its phosphorylated and active form. Treatment of activated T cells with HLA-G5 also reduced the amounts of cyclin D2, E, A, and B by >80%. In contrast, it induced an accumulation of p27kip, but not p21cip, in activated T cells. HLA-G does not induce apoptosis of alloreactive T cells, but induces p27kip1 and inhibits cell cycle progression.     

Soluble HLA-G induces NF–кB activation in natural killer cells

Human leukocyte antigen (HLA)-G is an immunomodulatory molecule discovered for the first time in the maternal–fetal interface. In cancer context, where high number of natural killer (NK) cells is described, the presence of HLA-G in its soluble form is thought to be essential for NK cells signaling. To evaluate intracellular signaling in NK cells upon HLA-G soluble forms stimulation, we investigate the role of soluble HLA-G (HLA-G5- and HLA-G1 shedding form) stimulation on classical nuclear factor (NF)–κB pathway activation. We reported that these two forms of soluble HLA-G could activate NF–κB in NK cells. NF–κB activation in NK cells does implicate neither phosphatidylinositol 3-kinase (PI3K) nor MEK (MAP kinase kinase) as demonstrated after specific inhibition experiments. We demonstrated elsewhere that NF–κB activation in NK cells is not implicated in cytotoxicity inhibition by HLA-G. Our findings may suggest the important role played by NF–κB activation after soluble HLA-G stimulation in other NK cells function.     

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.     

MiRNA-mediated control of HLA-G expression and function

HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT) of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3'UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3'UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G.     

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.     

Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.