RESUMEN
Ideally, a vaccine should provide life-long protection following a single administered dose. In our previous study, the immunopotentiator CVC1302, which contains pattern- recognition receptor (PRR) agonists, was demonstrated to prolong the lifetime of the humoral immune response induced by killed foot-and-mouth disease virus (FMDV) vaccine. To elucidate the mechanism by which CVC1302 induces long-term humoral immunity, we used 4-hydroxy-3-nitrophenylacetyl (NP)-OVA as a pattern antigen and administered it to mice along with CVC1302, emulsified together with Marcol 52 mineral oil (NP-CVC1302). From the results of NP-specific antibody levels, we found that CVC1302 could induce not only higher levels of NP-specific antibodies but also high-affinity NP-specific antibody levels. To detect the resulting NP-specific immune cells, samples were taken from the injection sites, draining lymph nodes (LNs), and bone marrow of mice injected with NP-CVC1302. The results of these experiments show that, compared with mice injected with NP alone, those injected with NP-CVC1302 had higher percentages of NP+ antigen-presenting cells (APCs) at the injection sites and draining LNs, higher percentages of follicular helper T cells (TFH), germinal center (GC) B cells, and NP+ plasma-blasts in the draining LNs, as well as higher percentages of NP+ long-lived plasma cells (LLPCs) in the bone marrow. Additionally, we observed that the inclusion of CVC1302 in the immunization prolonged the lifetime of LLPCs in the bone marrow by improving the transcription expression of anti-apoptotic transcription factors such as Mcl-1, Bcl-2, BAFF, BCMA, Bax, and IRF-4. This research provides a blueprint for designing new generations of immunopotentiators.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos/administración & dosificación , Inmunidad Humoral/efectos de los fármacos , Nitrofenoles/administración & dosificación , Ovalbúmina/administración & dosificación , Fenilacetatos/administración & dosificación , Receptores de Reconocimiento de Patrones/agonistas , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Linfocitos B/inmunología , Femenino , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Nitrofenoles/inmunología , Ovalbúmina/inmunología , Fenilacetatos/inmunología , Linfocitos T/inmunologíaRESUMEN
Ageing is a complex multifactorial process associated with a plethora of disorders, which contribute significantly to morbidity worldwide. One of the organs significantly affected by age is the gut. Age-dependent changes of the gut-associated microbiome have been linked to increased frailty and systemic inflammation. This change in microbial composition with age occurs in parallel with a decline in function of the gut immune system; however, it is not clear whether there is a causal link between the two. Here we report that the defective germinal centre reaction in Peyer's patches of aged mice can be rescued by faecal transfers from younger adults into aged mice and by immunisations with cholera toxin, without affecting germinal centre reactions in peripheral lymph nodes. This demonstrates that the poor germinal centre reaction in aged animals is not irreversible, and that it is possible to improve this response in older individuals by providing appropriate stimuli.
Asunto(s)
Envejecimiento/inmunología , Disbiosis/inmunología , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal/inmunología , Centro Germinal/inmunología , Ganglios Linfáticos Agregados/inmunología , Animales , Toxina del Cólera/inmunología , Femenino , Inmunización , Inmunoglobulina A/inmunología , Ratones , Nitrofenoles/inmunología , Fenilacetatos/inmunologíaRESUMEN
An electrochemical immunosensor for ultrasensitive detection of acrylamide (AA) in water and food samples was developed. SnO2-SiC hollow sphere nanochains with high surface area and gold nanoparticles with good electroconductivity were fabricated onto the surface of a glassy carbon electrode pre-coated with chitosan. The coating antigen (AA-4-mercaptophenylacetic acid-ovalbumin conjugate, AA-4-MPA-OVA) was immobilized on the electrode. Polyclonal antibody specific for AA-4-MPA was conjugated to gold nanorod (AuNR) as primary antibody (AuNR-Ab1). Horseradish peroxidase labelled anti-rabbit antibody produced in goat was conjugated to AuNR as secondary antibody (HRP-AuNR-Ab2). For detection, the analyte (AA-4-MPA) in sample competed with coating antigen for binding with AuNR-Ab1. After washing, HRP-AuNR-Ab2 was added to capture the AuNR-Ab1, and the electrical signal was obtained by addition of hydroquinone and H2O2. After investigation of the binding ability on nanomaterials and optimization of competitive immunoassay conditions, the proposed immunosensor exhibited a sensitive response to AA with a detection limit of 45.9⯱â¯2.7â¯ngâ¯kg-1, and working range of 187⯱â¯12.3â¯ngâ¯kg-1 to 104⯱â¯8.2⯵gâ¯kg-1 for drinking water samples. Recoveries of AA from spiked samples were ranged from 86.0% to 115.0%. The specificity, repeatability and stability of the immunosensor were also proved to be acceptable, indicating its potential application in AA monitoring.
Asunto(s)
Acrilamida/análisis , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Nanotubos/química , Acrilamida/inmunología , Anticuerpos/inmunología , Técnicas Biosensibles/métodos , Compuestos Inorgánicos de Carbono/química , Quitosano/química , Café/química , Agua Potable/análisis , Contaminación de Alimentos/análisis , Oro/química , Límite de Detección , Ovalbúmina/inmunología , Fenilacetatos/inmunología , Compuestos de Silicona/química , Solanum tuberosum/química , Compuestos de Sulfhidrilo/inmunología , Compuestos de Estaño/químicaRESUMEN
Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response.
Asunto(s)
Nitrofenoles , Fenilacetatos , Hipermutación Somática de Inmunoglobulina , Animales , Formación de Anticuerpos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Reordenamiento Génico de Linfocito B , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunización , Región Variable de Inmunoglobulina/genética , Memoria Inmunológica , Inmunofenotipificación , Ratones , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains. We examined whether any biophysical properties of antigen-combing sites relate to this shift in B-cell clones by preparing single-chain Fv (scFv). Thermodynamic and kinetic parameters of the interaction of scFv with various haptens are in accordance with those of intact antibodies, indicating that scFvs are appropriate models for the study on structure and function of antibodies. Next, we measured thermal stability of scFvs using differential scanning calorimetry and found that the apparent melting temperature of free Tyr95-type was 64-66°C,while that of Gly95-type was 47-48°C, indicating that the latter was highly unstable. However, Gly95-type greatly gained thermal stability because of hapten binding. We discussed the relationship between thermal stability resulted by hapten binding and dynamism of antibody response during immunization.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Receptores de Antígenos de Linfocitos B/química , Anticuerpos de Cadena Única/química , Animales , Sitios de Unión de Anticuerpos/inmunología , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Glicina/química , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cinética , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Estabilidad Proteica , Receptores de Antígenos de Linfocitos B/inmunología , Anticuerpos de Cadena Única/inmunología , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.
Asunto(s)
Nitrofenoles/inmunología , Fenilacetatos/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Modelos Moleculares , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de Proteína , Anticuerpos de Cadena Única/aislamiento & purificación , TemperaturaRESUMEN
Polysaccharopeptide (PSP), from Coriolus versicolor, has been used widely as an adjuvant to chemotherapy with demonstrated anti-tumor and broad immunomodulating effects. While PSP's mechanism of action still remains unknown, its enhanced immunomodulatory potential with acacia gum is of great interest. Acacia gum, which also contains polysaccharides and glycoproteins, has been demonstrated to be immunopotentiating. To elucidate whether PSP directly activates T-cell-dependent B-cell responses in vivo, we used a well-established hapten carrier system (Nitrophenyl-chicken gamma globulin (NP-CGG)). 6-week C57BL/6 male mice were immunised with 50 µg of NP25-CGG alum precipitate intraperitoneally. Mice were gavaged daily with 50 mg/kg PSP in a vehicle containing acacia gum and sacrificed at days 0, 4, 7, 10, 14 and 21. ELISA was used to measure the total and relative hapten-specific anti-NP IgA, IgM and IgG titre levels compared to the controls. It was found that PSP, combined with acacia gum, significantly increased total IgG titre levels at day 4 (P< 0.05), decreased IgM titre levels at days 4 and 21 (P< 0.05) with no alterations observed in the IgA or IgE titre levels at any of the time points measured. Our results suggest that while PSP combined with acacia gum appears to exert weak immunological effects through specific T-cell dependent B-cell responses, they are likely to be broad and non-specific which supports the current literature on PSP. We report for the first time the application of a well-established hapten-carrier system that can be used to characterise and delineate specific T-cell dependent B-cell responses of potential immunomodulatory glycoprotein-based herbal medicines combinations in vivo.
Asunto(s)
Anticuerpos/sangre , Linfocitos B/inmunología , Goma Arábiga/farmacología , Proteoglicanos/inmunología , Linfocitos T/inmunología , gamma-Globinas/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos/inmunología , Haptenos/inmunología , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Inmunoterapia/métodos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/terapia , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Proteoglicanos/farmacología , Trametes/inmunologíaRESUMEN
OBJECTIVE: Caspase-1 is required for nephritis and robust autoantibody development in the pristane model of murine lupus. The objective of this study was to evaluate the immune response and to study the splenic B and T cell populations in wild-type (WT) and caspase-1-/- mice following pristane injection in order to develop an understanding of why absence of caspase-1 is protective in pristane-induced lupus. METHODS: Immunization responses to NP-Ficoll and NP-ovalbumin were assessed in WT and caspase-1-/- mice. In vitro IgM and IgG responses to R848 were measured by ELISA. Serum IgM anti-dsDNA and IL-1ß were also measured by ELISA. B and T cell populations 2 weeks and 6 months following pristane injection were measured by flow cytometry in WT and caspase-1-/- mice. RESULTS: Caspase-1-/- mice generate equivalent IgG responses to NP-Ficoll and NP-ova antigens when compared to wild-type mice. Additionally, they secrete IgM and IgG in response to TLR7 activation. Pristane injected WT and caspase-1-/- mice generate robust IgM anti-dsDNA responses. Caspase-1-/- mice have a significant reduction in marginal zone B cell populations compared to WT 6 months after pristane exposure whereas T cell responses are intact in these mice. CONCLUSIONS: Caspase-1-/- mice have intact immune responses but do not develop an expanded marginal zone B cell population in response to pristane-induced lupus. This may be one explanation for reduced IgG autoantibody production in these mice.
Asunto(s)
Linfocitos B/enzimología , Caspasa 1/deficiencia , Lupus Eritematoso Sistémico/enzimología , Bazo/enzimología , Terpenos , Animales , Anticuerpos Antinucleares/sangre , Linfocitos B/inmunología , Caspasa 1/genética , Células Cultivadas , Modelos Animales de Enfermedad , Ficoll/administración & dosificación , Ficoll/análogos & derivados , Ficoll/inmunología , Predisposición Genética a la Enfermedad , Imidazoles/administración & dosificación , Imidazoles/inmunología , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos BALB C , Ratones Noqueados , Nitrofenoles/administración & dosificación , Nitrofenoles/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Fenotipo , Fenilacetatos/administración & dosificación , Fenilacetatos/inmunología , Bazo/inmunología , Linfocitos T/enzimología , Linfocitos T/inmunología , Factores de TiempoRESUMEN
Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP1-, NIP1-, or NNP1-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Técnicas Biosensibles , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Nitrofenoles/química , Fenilacetatos/química , Hipermutación Somática de Inmunoglobulina , Resonancia por Plasmón de SuperficieRESUMEN
Studies on the structural basis of antibody affinity maturation have been carried out by measuring the affinity of secreted antibodies, and information on structures has often been obtained from nucleotide sequences of BCRs of memory B cells. We considered it important to establish whether the repertoire of secreted antibodies from plasma cells is really in accord with that of BCRs on memory B cells at the same time points post-immunization. We isolated plasma cells secreting antibodies specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten by affinity matrix technology using biotin-anti-CD138 and streptavidin-NP-allophycocyanin, to which anti-NP antibodies secreted by autologous plasma cells bound preferentially. We found that plasmablasts occupied >90% of the antibody-secreting cell compartment in the primary response and that they secreted antibodies whose VH regions were encoded by V186.2(+)Tyr95(+) sequences, which provided an increase in the medium level of affinity by somatic hypermutation (SHM) of heavy chains at position 33. After secondary immunization, a further increase in antibody affinity was observed, which was explained by the appearance of a number of plasma cells secreting V186.2(+)Gly95(+) antibodies that acquired high affinity by multiple SHMs as well as plasmablasts secreting V186.2(+)Tyr95(+) antibodies. However, we did not detect any plasmablasts secreting V186.2(+)Gly95(+) antibodies, showing that plasmablasts and plasma cells have a different antibody repertoire, i.e. their respective repertoires are asymmetric. On the basis of these findings, we discussed the relationship between the BCR affinity of memory B cells and plasmablasts as well as plasma cells as pertaining to their ontogeny.
Asunto(s)
Anticuerpos/metabolismo , Células Productoras de Anticuerpos/inmunología , Linfocitos B/inmunología , Memoria Inmunológica , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Anticuerpos/genética , Diversidad de Anticuerpos/genética , Diferenciación Celular , Células Cultivadas , Pollos , Inmunización Secundaria , Activación de Linfocitos , Nitrofenoles/química , Nitrofenoles/inmunología , Fenilacetatos/química , Fenilacetatos/inmunología , Receptores de Antígenos de Linfocitos B/genética , Hipermutación Somática de Inmunoglobulina , gammaglobulinas/química , gammaglobulinas/inmunologíaRESUMEN
BACKGROUND: Homeostatic B Cell-Attracting chemokine 1 (BCA-1) otherwise known as CXCL13 is constitutively expressed in secondary lymphoid organs by follicular dendritic cells (FDC) and macrophages. It is the only known ligand for the CXCR5 receptor, which is expressed on mature B cells, follicular helper T cells (Tfh), Th17 cells and regulatory T (Treg) cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune disorders (e.g. Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosis). We, therefore, hypothesized that antibody-mediated disruption of the CXCL13 signaling pathway would interfere with the formation of ectopic lymphoid follicles in the target organs and inhibit autoimmune disease progression. This work describes pre-clinical development of human anti-CXCL13 antibody MAb 5261 and includes therapeutic efficacy data of its mouse counterpart in murine models of autoimmunity. RESULTS: We developed a human IgG1 monoclonal antibody, MAb 5261 that specifically binds to human, rodent and primate CXCL13 with an affinity of approximately 5 nM and is capable of neutralizing the activity of CXCL13 from these various species in in vitro functional assays. For in vivo studies we have engineered a chimeric antibody to contain the same human heavy and light chain variable genes along with mouse constant regions. Treatment with this antibody led to a reduction in the number of germinal centers in mice immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) and, in adoptive transfer studies, interfered with the trafficking of B cells to the B cell areas of mouse spleen. Furthermore, this mouse anti-CXCL13 antibody demonstrated efficacy in a mouse model of Rheumatoid arthritis (Collagen-Induced Arthritis (CIA)) and Th17-mediated murine model of Multiple Sclerosis (passively-induced Experimental Autoimmune Encephalomyelitis (EAE)). CONCLUSIONS: We developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.
Asunto(s)
Anticuerpos Bloqueadores/administración & dosificación , Artritis Experimental/terapia , Artritis Reumatoide/terapia , Linfocitos B/efectos de los fármacos , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/terapia , Inmunoglobulina G/administración & dosificación , Inmunoterapia/métodos , Macrófagos/efectos de los fármacos , Esclerosis Múltiple/terapia , Proteínas Recombinantes de Fusión/administración & dosificación , Células Th17/efectos de los fármacos , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Linfocitos B/inmunología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL13/inmunología , Células Dendríticas Foliculares/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Ingeniería Genética , Centro Germinal/efectos de los fármacos , Hemocianinas/química , Hemocianinas/inmunología , Humanos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Esclerosis Múltiple/inmunología , Nitrofenoles/química , Nitrofenoles/inmunología , Fenilacetatos/química , Fenilacetatos/inmunología , Receptores CXCR5/metabolismo , Transducción de Señal/efectos de los fármacos , Células Th17/inmunologíaRESUMEN
We developed a method to detect and isolate plasma cells that produce antigen-specific antibodies. An affinity matrix of hapten was constructed on a cell surface, and subsequent incubation allowed cells to secrete antibodies. Anti-hapten antibodies preferentially bound to the affinity matrix on the cells from which they were secreted. We showed that the combination of surface biotinylation and streptavidin which was conjugated with a high valence of hapten was suitable for sensitive detection of antibody binding. Using this protocol, anti-hapten plasma cells from immunized mouse spleen were detected and enriched by flow cytometry. This method allows for isolation of intact plasma cells according to the antibody specificity and may be useful for highly efficient and precise analysis of an antibody repertoire.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Especificidad de Anticuerpos/inmunología , Haptenos/inmunología , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Células Plasmáticas/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Células Cultivadas , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Bazo/citología , Bazo/inmunologíaRESUMEN
Whereas NO is known to regulate T cell responses, its role in regulating B cell responses remains unclear. Previous studies suggested that inducible NO synthase 2 (NOS2/iNOS) is required for normal IgA Ab responses but inhibits antiviral IgG2a Ab responses. In this study we used NOS2(-/-) mice to determine the role of NO in T cell-dependent and T cell-independent (TI)-2 Ab responses. Whereas T cell-dependent Ab responses were only modestly increased in NOS2(-/-) mice, IgM and IgG3 Ab responses as well as marginal zone B cell plasma cell numbers and peritoneal B1b B cells were significantly elevated after immunization with the TI-2 Ag 4-hydroxy-3-nitrophenyl acetyl (NP)-Ficoll. The elevated TI-2 responses in NOS2(-/-) mice were accompanied by significant increases in serum levels of BAFF/BLyS and by increases in BAFF-producing Ly6C(hi) inflammatory monocytes and monocyte-derived dendritic cells (DCs), suggesting that NO normally inhibits BAFF expression. Indeed, we found that NOS2(-/-) DCs produced more BAFF than did wild-type DCs, and addition of a NO donor to NOS2(-/-) DCs reduced BAFF production. Bone marrow chimeric mice that lack NOS2 in either nonhematopoietic or hematopoietic cells had intermediate IgM and IgG3 Ab responses after NP-Ficoll immunization, suggesting that NOS2 from both hematopoietic and nonhematopoietic sources regulates TI-2 Ab responses. Similar to NOS2(-/-) mice, depletion of Ly6C(hi) inflammatory monocytes and monocyte-derived DCs enhanced NP-specific IgM and IgG3 responses to NP-Ficoll. Thus, NO produced by inflammatory monocytes and their derivative DC subsets plays an important role in regulating BAFF production and TI-2 Ab responses.
Asunto(s)
Formación de Anticuerpos/inmunología , Factor Activador de Células B/biosíntesis , Factor Activador de Células B/genética , Óxido Nítrico Sintasa de Tipo II/fisiología , Óxido Nítrico/química , Subgrupos de Linfocitos T/inmunología , Animales , Formación de Anticuerpos/genética , Factor Activador de Células B/inmunología , Subgrupos de Linfocitos B/química , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Ficoll/química , Ficoll/inmunología , Haptenos/inmunología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Subgrupos de Linfocitos T/químicaRESUMEN
B cells are required for follicular Th (Tfh) cell development, as is the ICOS ligand (ICOS-L); however, the separable contributions of Ag and ICOS-L delivery by cognate B cells to Tfh cell development and function are unknown. We find that Tfh cell and germinal center differentiation are dependent on cognate B cell display of ICOS-L, but only when Ag presentation by the latter is limiting, with the requirement for B cell expression of ICOS-L overcome by robust Ag delivery. These findings demonstrate that Ag-specific B cells provide different, yet compensatory, signals for Tfh cell differentiation, while reconciling conflicting data indicating a requirement for ICOS-L expression on cognate B cells for Tfh cell development with those demonstrating that the latter requirement could be bypassed in lieu of that tendered by noncognate B cells. Our findings clarify the separable roles of delivery of Ag and ICOS-L by cognate B cells for Tfh cell maturation and function, and have implications for using therapeutic ICOS blockade in settings of abundantly available Ag, such as in systemic autoimmunity.
Asunto(s)
Antígenos/inmunología , Linfocitos B/inmunología , Ligando Coestimulador de Linfocitos T Inducibles/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos CD19/genética , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Ligando Coestimulador de Linfocitos T Inducibles/genética , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Nitrofenoles/inmunología , Ovalbúmina/inmunología , Fenilacetatos/inmunología , Proteínas Proto-Oncogénicas c-bcl-6 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
The effect of a quinoline-3-carboxamide on the T cell-dependent B cell response was investigated in C57BL/6 mice after NP-CGG immunization. The primary serum response to the hapten was slightly inhibited by treatment with a quinoline-3-carboxamide. This inhibition was paralleled by reduced numbers of germinal centre (GC) B cells and follicular T cells in the spleen up to 21 days after immunization. Also, both the number of GCs formed and their size were reduced by quinoline-3-carboxamide treatment. In contrast to the observation in the primary immune response, there was no inhibitory effect on the secondary immune response. These data could help to explain how quinoline-3-carboxamides can modulate immune function in autoimmune diseases without being immunosuppressive.
Asunto(s)
Linfocitos B/efectos de los fármacos , Centro Germinal/inmunología , Haptenos/administración & dosificación , Quinolinas/administración & dosificación , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Animales , Linfocitos B/inmunología , Células Cultivadas , Pollos , Haptenos/inmunología , Inmunidad Humoral/efectos de los fármacos , Inmunización , Inmunoglobulina G/sangre , Inmunomodulación , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Nitrofenoles/química , Nitrofenoles/inmunología , Fenilacetatos/química , Fenilacetatos/inmunología , Quinolinas/inmunología , Linfocitos T/inmunología , gammaglobulinas/química , gammaglobulinas/inmunologíaRESUMEN
Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina M/metabolismo , Células Plasmáticas/inmunología , Animales , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Citidina Desaminasa/genética , Haptenos/inmunología , Hibridomas , Inmunización Secundaria , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Unión Proteica , Anticuerpos de Dominio Único/genética , Hipermutación Somática de InmunoglobulinaRESUMEN
Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.
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Autoanticuerpos/biosíntesis , Subgrupos de Linfocitos B/inmunología , Centro Germinal/inmunología , Interleucina-17/inmunología , Interleucinas/inmunología , Receptores de Interleucina-17/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Traslado Adoptivo , Animales , Movimiento Celular , Microambiente Celular/inmunología , Técnicas de Cocultivo , Cruzamientos Genéticos , Centro Germinal/ultraestructura , Haptenos/inmunología , Inmunoglobulina G/inmunología , Riñón/patología , Cooperación Linfocítica/inmunología , Linfopoyesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Receptores de Interleucina-17/deficienciaRESUMEN
Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP-specific or anti-Pn14-specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A-specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.
Asunto(s)
Diversidad de Anticuerpos , Formación de Anticuerpos , Linfocitos B/inmunología , Citidina Desaminasa/inmunología , Serina-Treonina Quinasas TOR/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Afinidad de Anticuerpos , Cápsulas Bacterianas/inmunología , Linaje de la Célula , Activación Enzimática , Técnicas de Sustitución del Gen , Centro Germinal/inmunología , Centro Germinal/patología , Haptenos/inmunología , Vivienda para Animales , Inmunización , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/inmunología , Ratones , Ratones Noqueados , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Transducción de Señal/inmunología , Hipermutación Somática de Inmunoglobulina , Bazo/inmunología , Bazo/patología , Infecciones Estreptocócicas/inmunología , Streptococcus pneumoniae/inmunologíaRESUMEN
Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7(+) B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Tracto Gastrointestinal/inmunología , Centro Germinal/inmunología , Inmunoglobulina A/inmunología , Ganglios Linfáticos Agregados/inmunología , Administración Oral , Traslado Adoptivo , Animales , Antígenos/administración & dosificación , Antígenos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Toxina del Cólera/inmunología , Tracto Gastrointestinal/metabolismo , Orden Génico , Centro Germinal/metabolismo , Inmunización , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Modelos Inmunológicos , Nitrofenoles/inmunología , Ganglios Linfáticos Agregados/metabolismo , Fenilacetatos/inmunología , Células Plasmáticas/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
This study was undertaken to isolate active secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3 and evaluate their activities using grass carp Ctenopharyngodon idella kidney (CIK) cells. By applying chromatography techniques and successive recrystallization, three purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: (1) phenylacetic acid, (2) p-hydroxyphenylacetylamide and (3) cyclo-(Gly-(L)-Pro). CIK cells were stimulated by different concentrations (1, 10 and 100 µg/ml) of the isolated compounds, and expression of MyD88, IL-1ß, TNF-α, type I-IFN and IL-8 genes at different time points (2, 8 and 24 h) post-stimulation was quantified by real-time PCR. The known immunostimulatory agent lipopolysaccharide (LPS) was used as a positive control. To analyse whether these compounds are toxic to the cells, the methyl tetrazolium assay was employed to measure changes in cell viability. The obtained results revealed that transcribing level of MyD88, an important adaptor molecule in toll-like receptor signalling pathway, was augmented remarkably by all the three isolated compounds and LPS as early as 2-h exposure. These compounds also induced gene expression of cytokines such as IL-1ß, TNF-α and type I-IFN. Under the experimental conditions, none of the test compounds is toxic to the CIK cells. These findings demonstrate that the immunostimulatory properties of the three metabolites [phenylacetic acid, p-hydroxyphenylacetylamide and cyclo-(Gly-(L)-Pro)] from A. faecalis FY-3 in CIK cells and highlight the potential of using these metabolites as immunostimulants in fish aquaculture.