RESUMEN
BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.
Asunto(s)
Enterotoxinas/inmunología , Pólipos Nasales/inmunología , Rinitis/inmunología , Sinusitis/inmunología , Asma Inducida por Aspirina/inmunología , Prueba de Desgranulación de los Basófilos , Basófilos/inmunología , Separación Celular , Enfermedad Crónica , Regiones Determinantes de Complementariedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Membrane-bound IgE (mIgE) on B lymphocytes is essential for IgE production. Earlier studies showed that the ε chain of mIgE (mε) on human B cells has a "long" isoform, with an extra "CεmX" domain of 52 amino acid (aa) residues between the CH4 domain and the membrane-anchor segment, as compared to the conventional "short" isoform. Because CεmX provides an antigenic site for targeting IgE-expressing B cells to down-regulate IgE production in patients with allergy, analysis of CεmX in various animals is of great interest. Hence, we analyzed the ε Ig gene, in particular, its membrane exon regions encoding the membrane anchor peptide segment and CεmX domain, of 26 species of the order Primates and 12 species of seven non-Primate orders using data obtained experimentally or retrieved from GenBank. Our analyses reveal the unexpected finding that the genes of three extant tarsier species do not contain the membrane exons for mIgE. Another striking finding is that early evolved Strepsirhini primates such as lemurs and lorises do not have gene segments for the long isoform, whereas New World monkeys such as marmosets and squirrel monkeys allow the transcription of only the long isoform. In Old World monkeys and apes, including humans, the ε gene allows the transcription of both isoforms. This work thus reveals the dramatic differences in the gene segment encoding the mε C terminal region among the four major primate lineages: the Strepsirhini primates, the tarsiers, New World monkeys, and Old World monkeys and apes/humans.
Asunto(s)
Evolución Molecular , Variación Genética , Inmunoglobulina E/genética , Primates/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Filogenia , Primates/clasificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Membrane-bound IgE (mIgE) is part of the IgE-BCR and is essential for generating isotype-specific IgE responses. On mIgE(+) B cells, the membrane-bound epsilon-chain (mepsilon) exists predominantly in the long isoform, mepsilon(L), containing an extra 52 aa CepsilonmX domain between CH4 and the C-terminal membrane-anchoring segment; the short isoform of mepsilon, mepsilon(S), exists in minor proportions. CepsilonmX thus provides an attractive site for immunologic targeting of mIgE(+) B cells. In this study, we show that nine newly prepared CepsilonmX-specific mAbs, as well as the previously reported a20, bound to mIgE.Fc(L)-expressing CHO cells, while only 4B12 and 26H2 bound to mIgE.Fc(L)-expressing B cell line Ramos cells. The mAb 4B12 bound to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CepsilonmX. Expression of Igalpha and Igbeta on the mIgE.Fc(L)-CHO cells reduces the binding of a20 to CepsilonmX as compared with that of 4B12 and 26H2. The chimeric mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, lysed mIgE.Fc(L)-Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 demonstrated Ab-dependent cellular cytotoxicity toward mIgE.Fc(L)-Ramos cells in a dose-dependent fashion. In cultures of PBMCs from atopic dermatitis patients, c4B12 and c26H2 inhibited the synthesis of IgE driven by anti-CD40 and IL-4. These results suggest that 4B12 and 26H2 and an immunogen using the peptide segments recognized by these mAbs are potentially useful for targeting mIgE(+) B cells to control IgE production.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/metabolismo , Marcación de Gen , Inmunoglobulina E/biosíntesis , Cadenas epsilon de Inmunoglobulina/biosíntesis , Receptores de Antígenos de Linfocitos B/metabolismo , Secuencia de Aminoácidos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Apoptosis/genética , Apoptosis/inmunología , Sitios de Unión de Anticuerpos/genética , Células CHO , Línea Celular , Línea Celular Tumoral , Células Clonales , Cricetinae , Cricetulus , Humanos , Inmunoglobulina E/genética , Inmunoglobulina E/metabolismo , Cadenas epsilon de Inmunoglobulina/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Anti-interferon (IFN)-γ autoantibodies (AIGAs) are a pathogenic factor in late-onset immunodeficiency with disseminated mycobacterial and other opportunistic infections. AIGAs block IFN-γ function, but their effects on IFN-γ signaling are unknown. Using a single-cell capture method, we isolated 19 IFN-γ-reactive monoclonal antibodies (mAbs) from patients with AIGAs. All displayed high-affinity (KD < 10-9 M) binding to IFN-γ, but only eight neutralized IFN-γ-STAT1 signaling and HLA-DR expression. Signal blockade and binding affinity were correlated and attributed to somatic hypermutations. Cross-competition assays identified three nonoverlapping binding sites (I-III) for AIGAs on IFN-γ. We found that site I mAb neutralized IFN-γ by blocking its binding to IFN-γR1. Site II and III mAbs bound the receptor-bound IFN-γ on the cell surface, abolishing IFN-γR1-IFN-γR2 heterodimerization and preventing downstream signaling. Site III mAbs mediated antibody-dependent cellular cytotoxicity, probably through antibody-IFN-γ complexes on cells. Pathogenic AIGAs underlie mycobacterial infections by the dual blockade of IFN-γ signaling and by eliminating IFN-γ-responsive cells.
Asunto(s)
Infecciones por Mycobacterium , Receptores de Interferón , Anticuerpos Monoclonales , Autoanticuerpos , Impedancia Eléctrica , Humanos , Interferón gamma , Infecciones por Mycobacterium/genética , Infecciones por Mycobacterium/microbiología , Receptores de Interferón/genéticaRESUMEN
Over the last 2 decades, omalizumab is the only anti-IgE antibody that has been approved for asthma and chronic spontaneous urticaria (CSU). Ligelizumab, a higher-affinity anti-IgE mAb and the only rival viable candidate in late-stage clinical trials, showed anti-CSU efficacy superior to that of omalizumab in phase IIb but not in phase III. This report features the antigenic-functional characteristics of UB-221, an anti-IgE mAb of a newer class that is distinct from omalizumab and ligelizumab. UB-221, in free form, bound abundantly to CD23-occupied IgE and, in oligomeric mAb-IgE complex forms, freely engaged CD23, while ligelizumab reacted limitedly and omalizumab stayed inert toward CD23; these observations are consistent with UB-221 outperforming ligelizumab and omalizumab in CD23-mediated downregulation of IgE production. UB-221 bound IgE with a strong affinity to prevent FcÔRI-mediated basophil activation and degranulation, exhibiting superior IgE-neutralizing activity to that of omalizumab. UB-221 and ligelizumab bound cellular IgE and effectively neutralized IgE in sera of patients with atopic dermatitis with equal strength, while omalizumab lagged behind. A single UB-221 dose administered to cynomolgus macaques and human IgE (ε, κ)-knockin mice could induce rapid, pronounced serum-IgE reduction. A single UB-221 dose administered to patients with CSU in a first-in-human trial exhibited durable disease symptom relief in parallel with a rapid reduction in serum free-IgE level.
Asunto(s)
Omalizumab , Urticaria , Animales , Anticuerpos Monoclonales Humanizados , Regulación hacia Abajo , Humanos , Inmunoglobulina E , Ratones , Omalizumab/farmacología , Omalizumab/uso terapéutico , Urticaria/tratamiento farmacológico , Urticaria/genéticaRESUMEN
The epsilon chain of membrane-bound IgE (mIgE) is expressed predominantly as a "long" isoform, containing an extra segment of 52 amino acid (a.a.) residues, referred to as C epsilon mX, between the CH4 domain and the C-terminal membrane-anchoring transmembrane peptide. C epsilon mX results from an alternative splicing of the epsilon RNA transcript at 156-bp upstream of the splicing acceptor site used by the "short" isoform. Here, based on an analysis of the C epsilon mX genomic DNA sequences of 320 subjects residing in Taiwan, we report that single-nucleotide polymorphisms have been found at two positions, namely, G/T at #46 and A/G at #93 (along the 156 bp of C epsilon mX), with the former creating an amino acid change from Val to Leu at #16 (along the 52 a.a. of C epsilon mX) and the latter resulting in no change (Gly). Among the 640 C epsilon mX sequences identified, the previously known 46G93A allelic form appeared 293 times, the newly discovered 46T93A allelic form (GeneBank accession no. GU208817) 26 times, and the 46G93G allelic form (GU208818) 321 times. No 46T93G allelic form was found. Serum IgE measurements showed that the polymorphisms did not correlate with the levels of serum IgE. The anti-C epsilon mX monoclonal antibody, 4B12, could bind equally well to mIgE.Fc(L)(16V) and mIgE.Fc(L)(16L). While genetic variation of C epsilon mX of broader populations should also be investigated, these newly discovered genetic variants of C epsilon mX in the Taiwanese population do not seem to affect the feasibility of using an anti-C epsilon mX mAb, such as 4B12, to target mIgE-expressing B cells.
Asunto(s)
Asma/genética , Asma/inmunología , Inmunoglobulina E/química , Inmunoglobulina E/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Taiwán , Adulto JovenRESUMEN
In humans, IgA exists as two subclasses, IgA1 and IgA2, which contain distinct alpha1 and alpha2 heavy chains, respectively. Both subclasses also have membrane-bound forms (mIgA1 and mIgA2) containing the corresponding malpha1 and malpha2 heavy chains, which differ from alpha1 and alpha2 by an additional "membrane-anchor" peptide segment extending from the CH3 domain of alpha1 and alpha2. The membrane-anchor segment has three parts: an extracellular, a transmembrane, and an intracellular segment. The heavy chain malpha1 exists in short and long isoforms, referred to as malpha1S and malpha1L, with the latter containing extra 6 amino acid residues, GSCSVA, at the N-terminus of the extracellular segment (residues 453-458). By studying the genomic and mRNA sequences of malpha1 and malpha2 from 30 individuals residing in Taiwan, we have found that, in addition to the known malpha1 allele, referred to as malpha1(456S), malpha1 also has a previously unknown allele, referred to as malpha1(456C) (GenBank accession no. EU431191). This newly identified allele is present in the donor population at a similar proportion to malpha1(456S), and appears to exist only as the long isoform, i.e. malpha1L, rather than the short isoform, malpha1S. Furthermore, we confirmed that malpha2 exists only as the short isoform. Future studies will examine whether these mIgA1 variations affect the regulation of IgA synthesis and whether mIgA1 can provide an antigenic site for the immunological targeting of IgA-expressing B cells.
Asunto(s)
Inmunoglobulina A/genética , Proteínas de la Membrana/genética , Polimorfismo Genético , Alelos , Empalme Alternativo/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Humanos , Inmunoglobulina A/metabolismo , Linfocitos/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/metabolismo , Inmunoglobulina E/química , Inmunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Linfocitos B/inmunología , Cristalografía por Rayos X , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Mastocitos/inmunología , Unión Proteica , Conformación ProteicaRESUMEN
Surface IgD and IgM doubly positive cells comprise the major population of B cells in the human immune system. The heavy chain of membrane-bound IgD (mδ) differs from that of IgD (δ) in that mδ contains a C-terminal membrane-anchor peptide. Our group previously proposed that the N-terminal extracellular segment of 27 aa residues of the membrane-anchor peptide of mδ, referred to as the mIg isotype-specific-δ (migis-δ) segment, may provide a unique antigenic site for isotype-specific targeting of mIgD(+) B cells. Here we report the preparation of mouse mAbs specific for human migis-δ. The mAbs bound to human migis-δ-containing recombinant proteins in an ELISA and to mIgD-expressing transfectants of a CHO cell line as analyzed by flow cytometry. MAb 20E6, which binds to an epitope toward the N-terminal of human migis-δ, could stain human B cell line MC116, which expressed mIgD and mIgM. MC116 cells could be induced to undergo apoptosis by treatment with 20E6 in the presence of a second crosslinking antibody. Chimeric 20E6 caused antibody-dependent cellular cytotoxicity of MC116 cells in the presence of human PBMCs as the source of effector cells. In cultures of PBMCs, 20E6 down-regulated the population of mIgD(+) B cells. The production of human IgM by transplanted MC116 cells in NOD-SCID (NOD.CB17-Prkdc(scid)/IcrCrlBltw) mice could be suppressed by 20E6. These results encourage further investigation of the potential of anti-migis-δ mAbs to control mIgD(+) B cells, when such a manipulation may alleviate a disease state.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales de Origen Murino/inmunología , Linfocitos B/inmunología , Inmunoglobulina D/metabolismo , Secuencia de Aminoácidos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos B/trasplante , Células CHO , Línea Celular , Cricetinae , Cricetulus , Epítopos de Linfocito B/genética , Humanos , Inmunoglobulina D/genética , Inmunoglobulina M/genética , Inmunoglobulina M/metabolismo , Cadenas delta de Inmunoglobulina/genética , Cadenas delta de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Resonancia por Plasmón de Superficie , Trasplante HeterólogoRESUMEN
CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/-) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3â¼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.
Asunto(s)
Antígenos de Diferenciación/genética , Macrófagos del Hígado/metabolismo , Lectinas Tipo C/genética , Listeriosis/genética , Hígado/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/inmunología , Animales , Antígenos de Diferenciación/inmunología , Sitios de Unión , Secuencia de Carbohidratos , Embrión de Mamíferos , Galactosa/química , Galactosa/inmunología , Galactosilceramidas/química , Galactosilceramidas/inmunología , Regulación del Desarrollo de la Expresión Génica , Glicosilación , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/microbiología , Lectinas Tipo C/química , Lectinas Tipo C/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/metabolismo , Listeriosis/microbiología , Hígado/inmunología , Hígado/microbiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/microbiología , Unión ProteicaRESUMEN
Type-I hypersensitivity reactions play a critical role in the pathogenesis of various allergic diseases. The successful development of the anti-IgE antibody, omalizumab, has validated IgE as an effective therapeutic target for the treatment of various IgE-mediated allergic diseases. Two research groups have reported that mAbs specific for certain parts of CÉmX, a domain of 52 aa residues in human membrane-bound IgE (mIgE), can cause the lysis of mIgE-B cells by apoptosis and antibody-dependent cellular cytotoxicity (ADCC). Herein, we explore virus-like particles formed by hepatitis B virus core antigen (HBcAg) that harbors the entire CÉmX peptide or its fragments as immunogens for inducing anti-CÉmX antibodies. The results showed that mice immunized subcutaneously with these immunogens produced antibodies that bind to recombinant CÉmX-containing human IgE.Fc in ELISA and Western blot analyses, and to genetically engineered human mIgE-expressing Ramos B cell line in fluorescence flow cytometric assays. The IgG antibodies purified from the sera of immunized mice were able to cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG could mediate ADCC in human mIgE-expressing A20 murine B-cell lymphoma. These studies suggest that HBcAg-CÉmX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Apoptosis , Linfocitos B/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Inmunoglobulina E/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos B/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Virus de la Hepatitis B/inmunología , Humanos , Inmunoglobulina E/química , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
A new monoclonal antibody (mAb), specific for human IgE, the central mediator of immediate-type hypersensitivity reactions, has been shown to possess a unique set of binding specificities. The mAb, 8D6, binds to a conformational epitope on the CH3 domain of human e immunoglobulin and can compete with omalizumab for binding to IgE. Like omalizumab, it does not bind to IgE bound by the high-affinity IgE.Fc receptor (FcÉRI) on basophils and mast cells. It also does not cause activation and degranulation of IgE-pulsed, human FcÉRI-expressing rat basophilic leukemic cells (RBL SX-38). The mAb can inhibit IgE binding to recombinant α chain of human FcÉRI in ELISA and to human FcÉRI-expressing RBL SX38 cells in fluorescence flow cytometric analysis. However, unlike omalizumab, 8D6 can bind to IgE already bound by the low-affinity IgE.Fc receptors (FcÉRII, or CD23), as revealed in ELISA with recombinant CD23 and in flow cytometric analysis with human B cells. Since earlier investigators have shown that anti-CD23 mAbs can inhibit the synthesis of IgE in lymphocyte culture in vitro and can down-regulate IgE production in treated patients, 8D6 may offer pharmacological mechanisms in addition to those mediated by omalizumab, for controlling IgE in patients with allergic diseases.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/inmunología , Receptores de IgE/inmunología , Animales , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Especificidad de Anticuerpos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/metabolismo , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática , Epítopos , Humanos , Hipersensibilidad Inmediata/tratamiento farmacológico , Hipersensibilidad Inmediata/metabolismo , Inmunoglobulina E/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Omalizumab , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Receptores de IgE/metabolismoRESUMEN
Membrane-bound IgA (mIgA) is associated with Igα/Igß as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα) contains a C-terminal membrane-anchor peptide, which encompasses extracellular, transmembrane and intracellular segments. The extracellular segment, referred to as the mIg isotype-specific (migis-α) segment or the extracellular membrane proximal domain of mα, has been proposed to be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-α. The mAbs bound strongly to synthetic peptides of migis-α and to various recombinant proteins containing migis-α as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-α region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-α-specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.