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1.
IEEE Trans Haptics ; 15(2): 267-279, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35130170

RESUMO

The tactile information to be presented to a user during interaction with a virtual object is calculated by simulating the contact between the object model and user model. In the simulation, a distributed force is applied to the contact area on the skin tissue of users' hands and results in deformation of the skin tissue. The skin deformation caused by the distributed force is the target contact state that should be presented by the device. However, most multipoint haptic displays do not have sufficient degrees of freedom (DoF) to represent the target contact state. This paper presents the concept and formulation of "deformation matching," whereby the output force is calculated to minimize the error between the target skin deformation and skin deformation that can be realized by the limited DoF device's output force. For comparison, the conventional concept of "force matching" was also formulated. The difference in human perception between these two concepts in the expression of friction was investigated through experiments using a pin-array tactile display capable of stimulating 128 points. It was demonstrated that the perception of the friction coefficient was more sensitive and the perception of the friction direction was more accurate in deformation matching than in force matching.


Assuntos
Percepção do Tato , Tato , Simulação por Computador , Fricção , Humanos , Pele
2.
J Immunol ; 205(1): 90-101, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32414809

RESUMO

BCR transgenic mice dominate studies of B cell tolerance; consequently, tolerance in normal mice expressing diverse sets of autoreactive B cells is poorly characterized. We have used single B cell cultures to trace self-reactivity in BCR repertoires across the first and second tolerance checkpoints and in tolerized B cell compartments of normal mice. This approach reveals affinity "setpoints" that define each checkpoint and a subset of tolerized, autoreactive B cells that is long-lived. In normal mice, the numbers of B cells avidly specific for DNA fall significantly as small pre-B become immature and transitional-1 B cells, revealing the first tolerance checkpoint. By contrast, DNA reactivity does not significantly change when immature and transitional-1 B cells become mature follicular B cells, showing that the second checkpoint does not reduce DNA reactivity. In the spleen, autoreactivity was high in transitional-3 (T3) B cells, CD93+IgM-/loIgDhi anergic B cells, and a CD93- anergic subset. Whereas splenic T3 and CD93+ anergic B cells are short-lived, CD93-IgM-/loIgDhi B cells have half-lives comparable to mature follicular B cells. B cell-specific deletion of proapoptotic genes, Bak and Bax, resulted in increased CD93-IgM-/loIgDhi B cell numbers but not T3 B cell numbers, suggesting that apoptosis regulates differently persistent and ephemeral autoreactive B cells. The self-reactivity and longevity of CD93-IgM-/loIgDhi B cells and their capacity to proliferate and differentiate into plasmacytes in response to CD40 activation in vitro lead us to propose that this persistent, self-reactive compartment may be the origin of systemic autoimmunity and a potential target for vaccines to elicit protective Abs cross-reactive with self-antigens.


Assuntos
Autoantígenos/imunologia , Autoimunidade , Linfócitos B/imunologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Autoantígenos/metabolismo , Linfócitos B/metabolismo , Células Cultivadas , Anergia Clonal , Reações Cruzadas , Meia-Vida , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Modelos Animais , Células Precursoras de Linfócitos B/metabolismo , Cultura Primária de Células , Análise de Célula Única , Baço/citologia , Baço/imunologia
3.
J Immunol ; 203(12): 3268-3281, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31732530

RESUMO

2F5 is an HIV-1 broadly neutralizing Ab that also binds the autoantigens kynureninase (KYNU) and anionic lipids. Generation of 2F5-like Abs is proscribed by immune tolerance, but it is unclear which autospecificity is responsible. We sampled the BCR repertoire of 2F5 knock-in mice before and after the first and second tolerance checkpoints. Nearly all small pre-B (precheckpoint) and 35-70% of anergic peripheral B cells (postcheckpoint) expressed the 2F5 BCR and maintained KYNU, lipid, and HIV-1 gp41 reactivity. In contrast, all postcheckpoint mature follicular (MF) B cells had undergone L chain editing that purged KYNU and gp41 binding but left lipid reactivity largely intact. We conclude that specificity for KYNU is the primary driver of tolerization of 2F5-expressing B cells. The MF and anergic B cell populations favored distinct collections of editor L chains; surprisingly, however, MF and anergic B cells also frequently expressed identical BCRs. These results imply that BCR autoreactivity is the primary determinant of whether a developing B cell enters the MF or anergic compartments, with a secondary role for stochastic factors that slightly mix the two pools. Our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1 and supports activation of anergic B cells as a potential method for HIV-1 vaccination.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Hidrolases/imunologia , Tolerância Imunológica/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Amplamente Neutralizantes/genética , Reações Cruzadas , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Anticorpos Anti-HIV/genética , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos B/imunologia
4.
Nat Commun ; 9(1): 928, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29500348

RESUMO

B cells expressing high affinity antigen receptors are advantaged in germinal centers (GC), perhaps by increased acquisition of antigen for presentation to follicular helper T cells and improved T-cell help. In this model for affinity-dependent selection, the density of peptide/MHCII (pMHCII) complexes on GC B cells is the primary determinant of selection. Here we show in chimeric mice populated by B cells differing only in their capacity to express MHCII (MHCII+/+ and MHCII+/-) that GC selection is insensitive to halving pMHCII density. Alone, both B cell types generate identical humoral responses; in competition, MHCII+/+ B cells are preferentially recruited to early GCs but this advantage does not persist once GCs are established. During GC responses, competing MHCII+/+ and MHCII+/- GC B cells comparably accumulate mutations and have indistinguishable rates of affinity maturation. We conclude that B-cell selection by pMHCII density is stringent in the establishment of GCs, but relaxed during GC responses.


Assuntos
Linfócitos B/metabolismo , Genes MHC da Classe II , Centro Germinativo/citologia , Animais , Feminino , Centro Germinativo/fisiologia , Imunidade Humoral , Camundongos Endogâmicos C57BL
5.
Methods Mol Biol ; 1623: 125-133, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28589353

RESUMO

In germinal centers (GCs), B cells undergo repeated cycles of proliferation and affinity-based selection, and differentiate into memory B cells or long-lived plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells has been limited. Here we describe a culture system in which we can induce massive expansion of naive B cells that exhibit GC B cell-like phenotype and acquire abilities to differentiate into memory B cells or bone marrow plasma cells depending on cytokine conditions. This system will allow us to elucidate the molecular mechanisms of GC B cell differentiation.


Assuntos
Linfócitos B/citologia , Centro Germinativo/citologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Centro Germinativo/imunologia , Memória Imunológica , Camundongos , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo
6.
Methods Mol Biol ; 1623: 243-251, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28589361

RESUMO

Expression of activation-induced cytidine deaminase (AID) is the hallmark of B cells engaged in an immune response in germinal centers. We designed an inducible fate-mapping reporter mouse in which AID-expressing B cells could be timely and irreversibly marked, by knockin at the Aicda locus of a tamoxifen-inducible Cre recombinase. This mouse model allows notably for the long-term follow-up of memory B cells and plasma cells engaged in an immune response. We describe here a protocol to generate hybridomas from small memory subsets that can be easily traced and identified in this mouse line through Cre-activated fluorescent reporters.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Citidina Desaminase/genética , Integrases/genética , Animais , Biomarcadores , Linhagem Celular , Linhagem Celular Tumoral , Citidina Desaminase/metabolismo , Expressão Gênica , Técnicas de Introdução de Genes , Marcação de Genes , Genes Reporter , Loci Gênicos , Hibridomas , Memória Imunológica , Integrases/metabolismo , Camundongos , RNA não Traduzido/genética
7.
Cell Rep ; 18(7): 1627-1635, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28199836

RESUMO

Activation-induced cytidine deaminase (AID) is required to purge autoreactive immature and transitional-1 (immature/T1) B cells at the first tolerance checkpoint, but how AID selectively removes self-reactive B cells is unclear. We now show that B cell antigen receptor (BCR) and endosomal Toll-like receptor (TLR) signals synergize to elicit high levels of AID expression in immature/T1 B cells. This synergy is restricted to ligands for endocytic TLR and requires phospholipase-D activation, endosomal acidification, and MyD88. The first checkpoint is significantly impaired in AID- or MyD88-deficient mice and in mice doubly heterozygous for AID and MyD88, suggesting interaction of these factors in central B cell tolerance. Moreover, administration of chloroquine, an inhibitor of endosomal acidification, results in a failure to remove autoreactive immature/T1 B cells in mice. We propose that a BCR/TLR pathway coordinately establishes central tolerance by hyper-activating AID in immature/T1 B cells that bind ligands for endosomal TLRs.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/imunologia , Tolerância Imunológica/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Animais , Endossomos/imunologia , Feminino , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/imunologia , Fosfolipase D/imunologia , Células Precursoras de Linfócitos B/imunologia
8.
J Immunol ; 198(3): 1066-1080, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28031341

RESUMO

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.


Assuntos
Linfócitos B/fisiologia , Caspases/fisiologia , Proteínas de Neoplasias/fisiologia , Animais , Apoptose , Linfócitos B/citologia , Diferenciação Celular , Sobrevivência Celular , Centro Germinativo/fisiologia , Ativação Linfocitária , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Receptores de Antígenos de Linfócitos B/fisiologia , Proteína bcl-X/análise
9.
Immunity ; 44(3): 542-552, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26948373

RESUMO

Germinal center (GC) B cells evolve toward increased affinity by a Darwinian process that has been studied primarily in genetically restricted, hapten-specific responses. We explored the population dynamics of genetically diverse GC responses to two complex antigens-Bacillus anthracis protective antigen and influenza hemagglutinin-in which B cells competed both intra- and interclonally for distinct epitopes. Preferred VH rearrangements among antigen-binding, naive B cells were similarly abundant in early GCs but, unlike responses to haptens, clonal diversity increased in GC B cells as early "winners" were replaced by rarer, high-affinity clones. Despite affinity maturation, inter- and intraclonal avidities varied greatly, and half of GC B cells did not bind the immunogen but nonetheless exhibited biased VH use, V(D)J mutation, and clonal expansion comparable to antigen-binding cells. GC reactions to complex antigens permit a range of specificities and affinities, with potential advantages for broad protection.


Assuntos
Linfócitos B/fisiologia , Seleção Clonal Mediada por Antígeno , Centro Germinativo/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Afinidade de Anticorpos/genética , Diversidade de Anticorpos , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Células Cultivadas , Feminino , Hemaglutininas Virais/imunologia , Humanos , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Orthomyxoviridae/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Anticorpos de Domínio Único/genética
10.
Biomaterials ; 63: 24-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26072995

RESUMO

Ex vivo engineered three-dimensional organotypic cultures have enabled the real-time study and control of biological functioning of mammalian tissues. Organs of broad interest where its architectural, cellular, and molecular complexity has prevented progress in ex vivo engineering are the secondary immune organs. Ex vivo immune organs can enable mechanistic understanding of the immune system and more importantly, accelerate the translation of immunotherapies as well as a deeper understanding of the mechanisms that lead to their malignant transformation into a variety of B and T cell malignancies. However, till date, no modular ex vivo immune organ has been developed with an ability to control the rate of immune reaction through tunable design parameter. Here we describe a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymphoid tissue that provides the basis to induce an accelerated germinal center (GC) reaction by continuously providing extracellular matrix (ECM) and cell-cell signals to naïve B cells. Compared to existing co-cultures, immune organoids provide a control over primary B cell proliferation with ∼100-fold higher and rapid differentiation to the GC phenotype with robust antibody class switching.


Assuntos
Linfócitos B/citologia , Materiais Biocompatíveis/química , Centro Germinativo/citologia , Nanocompostos/química , Organoides/citologia , Animais , Linfócitos B/imunologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Centro Germinativo/imunologia , Camundongos Endogâmicos C57BL , Nanocompostos/ultraestrutura , Técnicas de Cultura de Órgãos/métodos , Organoides/imunologia , Engenharia Tecidual/métodos
11.
J Immunol ; 194(4): 1480-8, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25601926

RESUMO

Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black × New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody-secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells.


Assuntos
Autoimunidade/imunologia , Linfócitos B/citologia , Células Precursoras de Linfócitos B/citologia , Proteína Fosfatase 2/imunologia , Transferência Adotiva , Animais , Autoanticorpos , Linfócitos B/imunologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/imunologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Imuno-Histoquímica , Subpopulações de Linfócitos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cavidade Peritoneal/citologia , Células Precursoras de Linfócitos B/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
J Immunol ; 193(2): 635-44, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24935931

RESUMO

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvß3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.


Assuntos
Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Memória Imunológica/imunologia , Tecido Linfoide/imunologia , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Animais , Linfócitos B/metabolismo , Células 3T3 BALB , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Galinhas , Ficoll/análogos & derivados , Ficoll/imunologia , Citometria de Fluxo , Imunização/métodos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/metabolismo , Tecido Linfoide/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Ligação Proteica/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , gama-Globulinas/imunologia
13.
PLoS One ; 9(3): e92732, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24647439

RESUMO

Immunotherapies such as adoptive transfer of T cells or natural killer cells, or monoclonal antibody (MoAb) treatment have recently been recognized as effective means to treat cancer patients. However, adoptive transfer of B cells or plasma cells producing tumor-specific antibodies has not been applied as a therapy because long-term culture and selective expansion of antigen-specific B cells has been technically very difficult. Here, we describe a novel cancer immunotherapy that uses B-cell adoptive transfer. We demonstrate that germinal-center-like B cells (iGB cells) induced in vitro from mouse naïve B cells become plasma cells and produce IgG antibodies for more than a month in the bone marrow of non-irradiated recipient mice. When transferred into mice, iGB cells producing antibody against a surrogate tumor antigen suppressed lung metastasis and growth of mouse melanoma cells expressing the same antigen and prolonged survival of the recipients. In addition, we have developed a novel culture system called FAIS to selectively expand antigen-specific iGB cells utilizing the fact that iGB cells are sensitive to Fas-induced cell death unless their antigen receptors are ligated by membrane-bound antigens. The selected iGB cells efficiently suppressed lung metastasis of melanoma cells in the adoptive immunotherapy model. As human blood B cells can be propagated as iGB cells using culture conditions similar to the mouse iGB cell cultures, our data suggest that it will be possible to treat cancer-bearing patients by the adoptive transfer of cancer-antigen-specific iGB cells selected in vitro. This new adoptive immunotherapy should be an alternative to the laborious development of MoAb drugs against cancers for which no effective treatments currently exist.


Assuntos
Imunoterapia Adotiva/métodos , Imunoterapia/métodos , Animais , Linfócitos B/imunologia , Melanoma/terapia , Camundongos , Camundongos Mutantes
14.
J Clin Invest ; 123(12): 5009-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24200695

RESUMO

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/enzimologia , Linfoma não Hodgkin/etiologia , Complexo Repressor Polycomb 2/fisiologia , Animais , Apoptose , Linfócitos B/patologia , Ciclo Celular , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Dano ao DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Inativação Gênica , Centro Germinativo/imunologia , Centro Germinativo/patologia , Imunidade Humoral , Memória Imunológica , Linfoma não Hodgkin/enzimologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Linfopoese , Metilação , Camundongos , Camundongos Transgênicos , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/fisiologia
15.
Nat Commun ; 2: 465, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21897376

RESUMO

In response to T cell-dependent antigens, B cells proliferate extensively to form germinal centres (GC), and then differentiate into memory B (B(mem)) cells or long-lived plasma cells (LLPCs) by largely unknown mechanisms. Here we show a new culture system in which mouse naïve B cells undergo massive expansion and isotype switching, and generate GC-phenotype B (iGB) cells. The iGB cells expressing IgG1 or IgM/D, but not IgE, differentiate into B(mem) cells in vivo after adoptive transfer and can elicit rapid immune responses with the help of cognate T cells. Secondary culture with IL-21 maintains the proliferation of the iGB cells, while shifting their in vivo developmental fate from B(mem) cells to LLPCs, an outcome that can be reversed by withdrawal of IL-21 in tertiary cultures. Thus, this system enables in vitro manipulation of B-cell fate, into either B(mem) cells or LLPCs, and will facilitate dissection of GC-B cell differentiation programs.


Assuntos
Linfócitos B/imunologia , Memória Imunológica , Plasmócitos/imunologia , Células 3T3 , Animais , Antígenos/imunologia , Linfócitos B/citologia , Proliferação de Células , Citometria de Fluxo , Técnicas In Vitro , Interleucina-4/fisiologia , Interleucinas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasmócitos/citologia
16.
J Immunol ; 186(10): 5620-8, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21490159

RESUMO

Memory B cells generated during a T cell-dependent immune response rapidly respond to a secondary immunization by producing abundant IgG Abs that bind cognate Ag with high affinity. It is currently unclear whether this heightened recall response by memory B cells is due to augmented IgG-BCR signaling, which has only been demonstrated in the context of naive transgenic B cells. To address this question, we examined whether memory B cells can respond in vivo to Ags that stimulate only through BCR, namely T cell-independent type II (TI-II) Ags. In this study, we show that the TI-II Ag (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll cannot elicit the recall response in mice first immunized with the T cell-dependent Ag NP-chicken γ-globulin. Moreover, the NP-Ficoll challenge in vivo as well as in vitro significantly inhibits a subsequent recall response to NP-chicken γ-globulin in a B cell-intrinsic manner. This NP-Ficoll-mediated tolerance is caused by the preferential elimination of IgG(+) memory B cells binding to NP with high affinity. These data indicate that BCR cross-linking with a TI-II Ag does not activate IgG(+) memory B cells, but rather tolerizes them, identifying a terminal checkpoint of memory B cell differentiation that may prevent autoimmunity.


Assuntos
Antígenos T-Independentes/imunologia , Linfócitos B/imunologia , Tolerância Imunológica , Imunoglobulina G/biossíntese , Memória Imunológica , Transferência Adotiva , Animais , Antígenos T-Independentes/metabolismo , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Ficoll/análogos & derivados , Ficoll/imunologia , Citometria de Fluxo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Receptores de Antígenos/imunologia , Receptores de Antígenos/metabolismo , Transdução de Sinais , gama-Globulinas/imunologia
17.
Blood ; 108(8): 2703-11, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16794253

RESUMO

The pre-B-cell receptor (pre-BCR) is thought to signal transcriptional activation of the immunoglobulin light (L) chain gene locus, proceeding to its V-J rearrangement. The pre-BCR signaling pathway for this process is largely unknown but may involve the adaptor protein BASH (BLNK/SLP-65). Here we report that the pre-B leukemia cell lines established from affected BASH-deficient mice rearrange kappaL-chain gene locus and down-regulate pre-BCR upon PMA treatment or BASH reconstitution. Analyses with specific inhibitors revealed that activation of novel PKC (nPKC) and MEK, but not Ras, is necessary for the rearrangement. Accordingly, retroviral transduction of active PKCeta, PKCepsilon, or Raf-1, but not Ras, induced the kappa gene rearrangement and expression in the pre-B-cell line. Tamoxifen-mediated BASH reconstitution resulted in the translocation of PKCeta to the plasma membrane and kappa chain expression. These data make evident that the Ras-independent BASH-nPKC-Raf-1 pathway of pre-BCR signaling induces the L-chain gene rearrangement and expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Rearranjo Gênico de Cadeia Leve de Linfócito B , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Leucemia de Células B/genética , Leucemia de Células B/imunologia , Leucemia de Células B/metabolismo , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Pré-Leucemia/genética , Pré-Leucemia/imunologia , Pré-Leucemia/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo
18.
Immunol Lett ; 105(1): 48-54, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16481047

RESUMO

The development and survival of mature B cells requires an antigen-independent signal from the B cell receptor (BCR) through an adaptor protein containing an SH2 domain, BASH (BLNK/SLP-65). It also requires signaling through BAFF and the BAFF receptor (BAFF-R), and is negatively regulated by protein kinase Cdelta (PKCdelta). In PKCdelta-deficient mice, B cell maturation occurs independently of the BAFF receptor (BAFF-R), indicating that BAFF-R signaling promotes maturation by inhibiting the negative function of PKCdelta. To clarify which of the two signaling pathways plays the primary role in B cell maturation, we crossed BASH-deficient mice with PKCdelta-deficient mice to generate BASH/PKCdelta-double knockout (DKO) mice. In the DKO mice, B cell maturation was blocked at the transitional type 1 (T1) stage and B cells were prone to apoptosis, in common with BASH-deficient mice. This indicates that BASH-mediated BCR signaling primarily controls B cell survival and maturation, with BAFF-R signaling and its inhibition of PKCdelta acting as a secondary regulator. By contrast, CD40-mediated proliferation and antibody production, which are low in BASH-deficient mice, were rescued in the DKO mice, indicating that the suppression of CD40-mediated B cell activation by PKCdelta is epistatic to BASH-mediated promotion. The physiological relevance of these opposing hierarchical effects of BASH and PKCdelta in the regulation of B cell maturation and activation is discussed.


Assuntos
Linfócitos B/imunologia , Fosfoproteínas/deficiência , Proteína Quinase C-delta/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fator Ativador de Células B , Receptor do Fator Ativador de Células B , Linfócitos B/citologia , Antígenos CD40/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Diferenciação Celular , Epistasia Genética , Ativação Linfocitária , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/imunologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
19.
J Immunol ; 173(10): 5980-8, 2004 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-15528332

RESUMO

The editing of B cell Ag receptor (BCR) through successive rearrangements of Ig genes has been considered to be a major mechanism for the central B cell tolerance, which precludes appearance of self-reactive B cells, through studies using anti-self-Ig transgenic/knock-in mouse systems. However, contribution of the receptor editing in the development of the normal B cell repertoire remains unclear. In addition, the signaling pathway directing this event is unknown. In this study, we demonstrate that receptor editing in anti-DNA Ig knock-in mice is impaired in the absence of an adaptor protein BASH (BLNK/SLP-65) that is involved in BCR signaling. Remarkably, the supposed hallmarks of receptor editing such as Iglambda chain expression, recombination sequence rearrangements at Igkappa loci, and presence of in-frame VkappaJkappa joins in the Igkappa loci inactivated by the recombination sequence rearrangements, were all diminished in BASH-deficient mice with unmanipulated Ig loci. BCR ligation-induced Iglambda gene recombination in vitro was also impaired in BASH-deficient B cells. Furthermore, the BASH-deficient mice showed an excessive Ab response to a DNA carrier immunization, suggesting the presence of unedited DNA-reactive B cells in the periphery. These results not only define a signaling pathway required for receptor editing but indicate that the BCR-signaled receptor editing indeed operates in the development of normal B cell repertoire and contributes to establishing the B cell tolerance.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Proteínas de Transporte/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Edição de RNA/imunologia , Receptores de Antígenos de Linfócitos B/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/metabolismo , Autoantígenos/imunologia , Subpopulações de Linfócitos B/imunologia , Proteínas de Transporte/fisiologia , Anergia Clonal/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Marcadores Genéticos/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfoproteínas/fisiologia , Edição de RNA/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia
20.
Int Immunol ; 16(8): 1161-71, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15237108

RESUMO

Signaling through the B cell antigen receptor (BCR) induces activation and proliferation of B cells, a response that requires the adaptor protein BASH (also known as BLNK/SLP-65). Although BASH and other molecules, such as Btk, PLCgamma2 and PKCbeta, are known to be essential for T cell-independent immune responses in vivo, their requirement during T cell-dependent immune responses, especially their role in antibody affinity-maturation and memory B cell generation remains unclear. In this study, we examined primary and memory immune responses to the T cell-dependent hapten antigen, (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to chicken gammaglobulin (CGG), in BASH-deficient mice on a C57BL/6 background. In the primary response, NP-specific IgM was barely produced and the typical anti-NP IgG1/lambda production was markedly attenuated, but kappa chain was unexpectedly over-represented in the anti-NP antibodies. In contrast, CGG-specific IgG1 was normally produced. In the memory response, IgG1/lambda antibody with high affinity to NP was produced at normal level in the mutant mice. The frequency and distribution of somatic mutations in the V(H)186.2 genes of the anti-NP IgG1/lambda antibody were also normal. These results indicate that BASH-mediated BCR signaling is dispensable for somatic hypermutation and affinity selection, as well as generation and response of memory B cells. Interestingly, mutated V(H) genes with the same clonal origin were prominent in the anti-NP antibodies of BASH-deficient mice, indicating that a limited number of original clones had been recruited into the memory compartment. Thus, the scarcity of specific clones in the primary repertoire and an impaired primary response is not detrimental to the quality and quantity of a memory response.


Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/imunologia , Diferenciação Celular/genética , Imunoglobulina G/genética , Imunoglobulina M/genética , Memória Imunológica/genética , Ativação Linfocitária/genética , Fosfoproteínas/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Proteínas de Transporte/genética , Diferenciação Celular/imunologia , Proliferação de Células , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Nitrofenóis/imunologia , Fosfoproteínas/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
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