Characterization of monoclonal antibodies recognizing HLA-G or HLA-E: New tools to analyze the expression of nonclassical HLA class I molecules

ArticleinHuman Immunology 64(3):315-26 · April 2003with12 Reads
DOI: 10.1016/S0198-8859(02)00821-2 · Source: PubMed
Nonclassical major histocompatibility complex (MHC) class I human leukocyte antigen E (HLA-E) and HLA-G molecules differ from classical ones by specific patterns of transcription, protein expression, and immunotolerant functions. The HLA-G molecule can be expressed as four membrane-bound (HLA-G1 to -G4) and three soluble (HLA-G5 to -G7) proteins upon alternative splicing of its primary transcript. In this study, we describe a new set of monoclonal antibodies (mAbs) called MEM-G/01, -G/04, -G/09, -G/13, MEM-E/02, and -E/06 recognizing HLA-G or HLA-E. The pattern of reactivity of these mAbs were analyzed on transfected cells by flow cytometry, Western blotting, and immunochemistry. MEM-G/09 and -G/13 mAbs react exclusively with native HLA-G1 molecules, as the 87G mAb. MEM-G/01 recognizes (similar to the 4H84 mAb) the denatured HLA-G heavy chain of all isoforms, whereas MEM-G/04 recognizes selectively denatured HLA-G1, -G2, and -G5 isoforms. MEM-E/02 and -E/06 mAbs bind the denatured and cell surface HLA-E molecules, respectively. These mAbs were then used to analyze the expression of HLA-G and HLA-E on freshly isolated cytotrophoblast cells, on the JEG-3 placental tumor cell line, and on cryopreserved and paraffin-embedded serial sections of trophoblast tissue. These new mAbs represent valuable tools to study the expression of HLA-G and HLA-E molecules in cells and tissues under normal and pathologic conditions.
    • "In contrast to classical HLA class I genes, which are very polymorphic and ubiquitously expressed, the HLA-G gene has a very low level of polymorphism and highly restricted distribution under non-pathological situations: trophoblast [44], thymus [45], cornea [46], pancreas [47], and erythroid and endothelial precursors [48]. Apart from its expression in adult immune privileged organs and in cells of the hematopoietic lineage, induction of HLA-G protein expression can be frequently observed in certain pathological situations such as cancer, transplantation, and viral infectious diseases [49][50][51][52][53]. Indeed, HLA-G has been detected in nearly thirty types of malignancies of distinct origin including melanoma, carcinoma (breast, renal, ovarian, lung, and colorectal), lymphoma and leuke- mia [54,55]. "
    [Show abstract] [Hide abstract] ABSTRACT: Radiotherapy has been employed for the treatment of oncological patients for nearly a century, and together with surgery and chemotherapy, radiation oncology constitutes one of the three pillars of cancer therapy. Ionizing radiation has complex effects on neoplastic cells and on tumor microenvironment: beyond its action as a direct cytotoxic agent, tumor irradiation triggers a series of alterations in tumoral cells, which includes the de novo synthesis of particular proteins and the up/down-regulation of cell surface molecules. Additionally, ionizing radiation may induce the release of "danger signals" which may, in turn lead to cellular and molecular responses by the immune system. This immunomodulatory action of ionizing radiation highlights the importance of the combined use (radiotherapy plus immunotherapy) for cancer healing. Major histocompatibility complex antigens (also called Human Leukocyte Antigens, HLA in humans) are one of those molecules whose expression is modulated after irradiation. This review summarizes the modulatory properties of ionizing radiation on the expression of HLA class I (classical and non-classical) and class II molecules, with special emphasis in non-classical HLA-I molecules.
    Full-text · Article · Apr 2016
    • "The anti-HLA-E specific monoclonal antibody (mAb) MEM-E/02 (MBL International, Woburn, MA, USA) was used. MEM-E/02 mAb recognizes the denatured form of HLA-E and its HLA-E specificity was previously shown to serve in immunoblotting and immunocytochemistry (Rabreau et al., 2003 ). The monoclonal and polyclonal antibodies employed in this study are listed inTable 1. "
    [Show abstract] [Hide abstract] ABSTRACT: The expression of endothelial HLA-E in the context of the systemic inflammatory response observed in preeclampsia has not been established. An experimental study was designed to determine the effect of the sera of pregnant women on the expression of HLA-E in EA.hy296 endothelial cells. First, measurements of protein fractions were performed in sera from early-onset, severely preeclamptic women without HELLP syndrome, in which there was no significant difference in total proteins between the groups, but a reduced level of plasma albumin and an increase in α1-globulin were observed in both groups of pregnant women compared with non-pregnant women. Measurements of colloid osmotic pressure (COP) using a recalculated albumin/globulin ratio formula determined only a significant decrease in COP in all pregnant groups compared with non-pregnant women. The expression of membrane HLA-E was increased in EA.hy296 endothelial cells stimulated with sera of early-onset, severely preeclamptic women, while recombinant interferon-γ (IFN-γ) significantly reduced the expression of membrane HLA-E. Pro-inflammatory cytokines were measured by Luminex in the serum samples, and increased levels of Tumor Necrosis Factor (TNF) and decreased levels of IFN-γ were observed in early-onset, severe preeclampsia compared with normal pregnancy. Moreover, soluble HLA-E was detected in these serum samples by Western blot and ELISA, but no significant difference was found. This raises the possibility that a systemic inflammatory response promotes a compensatory mechanism of COP balance in severe preeclampsia by release of inflammation-induced factors, including endothelial HLA-E. Evidence is now provided regarding HLA-E expression by EA.hy296 cells.
    Full-text · Article · Oct 2014
    • "Soluble HLA-G concentrations were evaluated by a specific sandwich ELISA in plasma using MEM-G/9 [23] and anti-human β2-microglobulin as capture and detection antibodies respectively [24]. Microtitration plates (Corning Incorporated, USA) were coated overnight at 4°C with 10 μg/mL MEM-G/9 Mouse-anti-human HLA-G mAb (ExbioPraha, Czech Republic). "
    [Show abstract] [Hide abstract] ABSTRACT: Background The immunosuppressive properties of HLA-G protein can create a tolerogenic environment that may allow Plasmodium falciparum to avoid host immune responses. There are known associations between high levels of circulating soluble HLA-G (sHLA-G) and either parasite or viral infections and it has been suggested that the induction of sHLA-G expression could be a mechanism via which infectious agents subvert host immune defence. The study presented here is the first to investigate the possible association between sHLA-G and malaria or malaria related risk factors in Benin. Methods A parasitological and clinical follow-up of 165 mothers and their newborns from delivery through to one year of age was conducted in the Tori Bossito area of southern Benin. Plasma levels of sHLA-G were determined by ELISA in maternal peripheral and cord blood and again in infants' peripheral blood at 3, 6, 9 and 12 months of age. The associations between the levels of sHLA-G and malaria risk factors were investigated through multivariate mixed models. Results Strong correlations were observed between the maternal and cord plasma concentrations of sHLA-G. In multivariate analyses, high cord plasma levels of sHLA-G were independently associated with (i) low birth weight and (ii) an increased risk of P. falciparum infection in infancy. Conclusion These results show for the first time the possible involvement of sHLA-G in generating immune tolerance during pregnancy-associated malaria. Soluble HLA-G may represent a useful marker of susceptibility to malaria in infants and be associated with the higher susceptibility to infection observed for LBW children.
    Full-text · Article · Aug 2014
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