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This study aims to determine if genetic polymorphisms in the HLA‐G gene are associated with age‐related macular degeneration (AMD). HLA‐G is important for immunological tolerance, and it is also known to have angiogenic effects. Polymorphisms in the 5′‐upstream regulatory region and 3′‐untranslated region (UTR) of HLA‐G have been associated with a number of diseases, especially with respect to a 14 bp insertion/deletion (ins/del) polymorphism in the 3′UTR. Full gene sequencing was performed on a cohort of 146 AMD patients and 63 healthy controls aged ≥60 and HLA‐G haplotypes were determined. Analyses were performed on a publicly available gene expression dataset from the NCBI GEO database (accession number GSE29801) from which expression data for HLA‐G, ‐C, and ‐A were extracted. Analysis of the GEO dataset showed that both HLA‐G and ‐C were expressed in the back of the eye and that expression was upregulated in the macular area of AMD. No differences were observed between patients and controls when analyzing the distribution of haplotypes in the HLA‐G promoter, coding region, 3′UTR, or the 14 bp ins/del polymorphism of the 3′UTR. The increased expression of HLA‐G in the macula of AMD patients indicates a role of HLA‐G in the micro environment as part of the AMD pathogenesis. This is supported by the expression of HLA‐C, which has previously been shown to play a role in AMD. The HLA‐G haplotype distribution did not display any differences between AMD patients and controls. [ABSTRACT FROM AUTHOR]

Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule from the major histocompatibility complex, which was initially shown to confer protection to the fetus from her mother’s immune system. The Third International Conference on HLA-G, held in 2003, showed that beyond its role in fetal–maternal tolerance, HLA-G exerts tolerogenic functions involved in transplant acceptance as well as in tumoral and viral immune escape. The Fourth International Conference, which took place in Paris on July 2006, counted 72 oral presentations and about 200 attendees from 25 countries. The reports presented brought new insight into HLA-G research, and we summarize here the major advances on the HLA-G biology that were reported. Abstracts for all presentations can be found in volume 68 issue number 4 of Tissue Antigens. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of wortmannin and 3-aminobenzamide (3-AB) on telomerase activity and apoptosis in two human leukaemia cells. Materials and methods: MOLT-4 (p53-wild type) and KG1a (p53-null) cells were irradiated with γ-rays (3 Gy at 1.57 Gy min-1) and the effects of wortmannin and 3-AB were evaluated. Telomerase activity was measured by polymerase chain reaction and the expression of human telomerase reverse transcriptase, human telomerase RNA and telomerase-associated protein 1 was assessed by reverse transcriptase-polymerase chain reaction. Apoptosis was evaluated by fluorescence microscopy and flow cytometry. Results: A radiation-induced up-regulation of telomerase activity was observed from 4 h post-irradiation in both cell lines. This up-regulation was abrogated by wortmannin and 3-AB. Telomerase activity was maximal 24 h post-irradiation, coinciding with an accumulation of human telomerase reverse transcriptase mRNA. Apoptosis and G2/M arrest were evident from 4 h post-irradiation in MOLT-4 cells. KG1a cells exhibited a G2/M block at 24 h post-irradiation and apoptosis increased between 24 and 48 h post-irradiation. 3-AB abolished G2/M blockage and enhanced radiation-induced apoptosis in both cell lines, while wortmannin increased apoptosis only in MOLT-4 cells. Conclusions: 3-AB inhibits the radiation-associated telomerase activity increase and enhances apoptosis in MOLT-4 and KG1a cells. Wortmannin, which also inhibits the radiation-associated telomerase activity increase in both cell lines, does not modify radiation-induced apoptosis in KG1a cells. DNA repair enzymes might be selective targets for enhancing radiosensitivity in certain tumour cells. [ABSTRACT FROM AUTHOR]

Purpose: To examine the influence of dose, dose-rate and radiation quality on telomerase activity (TA) in the KG1a hematopoietic cell line. Materials and methods: KG1a cells were irradiated with γ-rays (0.5-5 Gy) at 0.025 Gy/min, 0.30 Gy/min and 1.57 Gy/min and with a neutron/γ-ray field (5 Gy). Cell viability was determined by trypan blue exclusion. Apoptosis and cell cycle distribution were evaluated by flow cytometry. Proliferative capacity was studied by MTS assay and TA by PCR. Following 3Gy γ-irradiation, the expression of hTERT, hTR and TP1 genes was evaluated by RT-PCR. Results: Dose- and dose-rate-dependent telomerase activation with an increase in hTERT mRNA and a drop in hTP1 mRNA were observed after irradiation. Down-regulation of telomerase activity occurred in a dose-dependent manner. Although non-significant changes in short-term survival were observed after irradiation, late apoptosis became evident after G2/M arrest. Early repression of TA preceded telomerase activation in samples irradiated with a neutron/γ-ray field, in which short-term survival was affected. Conclusions: Radiation-induced telomerase activation depends on dose-rate. High-LET and low-LET irradiations induce similar changes in TA that differ mainly in their kinetics and their magnitude. Changes in TA are not related to cell-cycle redistribution nor to the induction of cell death; they are the consequence of specific regulatory responses to ionizing radiation. Mechanisms including both transcriptional and post-translational control may be involved in this regulation. [ABSTRACT FROM AUTHOR]

Data are presented on the intracellular trafficking of HLA-G protein, taking the unique features of this non-classical molecule into consideration: the existence of seven isoforms resulting from alternative splicing (HLA-G1 to G7), and reduced tail length compared with HLA class I antigens. Biochemical studies and analysis of viral strategies for escaping the host immune system led to the demonstration that (i) both the membrane-bound (HLA-G1) and the soluble (HLA-G5) forms of the molecule require peptide association for cell surface expression, using TAP-dependent or TAP-independent pathways; (ii) peptide loading onto the HLA-G protein plays a critical role in controlling the quality of the molecule reaching the cell surface; (iii) surface expression of truncated HLA-G molecules is possible, and (iv) HLA-G expression may be restricted to soluble HLA-G5. These data reveal that HLA-G presents specific cell trafficking pathways and strongly support the contention that the primary function of HLA-G is as of an inhibitor ligand for immune-competent cells. [ABSTRACT FROM AUTHOR]

Pre-eclampsia is a disorder of human pregnancy occuring in 5-10 % of all births, and represents the leading cause of infant morbidity and mortality and maternal death. In pre-eclampsia, invasion of fetal trophoblasts into maternal arteries during early pregnancy is shallow or absent. Here we examined the hypothesis that HLA-G, a non-classical class I HLA expressed in cytotrophoblasts, may act as a key gene in pre-eclampsia. We analysed HLA-G at the level of transcription and genotyped a silent CAC-CAT polymorphism in exon 3 and a 14-bp insertion/deletion in the 3′ untranslated region. A deficit in levels of the HLA-G3 transcript was observed in mild pre-eclampsia compared to normal placentas. The distribution of HLA-G polymorphisms was different between normal and pre-eclampsia samples. A correlation between the alteration in transcription of the HLA-G gene and certain HLA-G genotypes was also observed. Thus we provide the first evidence for a possible role of HLA-G in genetic susceptibility to, and pathogenesis of pre-eclampsia. [ABSTRACT FROM AUTHOR]

X-ray treatment induces a complex molecular response in hematopoietic cells leading to cell death. Using the mRNA differential display technique, we searched for genes whose expression was modified by ionizing radiation (IR) in the human p53-deficient leukemic cell line KG1a. We isolated a partial cDNA corresponding to the interferon (IFN)-inducible 1-8d gene. The expression of both 1-8d and 9-27, another gene from the same IFN-inducible family, was increased 24 and 48 h following irradiation. We did not find enhancement of either IFNgamma mRNA or interferon regulatory factor-1 (IRF-1) mRNA in irradiated KG1a cells, indicating that 1-8d and 9-27 enhancement was not due to an IFN activation. Thus, the induction of IFN-inducible genes by IR may provide a link between radiation-induced and IFN-mediated cell death. [ABSTRACT FROM AUTHOR]

Telomeric DNA protects chromosome ends from recombination events and its length serves as a mitotic clock that triggers exit from the cell cycle when telomeres become too short. Telomerase is the enzyme involved in telomere elongation, one of the events that permits unlimited cell proliferation. Variations in telomerase activity were quantified in hematopoietic cell lines after gamma-irradiation. Telomerase activity increased after irradiation of between 0 and 3 Gy in a dose-dependent manner, reaching a maximum at 3 Gy. The increase in telomerase activity was nearly maximum 8 h after irradiation, the peak being observed at around 24 h. Although this kinetics partly correlated with cell redistribution into the G2/M phase of the cell cycle, telomerase activity did not show significant variation over the cell cycle. Therefore, the activation of telomerase observed after gamma-irradiation may suggest the involvement of telomerase in DNA repair and chromosome healing. [ABSTRACT FROM AUTHOR]

HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8 + T cell cytotoxic functions. Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8 + T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition. This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2 + CD8 + T cell and ILT2 + NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Purpose: To examine the influence of dose, dose-rate and radiation quality on telomerase activity (TA) in the KG1a hematopoietic cell line., Materials and Methods: KG1a cells were irradiated with gamma-rays (0.5-5 Gy) at 0.025 Gy/min, 0.30 Gy/min and 1.57 Gy/min and with a neutron/gamma-ray field (5 Gy). Cell viability was determined by trypan blue exclusion. Apoptosis and cell cycle distribution were evaluated by flow cytometry. Proliferative capacity was studied by MTS assay and TA by PCR. Following 3Gy gamma-irradiation, the expression of hTERT, hTR and TP1 genes was evaluated by RT-PCR., Results: Dose- and dose-rate-dependent telomerase activation with an increase in hTERT mRNA and a drop in hTP1 mRNA were observed after irradiation. Down-regulation of telomerase activity occurred in a dose-dependent manner. Although non-significant changes in short-term survival were observed after irradiation, late apoptosis became evident after G2/M arrest. Early repression of TA preceded telomerase activation in samples irradiated with a neutron/gamma-ray field, in which short-term survival was affected., Conclusions: Radiation-induced telomerase activation depends on dose-rate. High-LET and low-LET irradiations induce similar changes in TA that differ mainly in their kinetics and their magnitude. Changes in TA are not related to cell-cycle redistribution nor to the induction of cell death; they are the consequence of specific regulatory responses to ionizing radiation. Mechanisms including both transcriptional and post-translational control may be involved in this regulation.

We recently reported that the nonclassical HLA class I molecule HLA-G was expressed in the endomyocardial biopsies and sera of 16% of heart transplant patients studied. The aim of the present report is to identify cells that may be responsible for HLA-G protein expression during the allogeneic reaction. Carrying out mixed lymphocyte cultures in which the responder cell population was depleted either in CD4(+) or CD8(+) T cells, we found that soluble HLA-G5 protein but not the membrane-bound HLA-G isoform was secreted by allo-specific CD4(+) T cells from the responder population, which suppressed the allogeneic proliferative T cell response. This inhibition may be reversed by adding the anti-HLA-G 87G antibody to a mixed lymphocyte culture. That may indicate a previously uncharacterized regulatory mechanism of CD4(+) T cell proliferative response.

Recent developments in the field of HLA-G research have revealed that, besides its involvement during pregnancy, HLA-G is expressed in peripheral tissues during pathological processes, such as viral infections, malignancies and organ transplantation. Here, we discuss recent findings regarding the influence of HLA-G on the T helper (Th) cytokine balance (favoring Th2-type responses), and the expression of HLA-G during chronic, cutaneous inflammatory diseases, such as psoriasis and atopic dermatitis. We propose a novel role for HLA-G as a tissue-protective molecule in inflammatory responses.

HLA-G is a nonclassical class I antigen mainly expressed at the maternofetal interface during pregnancy where it is thought to down-modulate maternal immune response against the semiallogeneic fetus. Recent studies indicate that ectopic up-regulation of HLA-G expression on melanoma cells may also favor their escape from antitumor immune response. HLA-G expression was here investigated on paraffin-embedded tumor and adjacent normal renal tissues of 18 renal cell carcinoma (RCC) patients. We provide evidence that HLA-G antigen is differentially expressed in carcinoma and normal renal cells and that up-regulation of this antigen in the tumor cells is more frequent than alterations of other MHC class I or class II antigens. We also demonstrated that HLA-G cell surface expression and secretion is maintained in a tumor cell line (DM) established from an HLA-G-positive RCC lesion. Furthermore, we show that type I (alpha and beta) and, in particular, type II (gamma) IFN treatment enhances steady-state mRNA levels and cell surface expression of HLA-G in the DM cell line. As several studies suggest that HLA-G displays various functional features that allow down-modulation of immune response in vitro, we propose that selective in vivo expression of HLA-G may participate in the impairment of antitumor immunity in RCC.

Recent data have suggested that in psoriasis, the T-infiltrating cells could be submitted to regulatory pathways, possibly through natural killer receptors. HLA-G binds to different natural killer receptors and is able to inhibit T-cell functions. Because this molecule is induced by interferon-gamma, a major cytokine in psoriasis, we asked whether HLA-G and its receptor might be expressed in this disease. Specific RNAs for HLA-G1 and HLA-G5 were consistently found in lesional skin specimens, soluble HLA-G5 transcripts being found only in psoriasis. HLA-G protein was found in all psoriatic sections, but never in normal skin controls. Double labeling demonstrated that HLA-G-positive cells were CD68(+), CD11c(+) macrophages. The NKR ILT2 was also present in psoriatic skin, the T CD4(+)-infiltrating cells expressing indeed ILT2. The demonstration of HLA-G and ILT2 expression in psoriatic skin suggests that this pathway may act as an inhibitory feed back aimed to down-regulate the deleterious effects of T-cell infiltrate in this disease.

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.

Type I interferons display a broad range of immunomodulatory functions. Interferon beta increases gene expression at the transcriptional level through binding of factors to the interferon-stimulated response element (ISRE) within the promoters of interferon-inducible genes, such as HLA class I. Despite mutation of the class I ISRE sequence within the nonclassical HLA-G class I gene promoter, we show that interferon beta enhances both transcription and cell surface expression of HLA-G in trophoblasts and amniotic and thymic epithelial cells that selectively express it in vivo. Deletion and mutagenesis analysis of a putative interferon-regulatory factor (IRF)-1 binding site within the HLA-G promoter show that HLA-G transactivation is mediated through an ISRE sequence 746 base pairs upstream from ATG, which is distinct from the interferon-responsive element described within proximal classical class I gene promoters. Electrophoretic mobility shift analysis and supershift analysis further demonstrate that interferon-responsive transcription factors, including IRF-1, specifically bind to the HLA-G ISRE. Our results provide evidence that IRF-1 binding to a functional ISRE within the HLA-G promoter mediates interferon beta-induced expression of the HLA-G gene. These observations are of general interest considering the implication of HLA-G in mechanisms of immune escape involved in fetal-maternal tolerance and other immune privilege situations.

It is now acknowledged that the pattern of HLA-G expression is not restricted to extravillous cytotrophoblast cells, as several studies described HLA-G in HLA class I+ cells, such as thymic epithelial cells, cytokine-activated monocytes and some tumors. In these situations, HLA-G may provide an additional inhibitory signal to escape from NK cell-mediated cytotoxicity. Accordingly, the aim of this study was to define the behavior of HLA-G once it is co-expressed into an HLA-A, -B, -C and -E+ cell line. For this purpose, HLA-G1 cDNA was transfected into an HLA class I+ melanoma cell line which was used as a target towards freshly isolated peripheral blood NK cells. Cytotoxic experiments using either anti-HLA-G1 or anti-HLA-G1 inhibitory receptor mAb show that HLA-G1 boosts the HLA class I-mediated inhibition of polyclonal NK cells through interaction with ILT-2, which appears as the major HLA-G1 inhibitory receptor involved. Nevertheless, HLA-G1 is also able to inhibit the cytolytic activity of an ILT-2- NK clone which otherwise expresses another HLA-G1 inhibitory receptor belonging to the KIR103 gene family. In order to more precisely define the relative role exerted by HLA-G1 versus -E on polyclonal NK cells, antibody-blocking assays were carried out using either anti-HLA class I or anti-CD94/NKG2A. Results demonstrate that in the absence of HLA-G1, the naturally expressed HLA class I-mediated NK inhibition is predominantly exerted by HLA-E through binding with CD94/NKG2A. In contrast, once HLA-G1 is expressed, it becomes the major NK inhibitory ligand.

HLA-G is a nonclassical major histocompatibility complex class I molecule that is selectively expressed on cytotrophoblasts at the feto-maternal interface where it may play a major role in maternal-fetal tolerance. In this study, we compared HLA-G expression in trophoblasts from normal and pathologic pregnancies by immunohistochemical analysis. First, we found a defective HLA-G expression in miscarriages associated with hypotrophic but normal eggs. Conversely, by studying molar pregnancies, we observed a high HLA-G expression in complete and partial hydatidiform moles. Finally, HLA-G expression could be visualized in extravillous trophoblasts that develop outside of their normal environment, as reported here in ectopic pregnancies. Taken together, these results suggest that HLA-G expression in extravillous trophoblasts is induced in an autonomous manner, independently of embryonic development, and may be an integral part of placental development allowing its tolerance from maternal immune system.

The nonclassical HLA-G primary transcript is alternatively spliced to generate several mRNAs that have the capacity to encode four membrane bound isoforms, namely HLA-G1, -G2, -G3, and -G4 and two soluble isoforms HLA-G5 and -G6. We aimed at defining the capacity of full length and truncated soluble HLA-G transcripts to be translated in human cell lines. Our study of HLA-G alternative transcripts in various human tissues led us to identify a new splice variant of the HLA-G mRNA, named G7, in which open reading frame continues in intron 2. Due to the presence of a stop codon within intron 2, HLA-G7 transcripts retain the capacity to be translated as soluble truncated HLA-G proteins bearing the alpha1 domain linked to two specific aminoacids encoded by intron 2. Expression vectors containing cDNAs encoding HLA-G5, -G6, and -G7 isoforms were transfected into human cell lines. The presence of translated HLA-G5, -G6, and -G7 proteins was detected in protein extracts of transfected cells by Western blot and immunoprecipitation, but only the full length HLA-G5 soluble isoform could be clearly detected as a secreted protein in both transfected cells supernatants and body fluids.