RESUMO
B cell antigen receptor (BCR) signaling is a key driver of growth and survival in both normal and malignant B cells. Several lines of evidence support an important pathogenic role of the BCR in chronic lymphocytic leukemia (CLL). The significant improvement of CLL patients' survival with the use of various BCR pathway targeting inhibitors, supports a crucial involvement of BCR signaling in the pathogenesis of CLL. Although the treatment landscape of CLL has significantly evolved in recent years, no agent has clearly demonstrated efficacy in patients with treatment-refractory CLL in the long run. To identify new drug targets and mechanisms of drug action in neoplastic B cells, a detailed understanding of the molecular mechanisms of leukemic transformation as well as CLL cell survival is required. In the last decades, studies of genetically modified CLL mouse models in line with CLL patient studies provided a variety of exciting data about BCR and BCR-associated kinases in their role in CLL pathogenesis as well as disease progression. BCR surface expression was identified as a particularly important factor regulating CLL cell survival. Also, BCR-associated kinases were shown to provide a crosstalk of the CLL cells with their tumor microenvironment, which highlights the significance of the cells' milieu in the assessment of disease progression and treatment. In this review, we summarize the major findings of recent CLL mouse as well as patient studies in regard to the BCR signalosome and discuss its relevance in the clinics.
RESUMO
The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Células Precursoras de Linfócitos B , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Proteína rhoA de Ligação ao GTPRESUMO
Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.
Assuntos
Leucemia Linfocítica Crônica de Células B , MicroRNAs , Animais , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genéticaRESUMO
Members of the RAF family of serine-threonine kinases are intermediates in the mitogen-activated protein kinase and extracellular signal-regulated kinase (MAPK-ERK) signaling pathway, which controls key differentiation processes in B cells. By analyzing mice with B cell-specific deletion of Raf1, Braf, or both, we showed that Raf-1 and B-Raf acted together in mediating the positive selection of pre-B and transitional B cells as well as in initiating plasma cell differentiation. However, genetic or chemical inactivation of RAFs led to increased ERK phosphorylation in mature B cells. ERK activation in the absence of Raf-1 and B-Raf was mediated by multiple RAF-independent pathways, with phosphoinositide 3-kinase (PI3K) playing an important role. Furthermore, we found that ERK phosphorylation strongly increased during the transition from activated B cells to pre-plasmablasts. This increase in ERK phosphorylation did not occur in B cells lacking both Raf-1 and B-Raf, which most likely explains the partial block of plasma cell differentiation in mice lacking both RAFs. Collectively, our data indicate that B-Raf and Raf-1 are not necessary to mediate ERK phosphorylation in naïve or activated B cells but are essential for mediating the marked increase in ERK phosphorylation during the transition from activated B cells to pre-plasmablasts.
Assuntos
Linfócitos B/citologia , MAP Quinases Reguladas por Sinal Extracelular , Plasmócitos/citologia , Proteínas Proto-Oncogênicas c-raf , Animais , Diferenciação Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases , Fosforilação , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
Central nervous system (CNS) involvement remains a challenge in the diagnosis and treatment of acute lymphoblastic leukemia (ALL). In this study, we identify CD79a (also known as Igα), a signaling component of the preB cell receptor (preBCR), to be associated with CNS-infiltration and -relapse in B-cell precursor (BCP)-ALL patients. Furthermore, we show that downregulation of CD79a hampers the engraftment of leukemia cells in different murine xenograft models, particularly in the CNS.
Assuntos
Antígenos CD79/metabolismo , Neoplasias do Sistema Nervoso Central/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ph+ acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph+ ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph+ ALL. Targeting this platform with anti-IL7R antibody eliminates Ph+ ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores CXCR4/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Feminino , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-7/farmacologia , Subunidade alfa de Receptor de Interleucina-7/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-7/genética , Camundongos , Camundongos Mutantes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores CXCR4/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+] i ), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+] i Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.
Assuntos
Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Fosfolipase C gama/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Adenina/farmacologia , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Fosfolipase C gama/metabolismo , Mutação Puntual/efeitos dos fármacosRESUMO
Developing B cells undergo defined maturation steps in the bone marrow and in the spleen. The timing and the factors that control these differentiation steps are not fully understood. By targeting the B cell-restricted mb-1 locus to generate an mb-1 allele that expresses a tamoxifen inducible Cre and another allele in which mb-1 expression can be controlled by Cre, we have established a mouse model with an inducible B cell compartment. With these mice, we studied in detail the kinetics of B cell development and the consequence of BCR activation at a defined B cell maturation stage. Contrary to expectations, transitional 1-B cells exposed to anti-IgM reagents in vivo did not die but instead developed into transitional 2 (T2)-B cells with upregulated Bcl-2 expression. We show, however, that these T2-B cells had an increased dependency on the B cell survival factor B cell activating factor when compared to non-stimulated B cells. Overall, our findings indicate that the inducible mb-1 mouse strain represents a useful model, which allows studying the signals that control the selection of B cells in greater detail.
Assuntos
Linfócitos B/citologia , Linfopoese , Animais , Anticorpos Anti-Idiotípicos/imunologia , Fator Ativador de Células B/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Medula Óssea/imunologia , Antígenos CD79/genética , Antígenos CD79/imunologia , Linhagem Celular , Separação Celular , Sobrevivência Celular , Genes bcl-2 , Linfopoese/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Baço/imunologia , Tamoxifeno/farmacologiaAssuntos
Sistema Nervoso Central/imunologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Infiltração Leucêmica/imunologia , Recidiva Local de Neoplasia/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Animais , Apoptose , Proliferação de Células , Sistema Nervoso Central/patologia , Criança , Humanos , Infiltração Leucêmica/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activation of phosphoinositide 3-kinase (PI3K) signaling plays a central role in regulating proliferation and survival of B cells. Here, we tested the hypothesis that B cell receptor (BCR)-mediated activation of PI3K induces the terminal differentiation factor Blimp-1 that interferes with proliferation and survival, thereby controlling the expansion of activated B cells. In fact, B-cell-specific inactivation of Pten, the negative regulator of PI3K signaling, leads to deregulated PI3K activity and elevated Blimp-1 expression. Combined deficiency for Pten and Blimp-1 results in abnormal expansion of B-1 B cells and splenomegaly. Interestingly, Blimp-1 also acts at early stages of B cell development to regulate B cell selection, as Blimp-1 deficiency results in an increased proportion of autoreactive B cells. Together, our data suggest that the combined requirement of deregulated PI3K signaling in addition to defective terminal differentiation represents the basis for proper selection and expansion of developing B cells.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Homeostase , Fosfatidilinositol 3-Quinases/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Animais , Compartimento Celular , Morte Celular , Diferenciação Celular , Proliferação de Células , Citoproteção , Feminino , Masculino , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge.
Assuntos
Antígenos/metabolismo , Citocinas/biossíntese , Imunoglobulina M/metabolismo , Plasmócitos/metabolismo , Animais , Células Produtoras de Anticorpos/metabolismo , Células da Medula Óssea/citologia , Dextranos/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
How the B-cell antigen receptor (BCR) is activated upon interaction with its cognate antigen or with anti-BCR antibodies is not fully understood. We have recently shown that B-cell activation is accompanied by the opening of the pre-organized BCR oligomers, an observation that strengthens the role of receptor reorganization in signalling. We have now analysed the BCR oligomer opening and signalling upon treatment with different monovalent stimuli. Our results indicate that monovalent antigens are able to disturb and open the BCR oligomer, but that this requires the presence and activity of the Src family kinase (SFK) Lyn. We have also shown that monovalent Fab fragments of anti-BCR antibodies can open the BCR oligomers as long as they directly interact with the antigen-binding site. We found that monovalent antigen binding opens both the IgM-BCR and IgD-BCR, but calcium signalling is only seen in cells expressing IgM-BCR; this provides a molecular basis for IgM- and IgD-BCR functional segregation.
Assuntos
Nitro-Hidroxi-Iodofenilacetato/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Soroalbumina Bovina/metabolismo , Quinases da Família src/metabolismo , Animais , Linfócitos B/metabolismo , Sinalização do Cálcio , Linhagem Celular , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Camundongos Transgênicos , Peptídeos/metabolismo , Baço/citologiaRESUMO
Expression of a functional BCR is essential for the development of mature B cells and has been invoked in the control of their maintenance. To test this maintenance function in a new experimental setting, we used the tamoxifen-inducible mb1-CreER(T2) mouse strain to delete or truncate either the mb-1 gene encoding the BCR signaling subunit Igα or the VDJ segment of the IgH (H chain [HC]). In this system, Cre-mediated deletion of the mb-1 gene is accompanied by expression of a GFP reporter. We found that, although the Igα-deficient mature B cells survive for >20 d in vivo, the HC-deficient or Igα tail-truncated B cell population is short-lived, with the HC-deficient cells displaying signs of an unfolded protein response. We also show that Igα-deficient B cells still respond to the prosurvival factor BAFF in culture and require BAFF-R signaling for their in vivo maintenance. These results suggest that, under certain conditions, the loss of the BCR can be tolerated by mature B cells for some time, whereas HC-deficient B cells, potentially generated by aberrant somatic mutations in the germinal center, are rapidly eliminated.
Assuntos
Receptor do Fator Ativador de Células B/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Animais , Receptor do Fator Ativador de Células B/antagonistas & inibidores , Receptor do Fator Ativador de Células B/química , Receptor do Fator Ativador de Células B/metabolismo , Sobrevivência Celular/genética , Estresse do Retículo Endoplasmático , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Deleção de Sequência , Transdução de SinaisRESUMO
B cell antigen receptor (BCR) signaling is critical for B cell development and activation. Using mass spectrometry, we identified a protein kinase D-interacting substrate of 220 kD (Kidins220)/ankyrin repeat-rich membrane-spanning protein (ARMS) as a novel interaction partner of resting and stimulated BCR. Upon BCR stimulation, the interaction increases in a Src kinase-independent manner. By knocking down Kidins220 in a B cell line and generating a conditional B cell-specific Kidins220 knockout (B-KO) mouse strain, we show that Kidins220 couples the BCR to PLCγ2, Ca(2+), and extracellular signal-regulated kinase (Erk) signaling. Consequently, BCR-mediated B cell activation was reduced in vitro and in vivo upon Kidins220 deletion. Furthermore, B cell development was impaired at stages where pre-BCR or BCR signaling is required. Most strikingly, λ light chain-positive B cells were reduced sixfold in the B-KO mice, genetically placing Kidins220 in the PLCγ2 pathway. Thus, our data indicate that Kidins220 positively regulates pre-BCR and BCR functioning.
Assuntos
Linfócitos B/fisiologia , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Linfócitos B/imunologia , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfolipase C gama/metabolismo , Baço/citologiaRESUMO
The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B-cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B-cell-specific deletion of the Syk gene and found that a considerable fraction of mature Syk-negative B cells can survive in the periphery for an extended time. Syk-negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL-4, anti-CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk-deficient B cells require BAFF receptor and CD19/PI3K signaling for their long-term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B-cell pool.
Assuntos
Antígenos CD19/imunologia , Linfócitos B/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/imunologia , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/imunologia , Animais , Anticorpos/farmacologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD19/genética , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/imunologia , Linfócitos B/citologia , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/genética , Antígenos CD40/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Interleucina-4/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Oligodesoxirribonucleotídeos/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/genética , Quinase SykRESUMO
Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.
Assuntos
Linfócitos B/metabolismo , Centro Germinativo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Alelos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Teste de Complementação Genética , Centro Germinativo/citologia , Centro Germinativo/imunologia , Integrases/genética , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Camundongos Transgênicos , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Quinase Syk , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Plasmócitos/citologia , Plasmócitos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Animais , Apoptose/genética , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/ultraestrutura , Diferenciação Celular/imunologia , Regulação da Expressão Gênica , Imunidade Humoral , Imunomodulação , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Plasmócitos/imunologia , Plasmócitos/ultraestrutura , Fator 1 de Ligação ao Domínio I Regulador Positivo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Successful B cell differentiation and prevention of cell transformation depends on balanced and fine-tuned activation of cellular signaling pathways. The phosphatidyl inositol-3 kinase (PI3K) signaling pathway has emerged as a major regulator of B lymphocyte homeostasis and function. Phosphoinositide-dependent protein kinase-1 (PDK1) is the pivotal node in the PI3K pathway, regulating the stability and activity of downstream AGC kinases (including Akt, RSK, S6K, SGK, and PKC). Although the importance of PI3K activity in B cell differentiation is well documented, the role of PDK1 and other downstream effectors is underexplored. Here we used inducible and stage-specific gene targeting approaches to elucidate the role of PDK1 in early and peripheral B cell differentiation. PDK1 ablation enhanced cell cycle entry and apoptosis of IL-7-dependent pro-B cells, blocking Ig synthesis and B cell maturation. PDK1 also was essential for the survival and activation of peripheral B cells via regulation of PKC and Akt-dependent downstream effectors, such as GSK3α/ß and Foxo1. We found that PDK1 deletion strongly impaired B cell receptor (BCR) signaling, but IL-4 costimulation was sufficient to restore BCR-induced proliferation. IL-4 also normalized PKCß activation and hexokinase II expression in BCR-stimulated cells, suggesting that this signaling pathway can act independent of PDK1 to support B cell growth. In summary, our results demonstrate that PDK1 is indispensable for B cell survival, proliferation, and growth regulation.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Linfócitos B/imunologia , Medula Óssea/imunologia , Diferenciação Celular/imunologia , Homeostase/imunologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Citocinas/metabolismo , Estatísticas não ParamétricasRESUMO
Signal transduction from the BCR is regulated by the equilibrium between kinases (e.g., spleen tyrosine kinase [Syk]) and phosphatases (e.g., Shp-1). Previous studies showed that Syk-deficient B cells have a developmental block at the pro/pre-B cell stage, whereas a B cell-specific Shp-1 deficiency promoted B-1a cell development and led to autoimmunity. We generated B cell-specific Shp-1 and Syk double-knockout (DKO) mice and compared them to the single-knockout mice deficient for either Syk or Shp-1. Unlike Syk-deficient mice, the DKO mice can generate mature B cells, albeit at >20-fold reduced B cell numbers. The DKO B-2 cells are all Syk-negative, whereas the peritoneal B1 cells of the DKO mice still express Syk, indicating that they require this kinase for their proper development. The DKO B-2 cells cannot be stimulated via the BCR, whereas they are efficiently activated via TLR or CD40. We also found that in DKO pre-B cells, the kinase Zap70 is associated with the pre-BCR, suggesting that Zap70 is important to promote B cell maturation in the absence of Syk and SHP-1. Together, our data show that a properly balanced kinase/phosphatase equilibrium is crucial for normal B cell development and function.