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-G2, -G3, and -G4 isoforms expressed as nonmature cell surface glycoproteins inhibit NK and antigen-specific CTL cytolysis

HLA-G is a nonclassical MHC class I molecule that plays a major role in maternal-fetal tolerance. Four membrane-bound (HLA-G1 to -G4) and two soluble (HLA-G5, and -G6) proteins are generated by alternative splicing. Only HLA-G1 has been extensively studied in terms of both expression and function. We provide evidence here that HLA-G2, -G3, and -G4 truncated isoforms reach the cell surface of transfected cells, as endoglycosidase H-sensitive glycoproteins, after a 2-h chase period. Moreover, cytotoxicity experiments show that these transfected cells are protected from the lytic activity of both innate (NK cells) and acquired (CTL) effectors. These findings highlight the immunomodulatory role that HLA-G2, -G3, and -G4 proteins will assume during physiologic or pathologic processes in which HLA-G1 expression is altered.     

The <Emphasis Type="Italic">HLA-G*0105N</Emphasis> null allele induces cell surface expression of HLA-E molecule and promotes CD94/NKG2A-mediated recognition in JAR choriocarcinoma cell line

HLA-G is a non-classical HLA class Ib molecule primarily expressed in trophoblast cells, and is thought to play a key role in the induction of materno-fetal tolerance during pregnancy. In addition, the HLA-G gene provides a suitable leader sequence peptide capable of binding to HLA-E. However, the existence of placentas homozygous for the HLA-G*0105N null allele suggests that HLA-G1 might not be essential for fetal survival. To investigate whether expression of the HLA-G*0105N allele supports HLA-E cell surface expression, we transfected the HLA-G*0105N gene into JAR trophoblast cells. Flow cytometry analysis showed that HLA-G*0105N-transfected cells express surface HLA-E to a similar extent as the unmutated HLA-G gene, whereas HLA-G1 cell surface expression was undetectable. Using the NKL cell line in a standard ⁵¹Cr release assay, the HLA-E molecules were found to inhibit natural killer lysis, through a mechanism partially dependent on CD94/NKG2A-mediated recognition.F.G. Sala and P-M. Del Moral contributed equally to this work.     

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.     

The role of HLA-G in human pregnancy

Pregnancy in mammals featuring hemochorial placentation introduces a major conflict with the mother's immune system, which is dedicated to repelling invaders bearing foreign DNA and RNA. Numerous and highly sophisticated strategies for preventing mothers from rejecting their genetically different fetus(es) have now been identified. These involve production of novel soluble and membrane-bound molecules by uterine and placental cells. In humans, the placenta-derived molecules include glycoproteins derived from the HLA class Ib gene, HLA-G. Isoforms of HLA-G saturate the maternal-fetal interface and circulate in mothers throughout pregnancy. Uteroplacental immune privilege for the fetus and its associated tissues is believed to result when immune cells encounter HLA-G. Unequivocally demonstration of this concept requires experiments in animal models. Both the monkey and the baboon express molecules that are similar but not identical to HLA-G, and may comprise suitable animal models for establishing a central role for these proteins in pregnancy.     

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.     

HLA-G promotes immune tolerance

HLA-G is a non-classical major histocompatibility complex class I molecule that differs from the classical HLA-A, -B and -C molecules by (i) alternative splicing of mRNAs encoding for at least four membrane-bound and two soluble HLA-G isoforms, (ii) a limited polymorphism, and (iii) a tissue-restricted distribution. Studies over the past few years have elucidated the function of HLA-G demonstrating inhibition of both NK cell- and T cell-mediated cytolysis. Furthermore, aside from its expression during pregnancy, we have shown that HLA-G is also expressed in solid tumor cells (i.e. human melanoma cell lines and ex vivo melanoma biopsies). Here we present a review of the current state of knowledge of the immunotolerant functions of HLA-G and their implications in materno-fetal tolerance and tumor immunosurveillance.     

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: 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.     

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.     

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.     

A functional role of HLA-G expression in human gliomas: an alternative strategy of immune escape

HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been attributed chiefly immune regulatory functions. Glioblastoma is paradigmatic for the capability of human cancers to paralyze the immune system. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cells and brain tissues. HLA-G mRNA expression was detected in six of 12 glioma cell lines in the absence of IFN-gamma and in 10 of 12 cell lines in the presence of IFN-gamma. HLA-G protein was detected in four of 12 cell lines in the absence of IFN-gamma and in eight of 12 cell lines in the presence of IFN-gamma. Immunohistochemical analysis of human brain tumors revealed expression of HLA-G in four of five tissue samples. Functional studies on the role of HLA-G in glioma cells were conducted with alloreactive PBMCs, NK cells, and T cell subpopulations. Expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG) rendered cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response, and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8 and CD4 T cells, but appeared to be NK cell independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumor cells exerted significant immune inhibitory effects. We conclude that the aberrant expression of HLA-G may contribute to immune escape in human glioblastoma.     

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.     

<Emphasis Type="Italic">HLA-G</Emphasis> allelic variants are associated with differences in the<Emphasis Type="Italic"> HLA-G</Emphasis> mRNA isoform profile and<Emphasis Type="Italic"> HLA-G</Emphasis> mRNA levels

During pregnancy, the human extra-villous trophoblast in the contact zone between maternal and fetal tissue in the placenta does not express the classical MHC class I and II molecules. Instead, HLA-G and -C, and possibly HLA-E, are expressed. HLA-G may modulate the immunological relationship between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed at a significantly lower level than the corresponding HLA-G mRNA isoforms with the 14-bp sequence deleted. Furthermore, characteristic HLA-G mRNA isoform expression patterns were associated with specific HLA-G genotypes and alleles. In the HLA-G*01012 and - G*01013 alleles that include the 14-bp sequence, an additional alternative splicing was observed, with the first 92-bp of exon 8 spliced out. This was most pronounced in HLA-G genotypes with G*01013. These findings may have functional implications for the recent reports of aberrant HLA-G expression and reproductive success.     

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与肿瘤研究进展

人类白细胞抗原-G（human leukocyte antigen G,HLA—G1属于非经典的HLAI类分子,是机体内重要的免疫耐受分子。HLA．G可以与表达在免疫细胞上的受体结合直接发挥免疫抑制功能,同时通过诱导产生调节性T细胞（regulatory Tcells,Treg）或“Trogocytosis”机制参与机体的免疫耐受,以协助肿瘤细胞实现免疫逃逸。近年来研究发现,HLA．G在多种恶性肿瘤中均存在异常表达并抑制宿主的抗肿瘤免疫反应。对HLA-G在肿瘤中的表达及可能的作用机制研究进展作一综述。     

Role of HLA-G and other immune mechanisms in pregnancy

Pregnancy loss (abortion) and pre-eclampsia represent the most common disorders in pregnant women. Besides infection, there are anatomical, endocrinological, genetic and immunological factors that can induce pregnancy disorders. Because the exact mechanisms of physiological pregnancy maintenance are still not clearly understood, the search for genes and proteins fulfilling this role is still in progress. One of the immune molecules that plays a beneficial role in pregnancy is the nonclassical HLA-G molecule. The molecule is mainly expressed on trophoblast cells in the foetal placenta and induces the immune tolerance of the foetus via its interaction with inhibitory receptors on maternal NK cells and CD8⁺ T lymphocytes. In relation to pregnancy disorders, associations between HLA-G polymorphism, HLA-G level and HLA-G function were described. Thus, the HLA-G molecule can be used as a new diagnostic marker and, potentially, for the future therapy of pregnancy disorders.     

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。