RESUMEN
We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with SjÓ§gren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Linfoma de Células B Grandes Difuso , Síndrome de Sjögren , Humanos , Síndrome de Sjögren/genética , Síndrome de Sjögren/complicaciones , Células B de Memoria , Factor Reumatoide , Mutación , Linfoma de Células B Grandes Difuso/genéticaRESUMEN
Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen-presenting cells for the discovery of tumor neoantigens.
Asunto(s)
Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Autorrenovación de las Células , Animales , Anticuerpos Monoclonales/biosíntesis , Formación de Anticuerpos/inmunología , Células Productoras de Anticuerpos/citología , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Descubrimiento de Drogas , Regulación de la Expresión Génica , Centro Germinal/citología , Centro Germinal/fisiología , Humanos , Memoria Inmunológica , Interleucinas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Vacunas/inmunología , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Química Clic , Virus de la Influenza A/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Linfocitos B/virología , Western Blotting , Células Cultivadas , Dimerización , Electroforesis en Gel de Poliacrilamida , Humanos , Virus de la Influenza A/clasificación , Resonancia por Plasmón de SuperficieRESUMEN
The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Influenza A/química , Animales , Anticuerpos/química , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Memoria Inmunológica , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Cinética , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Modelos Moleculares , Conformación Molecular , Especificidad de la EspecieRESUMEN
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Asunto(s)
Proteínas Bacterianas/inmunología , Glicosiltransferasas/inmunología , Interacciones Huésped-Patógeno/inmunología , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/fisiología , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catepsina G/genética , Catepsina G/inmunología , Catepsina G/metabolismo , Línea Celular Tumoral , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Secuencias Repetitivas de Aminoácido , Infecciones Estafilocócicas/enzimología , Infecciones Estafilocócicas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Antibody based therapies are increasingly applied to prevent and treat human disease. While the majority of antibodies currently on the market are chimeric or humanized antibodies from rodents, the focus has now shifted to the isolation and development of fully human antibodies. By retroviral transduction of B cell lymphoma-6 (BCL-6), which prevents terminal differentiation of B cells and, the anti-apoptotic gene B-cell lymphoma-extra large (Bcl-xL) into primary human B cells we efficiently immortalize antibody-producing B cells allowing the isolation of therapeutic antibodies. Selection of antigen-specific B cell clones was greatly facilitated because the transduced B cells retain surface immunoglobulin expression and secrete immunoglobulin into the culture supernatant. Surface immunoglobulin expression can be utilized to stain and isolate antigen specific B cell clones with labeled antigen. Immunoglobulins secreted in culture supernatant can directly be tested in functional assays to identify unique B cell clones. Here we describe the key features of our Bcl-6/Bcl-xL culture platform (AIMSelect).
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Linfocitos B/fisiología , Animales , Anticuerpos Monoclonales/biosíntesis , Técnicas de Cultivo de Célula , Separación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Descubrimiento de Drogas , Ingeniería Genética , Humanos , Proteínas Proto-Oncogénicas c-bcl-6 , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Proteína bcl-X/genéticaRESUMEN
Secondary T cell responses are enhanced because of an expansion in numbers of antigen-specific (memory) cells. Using major histocompatibility complex class II tetramers we have tracked peptide-specific endogenous (non-T cell receptor transgenic) CD4 memory T cells in normal and in costimulation-deficient mice. CD4 memory T cells were detectable after immunization for more than 200 days, although decay was apparent. Memory cells generated in CD40 knockout mice by immunization with peptide-pulsed wild-type dendritic cells survived in the absence of CD40 and proliferated when boosted with peptide (plus adjuvant) in a CD40-independent fashion. However, differentiation of the memory cells into cytokine-producing effector cells did not occur in the absence of CD40. The data indicate that memory cells can be generated without passing through the effector cell stage.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/fisiología , Diferenciación Celular , Memoria Inmunológica , Secuencia de Aminoácidos , Animales , Antígenos CD40/genética , Diferenciación Celular/inmunología , Proliferación Celular , Supervivencia Celular/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Línea Celular , Epítopos/genética , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Pruebas de Neutralización , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/terapia , Proteínas Proto-Oncogénicas c-bcl-6/genética , Porcinos , Proteína bcl-X/genéticaRESUMEN
OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen-specific B cells remains poorly understood in the context of this disease. This study set out to elucidate autoantigen-specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)-reactive B cell clones from RA patients. METHODS: B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody-positive RA patients were immortalized by genetic reprogramming with Bcl-6 and Bcl-xL. Enzyme-linked immunosorbent assay and flow cytometry were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing. RESULTS: Three unique CP-reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP-reactive memory B cells did not appear to be broadly cross-reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro- and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non-CP-reactive clones from the same patient. In addition, CP-reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding. CONCLUSION: CP-reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen-presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.
Asunto(s)
Anticuerpos Antiproteína Citrulinada/inmunología , Artritis Reumatoide/inmunología , Autoantígenos/inmunología , Linfocitos B/inmunología , Péptidos Cíclicos/inmunología , Autoanticuerpos/inmunología , Células Clonales/inmunología , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Líquido Sinovial/inmunologíaRESUMEN
CD97 is broadly expressed on hematopoietic cells and is involved in neutrophil migration. Since neutrophils are key regulators in HSC/HPC mobilization, we studied a possible role for CD97 in interleukin-8 and granulocyte-colony stimulating factor-induced HSC/HPC mobilization. Mobilization was absent in mice receiving CD97 mAb followed by interleukin-8, while granulocyte-colony stimulating factor-induced mobilization remained unaltered following anti-CD97 administration. Furthermore, combined administration of CD97 mAb and IL-8 induced a significant reduction in the neutrophilic compartment. We hypothesize that the absence of interleukin-8-induced HSC/HPC mobilization after CD97 mAb administration is due to its effect on neutrophil function.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética/métodos , Interleucina-8/farmacología , Glicoproteínas de Membrana/fisiología , Animales , Anticuerpos Monoclonales/administración & dosificación , Ensayo de Unidades Formadoras de Colonias , Esquema de Medicación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Interleucina-8/administración & dosificación , Recuento de Leucocitos , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Neutrófilos/fisiología , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The epidermal growth factor (EGF)-TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF-like domains, localized N-terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven-span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF-TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.
Asunto(s)
Evolución Molecular , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos , Animales , Callithrix , Perros , Macaca mulatta , Datos de Secuencia Molecular , Pan troglodytes , Papio hamadryas , Pongo pygmaeus , Conformación Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , SaguinusRESUMEN
Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence.
Asunto(s)
Linfocitos B/inmunología , ADN/inmunología , Interferón Tipo I/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Transformada , ADN/metabolismo , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/patología , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Masculino , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The sodium taurocholate co-transporting polypeptide (NTCP, SLC10A1) is the main hepatic transporter of conjugated bile acids, and the entry receptor for hepatitis B virus (HBV) and hepatitis delta virus (HDV). Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, effectively blocks HBV and HDV infection. In addition, Myrcludex B inhibits NTCP-mediated bile acid uptake, suggesting that also other NTCP inhibitors could potentially be a novel treatment of HBV/HDV infection. This study aims to identify clinically-applied compounds intervening with NTCP-mediated bile acid transport and HBV/HDV infection. 1280 FDA/EMA-approved drugs were screened to identify compounds that reduce uptake of taurocholic acid and lower Myrcludex B-binding in U2OS cells stably expressing human NTCP. HBV/HDV viral entry inhibition was studied in HepaRG cells. The four most potent inhibitors of human NTCP were rosiglitazone (IC50 5.1 µM), zafirlukast (IC50 6.5 µM), TRIAC (IC50 6.9 µM), and sulfasalazine (IC50 9.6 µM). Chicago sky blue 6B (IC50 7.1 µM) inhibited both NTCP and ASBT, a distinct though related bile acid transporter. Rosiglitazone, zafirlukast, TRIAC, sulfasalazine, and chicago sky blue 6B reduced HBV/HDV infection in HepaRG cells in a dose-dependent manner. Five out of 1280 clinically approved drugs were identified that inhibit NTCP-mediated bile acid uptake and HBV/HDV infection in vitro.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/metabolismo , Hepatitis B , Hepatitis D , Virus de la Hepatitis Delta/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Simportadores/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Animales , Perros , Hepatitis B/tratamiento farmacológico , Hepatitis B/metabolismo , Hepatitis B/patología , Hepatitis D/tratamiento farmacológico , Hepatitis D/metabolismo , Hepatitis D/patología , Humanos , Indoles , Lipopéptidos/farmacología , Células de Riñón Canino Madin Darby , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Fenilcarbamatos , Rosiglitazona/farmacología , Sulfasalazina/farmacología , Sulfonamidas , Simportadores/genética , Simportadores/metabolismo , Compuestos de Tosilo/farmacología , Triyodotironina/análogos & derivados , Triyodotironina/farmacología , Azul de Tripano/farmacologíaRESUMEN
The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.
Asunto(s)
Linfocitos B/metabolismo , Sulfatos de Condroitina/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Leucocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Anticuerpos Monoclonales/metabolismo , Antígenos CD , Antígenos CD55/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Humanos , Ligandos , Activación de Linfocitos , Macrófagos/metabolismo , Células Mieloides/metabolismo , Isoformas de Proteínas , Receptores Acoplados a Proteínas GRESUMEN
The EGF-TM7 family is a group of class B seven-span transmembrane (TM7) receptors expressed predominantly by cells of the immune system. Family members CD97, EMR1, EMR2, EMR3, and ETL are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain by a stalk. The EGF domain region of the recently identified EMR2 differs from that of CD97 in only 6 out of 236 amino acids. Although small, this difference has been shown to alter ligand specificity. To analyze the structure and cellular distribution of EMR2, a specific monoclonal antibody (2A1) was generated. Use of 2A1 has demonstrated EMR2, like CD97, to be expressed as a heterodimeric receptor consisting of an extracellular alpha part and a TM7/cytoplasmic beta part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16(+) blood monocytes, on macrophages, and on BDCA-3(+) myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.
Asunto(s)
Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Células Mieloides/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Anticuerpos Monoclonales/inmunología , Antígenos CD , Diferenciación Celular , Línea Celular , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/metabolismo , Dimerización , Factor de Crecimiento Epidérmico/inmunología , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Lectinas/análisis , Linfocitos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/análisis , Monocitos/inmunología , Monocitos/metabolismo , Receptores Acoplados a Proteínas G , Distribución TisularRESUMEN
EMR2 is a human myeloid-restricted member of the EGF-TM7 receptor family that contains a highly conserved G protein-coupled receptor proteolysis site (GPS) in the membrane-proximal region. Here the post-translational proteolytic cleavage of EMR2 at GPS was investigated. We show the cleavage occurs at Leu517-Ser518 and is independent of the transmembrane domains. The non-covalent association of the resulting extracellular alpha-subunit and transmembrane beta-subunit requires a minimum of eight amino acids in the beta-subunit. The GPS motif is necessary, but not sufficient for receptor cleavage, which requires the entire extracellular stalk. Thus, an alternatively spliced EMR2 isoform with a truncated stalk fails to undergo proteolytic cleavage. Alternative splicing therefore provides a means to regulate GPS cleavage, producing receptors with two distinct structures.
Asunto(s)
Endopeptidasas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Receptores Acoplados a Proteínas G , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)-positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Subgrupos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/biosíntesis , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Subgrupos de Linfocitos B/inmunología , Línea Celular , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Activación Enzimática , Citometría de Flujo , Humanos , Memoria Inmunológica , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-6 , Ratas , Receptores de Antígenos de Linfocitos B/genética , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Toxina Tetánica/inmunología , Transducción Genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.
Asunto(s)
Diferenciación Celular , Regulación hacia Abajo , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Humanos , Inmunoglobulinas/inmunología , Interleucinas/farmacología , Imitación Molecular , Fenotipo , Fosforilación/efectos de los fármacos , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-bcl-6 , Factor de Transcripción STAT3/genéticaRESUMEN
The EGF-TM7 F4/80 is a defining marker of murine macrophage populations. Applying flow cytometric analysis using the newly generated mAb A10, and quantitative real-time PCR, we here report the surprising observation that the human ortholog of F4/80, EGF-like module containing mucin-like hormone receptor (EMR)1, is absent on mononuclear phagocytic cells including monocytes, macrophages, and myeloid dendritic cells. Unexpectedly, we found that EMR1 expression is restricted to eosinophilic granulocytes, where expression is overlapping with the eotaxin receptor CCR3 and the immunoglobulin-like lectin Siglec-8. Absence on other leukocytes, including basophils, implies that EMR1 is a highly specific marker for eosinophils in humans.