RESUMO
Human decidual NK cells are massively recruited at the site of embryonic implantation (decidua basalis). They differ in many ways from their peripheral blood NK cell counterparts in terms of gene expression, phenotype and functionality. The major subpopulation of decidual NK cells is CD56(bright) whereas the minor subset is CD56(dim), contrasting with the peripheral blood NK cells whose major subpopulation is CD56(dim). Decidual NK cell cytolytic function is much reduced despite the presence of several activating receptors and the essential machinery required for lysis. Decidual NK cells produce a number of cytokines that are not normally secreted by peripheral blood NK cells. Human decidual NK cell potential functions at the maternal-fetal interface are not yet clearly established but several hypotheses are being evaluated, including control of extravillous invasion, control of uterine vascular remodeling, and local anti-viral activity.
Assuntos
Movimento Celular , Vilosidades Coriônicas/metabolismo , Decídua/citologia , Implantação do Embrião , Células Matadoras Naturais/fisiologia , Citocinas/metabolismo , Decídua/metabolismo , Feminino , Expressão Gênica , Humanos , Células Matadoras Naturais/metabolismo , Modelos Biológicos , Fenótipo , Pré-Eclâmpsia , Gravidez , Receptores Imunológicos/fisiologia , Receptores KIR , Útero/irrigação sanguíneaRESUMO
A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 x 10(9) 1/mol at pH 7.4 and 4 degrees C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor-cobalamin-receptor complex was inhibited by two ways: at 4 degrees C and at 37 degrees C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross-linked to [57Co]cobalamin-intrinsic factor and solubilized with Triton X-100. The cross-linked complex had a relative molecular mass of 330 kDa in native PAGE.
Assuntos
Endocitose , Fator Intrínseco/metabolismo , Vitamina B 12/metabolismo , Cloroquina/farmacologia , Cromatografia em Gel , Neoplasias do Colo , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Receptores de Superfície Celular/metabolismo , Células Tumorais CultivadasRESUMO
A method for screening monoclonal antibodies (McAbs) to neuropeptides was evaluated using 8-arginine-vasopressin (AVP) as a model. Mice were immunized with AVP-thyroglobulin conjugate and their spleen cells were fused with X 63-Ag8.653 mouse myeloma cells. The resulting hybridoma supernatants were screened for specific antibody production using 3 different assays: solid phase enzyme radioimmunoassay in Terasaki plates (Ter-ELISA), liquid phase radioimmunoassay (LPRIA) and an immunohistochemical technique. From 2 independent fusions, 7 McAbs specific for AVP were obtained. They belonged to the IgG1 subclass and reacted more strongly to the ring part of the nonapeptide. The screening strategy proposed relies upon a crude selection of conjugate-reacting hybridomas, followed by neuropeptide-specific hybridoma identification using both LPRIA (with radioiodinated synthetic peptide) and an immunohistochemical technique (to detect natural neuropeptide). During subcloning steps Ter-ELISA is then chosen, to select for specific clones and to eliminate those reacting with the carrier thyroglobulin.
Assuntos
Anticorpos Monoclonais/imunologia , Arginina Vasopressina/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RadioimunoensaioRESUMO
Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.
Assuntos
Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Processamento Alternativo/genética , Sequência de Bases , Northern Blotting , Linhagem Celular , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Queratinócitos/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
In contrast to HLA class Ia, the HLA-G class Ib transcripts can be alternativeley spliced to yield several isoforms including four potentially membrane-bound variants, namely HLA-G1, -G2, -G3 and G4. It is so far unclear whether each of these splice variants lacking one or two external domains is properly translated and expressed at the cell surface. We used targeted Enhanced Green Fluorescence Protein (EGFP)-HLA-G fusion cDNA to track HLA-G isoform expression in living murine (L-human beta2m) and human (JAR) transiently transfected cells. It was demonstrated that the four HLA-G1, -G2, -G3, and -G4 isoforms were translated in these transfectants by the means of (i) Western blotting analysis, using an anti-EGFP mAb; (ii) intracellular double labeling flow cytometry analysis, using the EGFP natural fluorescence and phycoerythrin-labeled HCA2 anti-HLA-G mAb; and (iii) immunocytochemistry on isolated acetone fixed transfectants with the use of different anti-HLA-G mAbs. Cell surface flow cytometry analysis using the HCA2 mAb revealed that only the HLA-G1 isoform was expressed as a membrane-bound protein. Two color confocal microscopy performed on fixed, permeabilized cells further showed that the EGFP green fluorescence co-localized with anti-calnexin rhodamine fluorescence in the four HLA-G isoform transfectants but only in HLA-G1 transfectant was the green EGFP fluorescence also detectable at the outer part of the cells, suggesting that the HLA-G2, -G3, and G4 were retained in the endoplasmic reticulum. Such intracellular retention of the three shorter forms of HLA-G suggest that they may play a role in regulating cell surface expression either of the full length HLA-G1 form or of HLA-E.
Assuntos
Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Proteínas de Membrana/biossíntese , Animais , Transporte Biológico , Retículo Endoplasmático/metabolismo , Antígenos HLA/genética , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Biossíntese de Proteínas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
This article presents an overview of the more recent data dealing with the constitutive, transcriptional, and translational expression of classical class Ia and nonclassical HLA-E and -G class Ib products in the different trophoblast cell subpopulations that constitute the maternofetal interface during human pregnancy. Of particular interest is the expression of alternatively spliced HLA-G transcriptional isoforms that may be translated in membrane-bound or soluble protein products. Molecular regulatory mechanisms that may control the differential expression of class Ia and class Ib molecules, according to the cell types, state of differentiation, and stages of gestation are also examined. They may operate at the levels of transcription, translation and/or transport of proteins to the cell surface. Functional significance of the absence of detectable cell surface expression of class Ia molecules in all trophoblast cell subpopulations, and of the presence of membrane-bound HLA-G products in extravillous cytotrophoblast cells is finally questioned.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/imunologia , Genes MHC Classe I/imunologia , Antígenos HLA/biossíntese , Antígenos HLA/genética , Placenta/imunologia , Placenta/metabolismo , Feminino , Humanos , GravidezRESUMO
PROBLEM: We investigated the possible negative regulatory mechanisms that repress classical human leukocyte antigen (HLA) class I gene expression in human spermatozoa and searched for novel testis-specific coding sequences that might be present in MHC class I chromosomal region. METHOD: We performed a comparative DNA methylation analysis of this genomic region in both purified human spermatozoa and mononuclear blood cells from the same donors, using methylation-sensitive restriction enzymes followed by classical or pulsed field gel electrophoresis and hybridization with HLA class I locus-specific probes. RESULTS: Unmethylated CpG sites were detected in the 3' part of HpaII tiny fragments of the HLA-F and HLA-G genes in spermatozoal DNA. In contrast, no difference was observed in the methylation status of the HLA-B, HLA-C, and HLA-E genes between germ and somatic cells. CpG unmethylation events were also detected in several parts of this chromosomal region (outside the known loci) in spermatozoal DNA. CONCLUSIONS: These results suggest that this genomic region undergoes changes in its DNA methylation pattern during the developmental process. We hypothesize that these dynamic changes have functional importance, including a possible transcriptional activity of nonclassical class I genes and/or as yet undescribed testis-specific coding sequences.
Assuntos
DNA/química , DNA/genética , Genes MHC Classe I , Espermatozoides/química , Espermatozoides/imunologia , Sequência de Bases , Mapeamento Cromossômico , Sondas de DNA , Eletroforese em Gel de Campo Pulsado , Antígenos HLA/genética , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Masculino , Metilação , Hibridização de Ácido NucleicoRESUMO
An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.
Assuntos
Neoplasias do Colo/metabolismo , Transcobalaminas/biossíntese , Vitamina B 12/metabolismo , Western Blotting , Cromatografia de Afinidade , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Metionina/metabolismo , Transcobalaminas/isolamento & purificação , Transcobalaminas/metabolismo , Células Tumorais CultivadasRESUMO
Human placental trophoblast cells that constitute the materno-fetal interface during pregnancy escape maternal alloimmune attack. The different trophoblast cell subpopulations have developed efficient regulatory mechanisms to prevent expression of beta2-microglobulin-associated HLA class Ia molecules at their cell surface. We previously reported the presence of HLA class Ia messages in villous cytotrophoblast cells and in the syncytiotrophoblast differentiated in vitro purified from term placenta. In this study, we found that these transcripts are translated in heavy chain proteins that are endoglycosidase H sensitive and thus retained in the endoplasmic reticulum or cis-Golgi. Moreover, these class Ia heavy chains can be co-immunoprecipitated with the chaperone protein calnexin resident in the endoplasmic reticulum. When these trophoblast cells are treated with interferon (IFN)-gamma, part of the class Ia heavy chains become endoglycosidase H resistant, demonstrating that they have left the endoplasmic reticulum. Furthermore, after such a treatment, these heavy chains are detectable at the cell surface of these trophoblast cells, as assessed by two-color flow cytometry analysis and immunoprecipitation of cell surface biotinylated proteins using the W6/32 anti-HLA class I monoclonal antibody (mAb). IFN-gamma treatment induces a significant enhancement of the transcription of transporters associated with antigen processing (TAP1 and TAP2) rather than an increase of HLA class I or beta2-microglobulin messages. Finally, we demonstrate that an anti-TAP1 mAb co-immunoprecipitates TAP1 proteins and HLA class Ia heavy chains in these IFN-gamma-treated trophoblast cells. Thus, the constitutive absence of HLA class Ia cell surface expression in term villous cytotrophoblast and syncytiotrophoblast is likely to be due to a lack of transporter proteins that participate in the proper assembly of these molecules in the endoplasmic reticulum. Such a defect can be modified upon IFN-gamma treatment.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Antígenos de Histocompatibilidade Classe I/metabolismo , Interferon gama/farmacologia , Linfonodos/citologia , Placenta/citologia , Timo/citologia , Trofoblastos/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Anticorpos Monoclonais , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Sobrevivência Celular , Células Cultivadas , Retículo Endoplasmático/metabolismo , Feminino , Antígenos HLA/metabolismo , Antígenos HLA-G , Hexosaminidases/farmacologia , Humanos , Imunofenotipagem , Gravidez , Biossíntese de Proteínas , Proteínas Recombinantes , Transcrição Gênica , Trofoblastos/citologiaRESUMO
A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.
Assuntos
Anticorpos Monoclonais , Antígenos HLA/análise , Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/biossíntese , Trofoblastos/imunologia , Animais , Especificidade de Anticorpos , Linhagem Celular , Membrana Celular/imunologia , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Antígenos HLA-G , Humanos , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Placenta , Gravidez , Primeiro Trimestre da Gravidez , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Células Tumorais CultivadasRESUMO
Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated syncytiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane.
Assuntos
Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Transcrição Gênica/genética , Trofoblastos/imunologia , Sequência de Bases , Northern Blotting , Southern Blotting , Linhagem Celular , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Técnicas Imunoenzimáticas , Metilação , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.
Assuntos
Regulação da Expressão Gênica , Antígenos HLA/fisiologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Complexo Principal de Histocompatibilidade/imunologia , Placenta/imunologia , Líquido Amniótico/imunologia , Líquido Amniótico/metabolismo , Vilosidades Coriônicas/imunologia , Vilosidades Coriônicas/metabolismo , Feminino , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo Principal de Histocompatibilidade/fisiologia , Placenta/metabolismo , Gravidez , Isoformas de Proteínas , Trofoblastos/imunologia , Trofoblastos/metabolismoRESUMO
The nonpolymorphic soluble HLA-G1 (sHLA-G1) isoform has been reported to be secreted by trophoblast cells at the materno-fetal interface, suggesting that it may act as immunomodulator during pregnancy. In this paper, we report that affinity-purified beta2-microglobulin-associated sHLA-G1 triggered apoptosis in activated, but not resting CD8+ peripheral blood cells. We demonstrate by Western blotting that sHLA-G1 enhanced CD95 ligand expression in activated CD8+ cells. Cytotoxicity was inhibited by preincubation of the cells with a CD95 antagonist mAb (ZB4) or a soluble recombinant CD95-Fc, indicating that apoptosis is mediated through the CD95/CD95 ligand pathway. Finally, we show that such sHLA-G1-induced apoptosis depends on the interaction with CD8 molecules, with cell death being blocked by various CD8 mAbs.
Assuntos
Apoptose/imunologia , Antígenos CD8/metabolismo , Antígenos HLA/fisiologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptor fas/fisiologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Antígenos CD8/fisiologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta Imunológica , Proteína Ligante Fas , Antígenos HLA/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , Solubilidade , Receptor fas/metabolismoRESUMO
Human trophoblast cells have developed various efficient regulatory mechanisms to prevent cell surface expression of the classical HLA-A, -B, and (but not always) -C class I molecules. This allows them to escape maternal alloimmune attack during pregnancy. However, recent results have demonstrated that such a lack of expression could be reversed in villous cytotrophoblast cells purified from term placenta by in vitro IFN-gamma treatment. In this context, we investigated whether both maternal and paternal HLA class Ia antigens were co-dominantly expressed in such trophoblast cells. Using polymerase chain reaction sequence-specific primers for HLA-A and HLA-C alleles, we detected transcripts of both paternal and maternal origins, showing that these genes were not affected by genomic imprinting, at least in term placenta. After in vitro IFN-gamma treatment, the polymorphic HLA-A and HLA-B antigens of both parental origins become detectable at the cell surface, as assessed by flow cytometry and/or complement-dependent microtoxicity test. Appearance of paternal antigens on trophoblast cells upon IFN-gamma induction raises the question of the in vivo biological consequences of this phenomena, in term of materno-fetal tolerance and in particular of a potential allogeneic cytotoxic immune response.
Assuntos
Alelos , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Genes MHC Classe I , Impressão Genômica/imunologia , Antígenos HLA/genética , Interferon gama/biossíntese , Trofoblastos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Dominantes/imunologia , Antígenos HLA-A/biossíntese , Antígenos HLA-A/genética , Antígenos HLA-B/biossíntese , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Humanos , Interferon gama/farmacologia , Masculino , Gravidez , Transcrição Gênica/imunologia , Trofoblastos/efeitos dos fármacosRESUMO
PROBLEM: The detection of soluble forms of human leukocyte antigen-G molecule (sHLA-G) at the maternal-fetal interface suggest that sHLA-G may play a role during pregnancy. To study the potential functions of sHLA-G, we developed a procedure to detect and produce such HLA-G isoforms. METHOD OF STUDY: Transfected cell lines expressing either sHLA-G1s cDNA (JAR-G1s) or an sHLA-G monochain DNA (Fox-G-mono) containing extracellular domains of HLA-G linked to the human beta 2-microglobulin were used. Specific sHLA-G enzyme-linked immunosorbent assay (ELISA), using anti-HLA-G monoclonal antibodies (mAbs) (87G and BFL.1) as coating antibodies and the biotinylated HLA class I mAb, W6/32, to reveal the bound molecules, was then developed. RESULTS: To assess the specificity of the ELISA, we tested cell culture supernatants from the trophoblast-derived JEG-3 cell line and the HLA-G1s-transfected JAR cells, and we detected sHLA-G in both supernatants. sHLA-G monochain was also detected by ELISA in transfected cell supernatants using the conformational mAb, W6/32, showing that the conformation of sHLA-G monochain was proper. Using the same ELISA, sHLA-G was detected in various samples of amniotic fluid. To test the potential role of sHLA-G, sHLA-G has been purified by immunoaffinity columns, using W6/32 mAb, from culture supernatants of HLA-G1s or sHLA-G monochain-transfected cells. CONCLUSION: These important tools will be useful both for the detection of sHLA-G in various biological fluids and in functional tests.
Assuntos
Líquido Amniótico/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos HLA/análise , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Anticorpos Monoclonais/imunologia , Western Blotting , Linhagem Celular , Feminino , Antígenos HLA-G , Humanos , Testes de Precipitina , Gravidez , Sensibilidade e Especificidade , Solubilidade , Transfecção , Trofoblastos/imunologia , Células Tumorais CultivadasRESUMO
Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.