RESUMO
The transcription factors Foxo1, Foxo3 and Foxo4 modulate cell fate 'decisions' in diverse systems. Here we show that Foxo1-dependent gene expression was critical at many stages of B cell differentiation. Early deletion of Foxo1 caused a substantial block at the pro-B cell stage due to a failure to express interleukin 7 receptor-alpha. Foxo1 inactivation in late pro-B cells resulted in an arrest at the pre-B cell stage due to lower expression of the recombination-activating genes Rag1 and Rag2. Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to lower expression of L-selectin and failed class-switch recombination due to impaired upregulation of the gene encoding activation-induced cytidine deaminase. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/imunologia , Animais , Linfócitos B/imunologia , Southern Blotting , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/imunologia , Rearranjo Gênico do Linfócito B/genética , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/imunologia , Células-Tronco/metabolismo , Transcrição Gênica/imunologiaRESUMO
Although the B-cell antigen receptor (BCR) factors most prominently in the maintenance and differentiation of mature B cells, it is now appreciated that co-receptor molecules can positively or negatively modulate signals through the BCR. Co-receptors are functionally defined as modifiers of BCR engagement and signal transduction, and are distinct from other accessory molecules that act independently to regulate B-cell growth. The co-receptor CD19 functions to augment signals by the pre-BCR/BCR and in doing so can modulate B-cell fate decisions at multiple stages of development. In mature B cells, CD19 also associates with complement receptor 2 (CR2/CD21) and is pivotal for transducing signals induced by co-recognition of complement C3d-fixed antigens by the BCR and CD21. In this article, we focus on recent progress in the understanding of CD19 function through the characterization of mouse models that relate in vivo function to biochemical properties of CD19.
Assuntos
Antígenos CD19/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/fisiologia , Animais , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Humanos , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Ciclina D3/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Animais , Linfócitos B/metabolismo , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Ciclina D2/genética , Ciclina D2/imunologia , Ciclina D3/deficiência , Ciclina D3/genética , Primers do DNA/genética , Expressão Gênica , Centro Germinativo/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Imunização , Camundongos , Camundongos Knockout , Modelos Imunológicos , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
The inositol phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP) negatively regulate phosphatidylinositol-3-kinase (PI3K)-mediated growth, survival, and proliferation of hematopoietic cells. Although deletion of PTEN in mouse T cells results in lethal T cell lymphomas, we find that animals lacking PTEN or SHIP in B cells show no evidence of malignancy. However, concomitant deletion of PTEN and SHIP (bPTEN/SHIP(-/-)) results in spontaneous and lethal mature B cell neoplasms consistent with marginal zone lymphoma or, less frequently, follicular or centroblastic lymphoma. bPTEN/SHIP(-/-) B cells exhibit enhanced survival and express more MCL1 and less Bim. These cells also express low amounts of p27(kip1) and high amounts of cyclin D3 and thus appear poised to undergo proliferative expansion. Unlike normal B cells, bPTEN/SHIP(-/-) B cells proliferate to the prosurvival factor B cell activating factor (BAFF). Interestingly, although BAFF availability may promote lymphoma progression, we demonstrate that BAFF is not required for the expansion of transferred bPTEN/SHIP(-/-) B cells. This study reveals that PTEN and SHIP act cooperatively to suppress B cell lymphoma and provides the first direct evidence that SHIP is a tumor suppressor. As such, assessment of both PTEN and SHIP function are relevant to understanding the etiology of human B cell malignancies that exhibit augmented activation of the PI3K pathway.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/metabolismo , Linfócitos B/enzimologia , Linfócitos B/imunologia , Proteína 11 Semelhante a Bcl-2 , Proliferação de Células , Sobrevivência Celular , Ciclina D3/genética , Ciclina D3/imunologia , Ciclina D3/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/imunologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Deleção de Genes , Humanos , Inositol Polifosfato 5-Fosfatases , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.
Assuntos
Linfócitos B/citologia , Linfócitos T/citologia , Animais , Células da Medula Óssea/citologia , Células CHO , Diferenciação Celular , Proliferação de Células , Cricetinae , Cricetulus , Células-Tronco Hematopoéticas/citologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Desnaturação Proteica , Transdução de SinaisRESUMO
The humoral immune response is characterized by the early secretion of IgM antibodies followed by the generation of IgG, IgA or IgE antibodies as a result of class switch recombination. In recent years, progress had been made in understanding the molecular events of CSR as well as transcriptional control of B cell differentiation into germinal center or antibody secreting cells. However, the biochemical signals downstream of cell surface receptors that regulate CSR during B cell differentiation remain ill-defined. We have recently identified PI3K as a critical regulator of both antibody secreting cell formation and class switch recombination. How PI3K activity may be regulating CSR during antigen-driven B cell differentiation and its potential contribution to humoral immunodeficiencies will be discussed.
Assuntos
Formação de Anticorpos/fisiologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Switching de Imunoglobulina , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Animais , Linfócitos B/citologia , Diferenciação Celular , Centro Germinativo/citologia , Centro Germinativo/enzimologia , Centro Germinativo/imunologia , Humanos , Doenças do Sistema Imunitário/metabolismo , Imunoglobulina M/imunologia , Camundongos , Modelos Imunológicos , Plasmócitos/citologia , Plasmócitos/enzimologia , Plasmócitos/imunologiaRESUMO
Class-switch recombination (CSR) is essential for humoral immunity. However, the regulation of CSR is not completely understood. Here we demonstrate that phosphatidylinositol 3-kinase (PI3K) actively suppressed the onset and frequency of CSR in primary B cells. Consistently, mice lacking the lipid phosphatase, PTEN, in B cells exhibited a hyper-IgM condition due to impaired CSR, which could be restored in vitro by specific inhibition of PI3Kdelta. Inhibition of CSR by PI3K was partially dependent on the transcription factor, BLIMP1, linking plasma cell commitment and cessation of CSR. PI3K-dependent activation of the serine-threonine kinase, Akt, suppressed CSR, in part, through the inactivation of the Forkhead Box family (Foxo) of transcription factors. Reduced PI3K signaling enhanced the expression of AID (activation-induced cytidine deaminase) and accelerated CSR. However, ectopic expression of AID could not fully overcome inhibition of CSR by PI3K, suggesting that PI3K regulates both the expression and function of AID.
Assuntos
Switching de Imunoglobulina , Ativação Linfocitária , Fosfatidilinositol 3-Quinases/metabolismo , Plasmócitos/enzimologia , Plasmócitos/imunologia , Animais , Diferenciação Celular , Citidina Desaminase/metabolismo , Ativação Enzimática , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Hidrólise , Switching de Imunoglobulina/genética , Imunoglobulina M/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Mutantes , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Plasmócitos/citologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Recombinação Genética/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
NF-kappaB activity in mammalian cells is regulated through the IkappaB kinase (IKK) complex, consisting of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma). Targeted deletion of Ikkbeta results in early embryonic lethality, thus complicating the examination of IKKbeta function in adult tissues. Here we describe the role of IKKbeta in B lymphocytes made possible by generation of a mouse strain that expresses a conditional Ikkbeta allele. We find that the loss of IKKbeta results in a dramatic reduction in all peripheral B cell subsets due to associated defects in cell survival. IKKbeta-deficient B cells are also impaired in mitogenic responses to LPS, anti-CD40, and anti-IgM, indicating a general defect in the ability to activate the canonical NF-kappaB signaling pathway. These findings are consistent with a failure to mount effective Ab responses to T cell-dependent and independent Ags. Thus, IKKbeta provides a requisite role in B cell activation and maintenance and thus is a key determinant of humoral immunity.
Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Antígenos CD19/genética , Antígenos T-Independentes/administração & dosagem , Antígenos T-Independentes/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Haptenos/administração & dosagem , Haptenos/imunologia , Quinase I-kappa B , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Integrases/genética , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Linfopenia/enzimologia , Linfopenia/genética , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitógenos/farmacologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Baço/imunologia , Baço/metabolismo , Baço/patologia , Trinitrobenzenos/administração & dosagem , Trinitrobenzenos/imunologia , Proteínas Virais/genéticaRESUMO
TNF receptor (TNFR) superfamily members, CD40, and BAFFR play critical roles in B cell survival and differentiation. Genetic deficiency in a novel adaptor molecule, Act1, for CD40 and BAFF results in a dramatic increase in peripheral B cells, which culminates in lymphadenopathy and splenomegaly, hypergammaglobulinemia, and autoantibodies. While the B cell-specific Act1 knockout mice displayed a similar phenotype with less severity, the pathology of the Act1-deficient mice was mostly blocked in CD40-Act1 and BAFF-Act1 double knockout mice. CD40- and BAFF-mediated survival is significantly increased in Act1-deficent B cells, with stronger IkappaB phosphorylation, processing of NF-kappaB2 (p100/p52), and activation of JNK, ERK, and p38 pathways, indicating that Act1 negatively regulates CD40- and BAFF-mediated signaling events. These findings demonstrate that Act1 plays an important role in the homeostasis of B cells by attenuating CD40 and BAFFR signaling.