Characterization of monoclonal antibodies recognizing HLA-G or HLA-E: New tools to analyze the expression of nonclassical HLA class I molecules
University of Zaragoza, Caesaraugusta, Aragon, Spain Human Immunology
(Impact Factor: 2.14).
04/2003; 64(3):315-26. DOI: 10.1016/S0198-8859(02)00821-2
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.
Available from: Peter J.K. Kuppen
- "For HLA-E and HLA-G identification mouse monoclonal antibodies against HLA-E (ab2216 clone MEM-E/02: AbCam) and HLA-G (4H84: Exbio, Czech Republic) were used
. MEM-E/02 recognizes denatured HLA-E
[37,38], while 4H84 recognizes denatured HLA-G molecules and also binds to free heavy chains of classical HLA class I molecules
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Evasion of immune surveillance and suppression of the immune system are important hallmarks of tumorigenesis. The goal of this study was to establish distinct patterns that reflect a rectal tumors’ immune-phenotype and to determine their relation to patient outcome.
The study population consisted of 495 Stage I-IV non-preoperatively treated rectal cancer patients of which a tissue micro array (TMA) was available. Sections of this TMA were immunohistochemically stained and quantified for presence of Foxp3+ cells (Tregs) and tumor expression of HLA Class I and non-classical HLA-E and HLA-G. All markers were, separate and combined, analyzed for clinical prognostic value.
Expression of HLA class I (DFS HR 0.637 (0.458-0.886), p = 0.013), Foxp3+ infiltration above median (OS HR 0.637 (0.500-0.813), p < 0.001 and DFS HR 0.624 (0.491-0.793), p < 0.001) and expression of HLA-G (DFS HR 0.753 (0.574-0.989), p = 0.042) were related to a better clinical prognosis. When these markers were combined, patients with 2 or 3 markers associated with poor prognosis (loss of HLA Class I, Foxp3+ below median, and weak HLA-G expression), showed a significantly worse survival (OS and DFS p < 0.001). This immune-phenotype was an independent predictor for DFS (HR 1.56 (1.14-2.14), p = 0.019).
In conclusion, rectal tumors showing loss of HLA class I expression, Foxp3+ infiltration below median and weak HLA-G expression were related to a worse OS and DFS. Combining these immune markers lead to the creation of tumor immune-phenotypes , which related to patient outcome and were significant independent clinical prognostic markers in rectal cancer.
Available from: Ikuo Konishi
- "FITC-and phycoerythrin (PE)-conjugated mouse anti-human HLA-G (clone MEM-G/9, IgG1) mAbs were both obtained from Abcam (Cambridge, UK). This HLA-G antibody can only react with native HLA-G1 isotype that is specifically expressed on human EVT (Menier et al., 2003). FITC-conjugated and non-conjugated mouse IgG1 (clone DAK-GO1), IgG2a (clone DAK-GO5) and IgG2b (clone DAK-GO9) for negative controls were obtained from Dako (Glostrup, Denmark). "
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ABSTRACT: In primate placenta, extravillous trophoblast (EVT) invades maternal tissue in temporally- and spatially-regulated fashions. We previously identified a novel placenta-specific cell-surface aminopeptidase, laeverin/aminopeptidase Q, which is expressed on EVT-lineage cells in the fetal membrane. Laeverin possesses a peptide-binding site that is evolutionally unique to primates, suggesting possible involvement of laeverin in a primate-specific phenomenon during placentation. Thus, this study was designed to elucidate the molecular characteristics and physiological roles of laeverin in human EVT.
Placental tissues of various developmental stages were subjected to immunostaining and western blotting. Effects of siRNA and a soluble form of recombinant laeverin on EVT cells isolated from primary villous explant cultures were examined using Matrigel invasion assays and cell proliferation assays.
Laeverin was specifically immunolocalized to HLA-G-positive EVT in placentas from early and term pregnancy. In primary villous explant cultures, laeverin expression was induced on the cell surface of the outgrowing EVT. In western blotting, laeverin protein was detected as two distinct bands at 130 and 160 kDa along with a broad band ranging from 200 to 270 kDa. De-glycosylation treatment showed that these native laeverin isotypes are N-linked glycoproteins sharing a common 115-kDa core protein. In invasion assays, the reduction of laeverin expression by siRNA suppressed migration of the isolated EVT, while the soluble form of recombinant laeverin enhanced its migration.
Laeverin is a specific cell-surface marker for human EVT and plays a regulatory role in EVT migration.
Available from: Elisa lo monaco
- "The mouse monoclonal antibodies MEM-E/02, MEM-E/06, MEM-E/07, and MEM-E/08    are all from Exbio, Prague, Czech Republic; 3D12, 4D12  , W6/32 , Namb-1 , L31 , and a polyclonal to ERp57 were used in previous publications of ours [22,26–29]. The reverse surface biotin labeling method is described . "
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ABSTRACT: The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.
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