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。     

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.     

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。     

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。     

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.     

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 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.     

Expression of human leukocyte antigen G is associated with prognosis in nasopharyngeal carcinoma

Human leukocyte antigen G (HLA-G) has multiple immune regulatory functions including the induction of immune tolerance in malignancies. The roles of HLA-G have not been investigated in nasopharyngeal carcinoma (NPC). This study is aimed to evaluate the role of HLA-G as prognostic factor for NPC patients as well as its role in the immune regulation. Western assays showed high HLA-G expression in NPC cell lines, but low in the immortalized nasopharyngeal epithelial cell line NP69. HLA-G protein was further detected in 79.2% of 552 NPC specimens with immunohistochemistry (IHC), but not in normal nasopharyngeal epithelium tissue. Moreover, high expression of HLA-G predicted poor survival of NPC patients and positively correlated with tumor N classification and recurrence or metastasis. Multivariate analysis indicated that HLA-G was an independent and unfavorable prognostic factor. Furthermore, the presence of CD68+ macrophages and IL-10 were also examined, which are two prognostic markers of NPC and important factors for regulating immune surveillance. The correlations of HLA-G with these two immune factors were revealed in NPC tissues. Taken together, our results suggest that HLA-G is an independent biomarker for NPC prognosis, and HLA-G might contribute to NPC progression, which might jointly regulate immune surveillance in NPC together with macrophages and IL-10.     

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.     

Do nonhuman primates comprise appropriate experimental models for studying the function of human leukocyte antigen-G?

The expression and function of the human major histocompatibility complex (MHC) class Ia genes, human leukocyte antigen (HLA)-A, -B, and -C, is well-established; they are expressed in most nucleated cells and present endogenous peptides to CD8+ T cells. However, MHC class Ib genes are poorly characterized and have unknown functions. In humans, the best-characterized class Ib gene is HLA-G. This gene has a restricted tissue expression of the mRNA and a unique pattern of protein expression; it is expressed mainly in the extravillous cytotrophoblast cells in the placenta. The function of HLA-G is not clear, but its presence at the maternal-fetal interface suggests a role in protection of the semiallogeneic fetus. Whereas functional studies using in vitro models and transgenic mice provide useful insights regarding the potential function of this molecule, in vivo studies cannot be performed in humans. Nonhuman primates that are closely related to humans phylogenetically contain homologues of HLA-G. The MHC-G loci in nonhuman primates appear to have diverged from the human HLA-G. However, in the rhesus monkey (Macaca mulatta) and olive baboon (Papio anubis), a novel class Ia-related locus has been described. This gene encodes glycoproteins with characteristics that resemble those of HLA-G, including restricted tissue distribution, alternative splicing of mRNA, truncated cytoplasmic domain, and limited polymorphism. Thus, this molecule may be the functional homologue of HLA-G, and these two species may comprise appropriate models for elucidating the function of HLA-G.     

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia

Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA-G expression in malignant haematopoietic diseases are controversial, and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B-ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA-G-negative patients (P < 0.01). Total T-cell percentage was dramatically decreased in HLA-G-positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA-G-positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA-G-negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA-G expression in AML is of unfavourable clinical implications, and that HLA-G could be a potential target for therapy.     

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.     

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.     

microRNAs在肿瘤免疫中的调控作用

microRNAs（miRNAs）是一类转录后调控基因表达的内源性非编码微小RNA。愈来愈多的研究显示,miRNAs在肿瘤免疫应答中发挥重要调控作用。一方面,miRNAs通过转录后调控ICAM（intercellular adhesion molecule）、B7（CD80／86）和HLA—G（human leucocyte antigen—G）等肿瘤表面分子的表达,影响肿瘤的免疫原性;另一方面,miRNAs通过平衡肿瘤局部的细胞因子微环境或调控肿瘤免疫相关细胞的分化、发育及功能发挥,调节机体抗肿瘤免疫应答。为后续深入研究肿瘤与宿主的相互作用机制,以及发展更有效的肿瘤生物治疗手段,就目前miRNAs在肿瘤免疫中的调控作用的研究进展做一综述。     

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.     

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.     

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.     

Detection of HLA-G on human extravillous cytotrophoblast and skeletal muscle with a new monoclonal antibody MEM-G/1

Using immunohistochemistry with the newly available monoclonal antibody MEM-G/1 the reaction patterns on frozen and formaldehyde-fixed paraffin-embedded sections on human placentas, lymph nodes, skeletal muscles, and kidney and liver allografts were compared. HLA-G (a nonclassical major histocompatibility complex class I molecule that is assumed to influence the immune response during pregnancy and some pathological conditions) was found within human extravillous cytotrophoblast but not within villous cytotrophoblast and placental mesenchymal tissue. No HLA-G expression on human lymph nodes, tonsils, and kidney and liver allografts was demonstrated. However, HLA-G expression was observed in all samples of skeletal muscle. The binding capacity of monoclonal antibody MEM-G/1 provides new possibilities to study physiological and pathophysiological roles of HLA-G in humans.     

Inhibitory NK Receptor Recognition of HLA-G: Regulation by Contact Residues and by Cell Specific Expression at the Fetal-Maternal Interface

The non-classical HLA-G protein is distinguished from the classical MHC class I molecules by its expression pattern, low polymorphism and its ability to form complexes on the cell surface. The special role of HLA-G in the maternal-fetal interface has been attributed to its ability to interact with specific receptors found on maternal immune cells. However this interaction is restricted to a limited number of receptors. In this study we elucidate the reason for this phenomenon by comparing the specific contact residues responsible for MHC-KIR interactions. This alignment revealed a marked difference between the HLA-G molecule and other MHC class I molecules. By mutating these residues to the equivalent classical MHC residues, the HLA-G molecule regained an ability of interacting with KIR inhibitory receptors found on NK cells derived either from peripheral blood or from the decidua. Functional NK killing assays further substantiated the binding results. Furthermore, double immunofluorescent staining of placental sections revealed that while the conformed form of HLA-G was expressed in all extravillous trophoblasts, the free heavy chain form of HLA-G was expressed in more distal cells of the column, the invasion front. Overall we suggest that HLA-G protein evolved to interact with only some of the NK inhibitory receptors thus allowing a control of inhibition, while permitting appropriate NK cell cytokine and growth factor production necessary for a viable maternal fetal interface.