RESUMO
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.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos/administração & dosagem , Imunidade Humoral/efeitos dos fármacos , Nitrofenóis/administração & dosagem , Ovalbumina/administração & dosagem , Fenilacetatos/administração & dosagem , Receptores de Reconhecimento de Padrão/agonistas , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Feminino , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Nitrofenóis/imunologia , Ovalbumina/imunologia , Fenilacetatos/imunologia , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Envelhecimento/imunologia , Disbiose/imunologia , Transplante de Microbiota Fecal , Microbioma Gastrointestinal/imunologia , Centro Germinativo/imunologia , Nódulos Linfáticos Agregados/imunologia , Animais , Toxina da Cólera/imunologia , Feminino , Imunização , Imunoglobulina A/imunologia , Camundongos , Nitrofenóis/imunologia , Fenilacetatos/imunologiaRESUMO
Malaria remains a significant worldwide public health problem. To address biological questions, researchers rely on the experimental murine model. For decades, chloroquine (CQ) and pyrimethamine (Pyr) have been used to clear Plasmodium infections in experimental animals using standardised accepted protocols and, because of this, drug-treated controls are rarely included. However, there is limited data available on the modulation of anti-malarial immunity, including generation of memory B cells, when these drugs are administered days after malaria infection. We investigated B cell responses to an important malaria glycolipid, glycosylphosphatidylinositol (GPI), and the hapten nitrophenol (NP), with or without standard CQ and Pyr treatment using the murine model. At day 14, CQ/Pyr treatment significantly suppressed the frequency of NP+IgG1+ memory B cells in NP-KLH-immunised mice. Furthermore, CQ/Pyr-treated NP-KLH-immunised mice did not have significantly higher cellular counts of NP+ B cells, germinal centre B cells, nor NP+IgG1+ memory B cells than naïve mice (CQ/Pyr treated and untreated). CQ/Pyr-treated GPI-KLH-immunised mice did not have significantly higher cellular counts of GPI+ B cells than naïve untreated mice. By day 28, this effect appeared to resolve since all immunised mice, whether treated or untreated, had significantly higher B cell proliferative responses than naïve mice (CQ/Pyr treated and untreated) for the majority of B cell phenotypes. The current study emphasises the potential for drug modulation of antigenic B cell responses when using standardised malaria treatment protocols in the experimental murine model. It is recommended that drug-treated controls are included when using experimental malaria infections to address biological questions.
Assuntos
Anticorpos Antiprotozoários/sangue , Antimaláricos/uso terapêutico , Linfócitos B/imunologia , Cloroquina/uso terapêutico , Glicosilfosfatidilinositóis/imunologia , Malária/tratamento farmacológico , Nitrofenóis/imunologia , Plasmodium/imunologia , Pirimetamina/uso terapêutico , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Antimaláricos/efeitos adversos , Cloroquina/efeitos adversos , Modelos Animais de Doenças , Combinação de Medicamentos , Feminino , Humanos , Imunização , Imunoglobulina G/imunologia , Malária/imunologia , Malária/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirimetamina/efeitos adversosRESUMO
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.
Assuntos
Nitrofenóis , Fenilacetatos , Hipermutação Somática de Imunoglobulina , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Rearranjo Gênico do Linfócito B , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Imunização , Região Variável de Imunoglobulina/genética , Memória Imunológica , Imunofenotipagem , Camundongos , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Afinidade de Anticorpos/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Receptores de Antígenos de Linfócitos B/química , Anticorpos de Cadeia Única/química , Animais , Sítios de Ligação de Anticorpos/imunologia , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Glicina/química , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cinética , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Estabilidade Proteica , Receptores de Antígenos de Linfócitos B/imunologia , Anticorpos de Cadeia Única/imunologia , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
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.
Assuntos
Nitrofenóis/imunologia , Fenilacetatos/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Modelos Moleculares , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Anticorpos de Cadeia Única/isolamento & purificação , TemperaturaRESUMO
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.
Assuntos
Anticorpos/sangue , Linfócitos B/imunologia , Goma Arábica/farmacologia , Proteoglicanas/imunologia , Linfócitos T/imunologia , gama-Globinas/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos/imunologia , Haptenos/imunologia , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/terapia , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Proteoglicanas/farmacologia , Trametes/imunologiaRESUMO
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.
Assuntos
Linfócitos B/enzimologia , Caspase 1/deficiência , Lúpus Eritematoso Sistêmico/enzimologia , Baço/enzimologia , Terpenos , Animais , Anticorpos Antinucleares/sangue , Linfócitos B/imunologia , Caspase 1/genética , Células Cultivadas , Modelos Animais de Doenças , Ficoll/administração & dosagem , Ficoll/análogos & derivados , Ficoll/imunologia , Predisposição Genética para Doença , Imidazóis/administração & dosagem , Imidazóis/imunologia , Imunização , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Nitrofenóis/administração & dosagem , Nitrofenóis/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fenótipo , Fenilacetatos/administração & dosagem , Fenilacetatos/imunologia , Baço/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
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.
Assuntos
Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Técnicas Biossensoriais , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Nitrofenóis/química , Fenilacetatos/química , Hipermutação Somática de Imunoglobulina , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
Anticorpos/metabolismo , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Memória Imunológica , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Anticorpos/genética , Diversidade de Anticorpos/genética , Diferenciação Celular , Células Cultivadas , Galinhas , Imunização Secundária , Ativação Linfocitária , Nitrofenóis/química , Nitrofenóis/imunologia , Fenilacetatos/química , Fenilacetatos/imunologia , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , gama-Globulinas/química , gama-Globulinas/imunologiaRESUMO
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.
Assuntos
Anticorpos Bloqueadores/administração & dosagem , Artrite Experimental/terapia , Artrite Reumatoide/terapia , Linfócitos B/efeitos dos fármacos , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/efeitos dos fármacos , Encefalomielite Autoimune Experimental/terapia , Imunoglobulina G/administração & dosagem , Imunoterapia/métodos , Macrófagos/efeitos dos fármacos , Esclerose Múltipla/terapia , Proteínas Recombinantes de Fusão/administração & dosagem , Células Th17/efeitos dos fármacos , Animais , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL13/imunologia , Células Dendríticas Foliculares/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Engenharia Genética , Centro Germinativo/efeitos dos fármacos , Hemocianinas/química , Hemocianinas/imunologia , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Esclerose Múltipla/imunologia , Nitrofenóis/química , Nitrofenóis/imunologia , Fenilacetatos/química , Fenilacetatos/imunologia , Receptores CXCR5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th17/imunologiaRESUMO
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.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Especificidade de Anticorpos/imunologia , Haptenos/imunologia , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Plasmócitos/imunologia , Animais , Afinidade de Anticorpos/imunologia , Células Cultivadas , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologiaRESUMO
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.
Assuntos
Formação de Anticorpos/imunologia , Fator Ativador de Células B/biossíntese , Fator Ativador de Células B/genética , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico/química , Subpopulações de Linfócitos T/imunologia , Animais , Formação de Anticorpos/genética , Fator Ativador de Células B/imunologia , Subpopulações de Linfócitos B/química , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Ficoll/química , Ficoll/imunologia , Haptenos/imunologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Subpopulações de Linfócitos T/químicaRESUMO
Germinal centers are the anatomic sites for the generation of high affinity immunoglobulin expressing plasma cells and memory B cells. The germinal center B cells that are precursors of these cells circulate between the light zone B cell population that interact with antigen laden follicular dendritic cells (FDC) and the proliferative dark zone B cell population. Antigen retention by follicular dendritic cells is dependent on Fc receptors and complement receptors, and complement receptor 1 (Cr1) is the predominant complement receptor expressed by FDC. The newly created Cr1KO mouse was used to test the effect of Cr1-deficiency on the kinetics of the germinal center reaction and the generation of IgM and switched memory B cell formation. Immunization of Cr1KO mice with a T cell-dependent antigen resulted in the normal initial expansion of B cells with a germinal center phenotype however these cells were preferentially lost in the Cr1KO animal over time (days). Bone marrow chimera animals documented the surprising finding that the loss of germinal center B cell maintenance was linked to the expression of Cr1 on B cells, not the FDC. Cr1-deficiency further resulted in antigen-specific IgM titer and IgM memory B cell reductions, but not antigen-specific IgG after 35-37 days. Investigations of nitrophenyl (NP)-specific IgG demonstrated that Cr1 is not necessary for affinity maturation during the response to particulate antigen. These data, along with those generated in our initial description of the Cr1KO animal describe unique functions of Cr1 on the surface of both B cells and FDC.
Assuntos
Linfócitos B/imunologia , Células Dendríticas Foliculares/imunologia , Centro Germinativo/imunologia , Imunoglobulina M/imunologia , Receptores de Complemento 3b/genética , Animais , Células da Medula Óssea/imunologia , Proliferação de Células , Quimera/imunologia , Imunoglobulina G/imunologia , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/imunologia , Receptores Fc/imunologia , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Antígenos/imunologia , Linfócitos B/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Nitrofenóis/imunologia , Ovalbumina/imunologia , Fenilacetatos/imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
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.
Assuntos
Linfócitos B/efeitos dos fármacos , Centro Germinativo/imunologia , Haptenos/administração & dosagem , Quinolinas/administração & dosagem , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Animais , Linfócitos B/imunologia , Células Cultivadas , Galinhas , Haptenos/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunização , Imunoglobulina G/sangue , Imunomodulação , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Nitrofenóis/química , Nitrofenóis/imunologia , Fenilacetatos/química , Fenilacetatos/imunologia , Quinolinas/imunologia , Linfócitos T/imunologia , gama-Globulinas/química , gama-Globulinas/imunologiaRESUMO
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.
Assuntos
Linfócitos B/imunologia , Imunoglobulina M/metabolismo , Plasmócitos/imunologia , Animais , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos/genética , Citidina Desaminase/genética , Haptenos/imunologia , Hibridomas , Imunização Secundária , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Ligação Proteica , Anticorpos de Domínio Único/genética , Hipermutação Somática de ImunoglobulinaRESUMO
The response of B cells to Ag targeted to Clec9A on dendritic cells was followed using the hapten nitrophenol (NP) conjugated to rat Ig carrier. Injection of small amounts of NP conjugated to anti-Clec9A in the absence of adjuvants gave high and very prolonged Ab responses, approaching those obtained by high doses of nontargeted NP-protein conjugates with alum adjuvant. The response to NP-anti-Clec9A included the transient formation of germinal centers, maturation of Ab affinity, and some memory B cell formation. Serum Ab titers remained high 35 wk postimmunization, well after the initial follicular response had faded. The results suggest Clec9A-targeting strategies for improving Ab responses to vaccine Ags.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Receptores Imunológicos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Formação de Anticorpos/imunologia , Feminino , Imunoglobulinas/imunologia , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Nitrofenóis/imunologiaRESUMO
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.
Assuntos
Autoanticorpos/biossíntese , Subpopulações de Linfócitos B/imunologia , Centro Germinativo/imunologia , Interleucina-17/imunologia , Interleucinas/imunologia , Receptores de Interleucina-17/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Movimento Celular , Microambiente Celular/imunologia , Técnicas de Cocultura , Cruzamentos Genéticos , Centro Germinativo/ultraestrutura , Haptenos/imunologia , Imunoglobulina G/imunologia , Rim/patologia , Cooperação Linfocítica/imunologia , Linfopoese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/imunologia , Fenilacetatos/imunologia , Receptores de Interleucina-17/deficiênciaRESUMO
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.