RESUMO
Transcription factors orchestrate T-lineage differentiation in the thymus. One critical checkpoint involves Notch1 signaling that instructs T-cell commitment at the expense of the B-lineage program. While GATA-3 is required for T-cell specification, its mechanism of action is poorly understood. We show that GATA-3 works in concert with Notch1 to commit thymic progenitors to the T-cell lineage via 2 distinct pathways. First, GATA-3 orchestrates a transcriptional "repertoire" that is required for thymocyte maturation up to and beyond the pro-T-cell stage. Second, GATA-3 critically suppresses a latent B-cell potential in proT cells. As such, GATA-3 is essential to sealing in Notch-induced T-cell fate in early thymocyte precursors by promoting T-cell identity through the repression of alternative developmental options.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Fator de Transcrição GATA3/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Timo/citologia , Animais , Linfócitos B/imunologia , Células Cultivadas , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Knockout , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Timo/embriologia , Timo/imunologia , Fatores de Transcrição/metabolismoRESUMO
Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA3/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Humanos , Interleucina-4/biossíntese , Interleucina-4/farmacologia , Cinética , Camundongos , Regiões Promotoras Genéticas/genética , Linfócitos T Reguladores/química , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Células Th2/efeitos dos fármacosRESUMO
The transcription factor GATA3 is essential at multiple stages of T cell development, including the earliest double-negative stages, beta-selection and CD4 single-positive thymocytes. Here, we show that in CD2-GATA3 transgenic mice, with enforced GATA3 expression driven by the CD2 promoter, thymocytes have reduced levels of CD5, which is a negative regulator of TCR signaling participating in TCR repertoire fine-tuning. Reduction of CD5 expression was most prominent in CD4(+)CD8(+) double-positive (DP) cells and was associated with increased levels of the transcription factor E2A. Conversely, GATA3-deficient DP thymocytes showed consistently higher CD5 levels and defective TCR up-regulation during their development towards the CD4(lo)CD8(lo) subpopulation. CD2-GATA3 transgenic mice carrying the MHC class II-restricted TCR DO11.10 also manifested decreased CD5 levels. As in these TCR-transgenic mice reduced CD5 expression cannot result from an effect of GATA3 on repertoire selection, we conclude that enforced GATA3 interferes with the developmentally regulated increase of CD5 levels. Enforced GATA3 expression in DO11.10 transgenic mice was also accompanied by enhanced TCR expression during CD4 positive selection. Because GATA3 is induced by TCR signaling in DP thymocytes, our findings indicate that GATA3 establishes a positive feedback loop that increases TCR surface expression in developing CD4 lineage cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Antígenos CD5/biossíntese , Antígenos CD5/genética , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Fator de Transcrição GATA3/fisiologia , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/genética , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Retroalimentação Fisiológica/genética , Retroalimentação Fisiológica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Control of integrin-mediated adhesion and migration by chemokines plays a critical role in B cell development, differentiation, and function; however, the underlying signaling mechanisms are poorly defined. Here we show that the chemokine SDF-1 induced activation of Bruton's tyrosine kinase (Btk) and that integrin-mediated adhesion and migration in response to SDF-1 or CXCL13, as well as in vivo homing to lymphoid organs, was impaired in Btk-deficient (pre-)B cells. Furthermore, SDF-1 induced tyrosine phosphorylation of Phospholipase Cgamma2 (PLCgamma2), which, unlike activation of the migration regulatory GTPases Rac or Rap1, was mediated by Btk. PLCgamma2-deficient B cells also exhibited impaired SDF-1-controlled migration. These results reveal that Btk and PLCgamma2 mediate chemokine-controlled migration, thereby providing insights into the control of B cell homeostasis, trafficking, and function, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulinemia (XLA).
Assuntos
Linfócitos B/metabolismo , Quimiocinas/imunologia , Quimiotaxia de Leucócito/imunologia , Fosfolipase C gama/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/imunologia , Adesão Celular/imunologia , Quimiocina CXCL12 , Quimiocina CXCL13 , Quimiocinas CXC/imunologia , Quimiocinas CXC/metabolismo , Ativação Enzimática/imunologia , Humanos , Immunoblotting , Imunoprecipitação , Integrinas/imunologia , Integrinas/metabolismo , Camundongos , Fosfolipase C gama/imunologia , Proteínas Tirosina Quinases/imunologiaRESUMO
Bruton's tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig lambda L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both kappa and lambda germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of kappa and lambda light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion.
Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/imunologia , Rearranjo Gênico de Cadeia Leve de Linfócito B , Fosfoproteínas/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Proteínas de Transporte/genética , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , DNA Complementar/genética , Interleucina-7/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição GênicaRESUMO
The proliferation and differentiation of lymphocytes are regulated by receptors localized on the cell surface. Engagement of these receptors induces the activation of intracellular signaling proteins that transmit the receptor signals to distinct targets and control the cellular responses. The first signaling proteins to be discovered in higher organisms were the products of oncogenes. For example, the kinases Src and Abelson (Abl) were originally identified as oncogenes and were later characterized as important proteins for signal transduction in various cell types, including lymphocytes. Now, as many cellular signaling molecules have been discovered and ordered into certain pathways, we can better understand why particular signaling proteins are associated with tumorigenesis. In this review, we discuss recent progress in unraveling the molecular mechanisms of signaling pathways that control the proliferation and differentiation of early B cells. We point out the concepts of auto-inhibition and subcellular localization as crucial aspects in the regulation of B cell signaling.
Assuntos
Linfócitos B/fisiologia , Leucemia de Células B/etiologia , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/etiologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Proteínas de Transporte/fisiologia , Humanos , Síndromes de Imunodeficiência/etiologia , Linfopoese , Camundongos , Modelos Biológicos , Fosfoproteínas/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais , VDJ Recombinases/fisiologiaRESUMO
During B-cell development in the mouse, Bruton tyrosine kinase (Btk) and the adaptor protein SLP-65 (Src homology 2 [SH2] domain-containing leukocyte protein of 65 kDa) limit the expansion and promote the differentiation of pre-B cells. Btk is thought to mainly function by phosphorylating phospholipase Cgamma2, which is brought into close proximity of Btk by SLP-65. However, this model was recently challenged by the identification of a role for Btk as a tumor suppressor in the absence of SLP-65 and by the finding that Btk function is partially independent of its kinase activity. To investigate if enzymatic activity is critical for the tumor suppressor function of Btk, we crossed transgenic mice expressing the kinase-inactive K430R-Btk mutant onto a Btk/SLP-65 double-deficient background. We found that K430R-Btk expression rescued the severe developmental arrest at the pre-B-cell stage in Btk/SLP-65 double-deficient mice. Moreover, K430R-Btk could functionally replace wild-type Btk as a tumor suppressor in SLP-65- mice: at 6 months of age, the observed pre-B-cell lymphoma frequencies were approximately 15% for SLP-65- mice, 44% for Btk/SLP-65-deficient mice, and 14% for K430R-Btk transgenic mice on the Btk/SLP-65-deficient background. Therefore, we conclude that Btk exerts its tumor suppressor function in pre-B cells as an adaptor protein, independent of its catalytic activity.
Assuntos
Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Catálise , Diferenciação Celular , Células Cultivadas , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Lisina/genética , Lisina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Taxa de SobrevidaRESUMO
Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.
Assuntos
Proteínas de Ciclo Celular/genética , Morte Fetal/genética , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , DNA/genética , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA , Feminino , Raios gama , Marcação de Genes , Genes Letais , Idade Gestacional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Mutagênicos/toxicidade , Gravidez , Tolerância a Radiação/genética , Recombinação Genética , Raios UltravioletaRESUMO
In the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in kappa(+) immature B cells, but was largely corrected in lambda(+) immature B cells. As lambda gene rearrangements are programmed to follow kappa rearrangements and lambda expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83 mu delta transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83 mu delta mice, the IgM(+) B cells in the bone marrow exhibited a very immature phenotype (pre-BCR(+)CD43(+)CD2(-)) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83 mu delta B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.
Assuntos
Subpopulações de Linfócitos B/patologia , Regulação para Baixo/imunologia , Rearranjo Gênico do Linfócito B/imunologia , Células-Tronco Hematopoéticas/enzimologia , Glicoproteínas de Membrana/biossíntese , Proteínas Tirosina Quinases/deficiência , Receptores de Antígenos de Linfócitos B/biossíntese , Regulação para Cima/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Deleção Clonal/genética , Regulação para Baixo/genética , Rearranjo Gênico do Linfócito B/genética , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Cadeias delta de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias mu de Imunoglobulina/genética , Linfopenia/enzimologia , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Células Precursoras de Linfócitos B , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Antígenos de Linfócitos B/genética , Baço/imunologia , Baço/patologia , Regulação para Cima/genéticaRESUMO
Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin alpha4beta1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton's tyrosine kinase (Btk), phospholipase C (PLC)gamma2, IP3R-mediated Ca2+ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin alpha4beta1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin alpha4beta1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).
Assuntos
Integrinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Fosfolipases Tipo C/metabolismo , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/metabolismo , Animais , Linfócitos B/metabolismo , Cálcio , Proteínas de Ligação ao Cálcio , Adesão Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Proteínas dos Microfilamentos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Proteína Quinase C/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of lambda L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of lambda L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted lambda usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-kappaB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of lambda L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity.
Assuntos
Linfócitos B/citologia , Linfócitos B/enzimologia , Domínio Catalítico/imunologia , Proteínas Tirosina Quinases/fisiologia , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/imunologia , Domínio Catalítico/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Rearranjo Gênico de Cadeia Leve de Linfócito B , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.
Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/fisiologia , Células-Tronco Hematopoéticas/imunologia , Linfoma de Células B/prevenção & controle , Fosfoproteínas/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/prevenção & controle , Proteínas Tirosina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Animais , Células Cultivadas , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/- mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre-B-cell proliferation and differentiation into immunoglobulin M-positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/- mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the null mutants precludes the analysis of adult hematopoiesis in Sp3-/- mice. We therefore investigated the ability of E12.5 Sp3-/- liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/- cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/- cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Eritrócitos/citologia , Perfilação da Expressão Gênica , Hematopoese/genética , Hepatócitos/citologia , Hepatócitos/transplante , Linfócitos/citologia , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Fator de Transcrição Sp3 , Baço/citologia , Fatores de Transcrição/genéticaRESUMO
Bruton's tyrosine kinase (Btk) is a cytoplasmic signaling molecule that is crucial for precursor (pre-B) cell differentiation in humans. In this study, we show that during the transition of large cycling to small resting pre-B cells in the mouse, Btk-deficient cells failed to efficiently modulate the expression of CD43, surrogate L chain, CD2, and CD25. In an analysis of the kinetics of pre-B cell differentiation in vivo, Btk-deficient cells manifested a specific developmental delay within the small pre-B cell compartment of about 3 h, when compared with wild-type cells. Likewise, in in vitro bone marrow cultures, Btk-deficient large cycling pre-B cells showed increased IL-7 mediated expansion and reduced developmental progression into noncycling CD2(+)CD25(+) surrogate L chain-negative small pre-B cells and subsequently into Ig-positive B cells. Furthermore, the absence of Btk resulted in increased proliferative responses to IL-7 in recombination-activating gene-1-deficient pro-B cells. These findings identify a novel role for Btk in the regulation of the differentiation stage-specific modulation of IL-7 responsiveness in pro-B and pre-B cells. Moreover, our results show that Btk is critical for an efficient transit through the small pre-B cell compartment, thereby regulating cell surface phenotype changes during the developmental progression of cytoplasmic mu H chain expressing pre-B cells into immature IgM(+) B cells.
Assuntos
Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/patologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/patologia , Camundongos Knockout/imunologia , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/patologia , Animais , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/genética , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Ciclo Celular/genética , Ciclo Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Células Cultivadas , Citoplasma/enzimologia , Citoplasma/imunologia , Células-Tronco Hematopoéticas/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias mu de Imunoglobulina/biossíntese , Cadeias mu de Imunoglobulina/genética , Imunofenotipagem , Interleucina-7/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Proteínas Tirosina Quinases/biossínteseRESUMO
Although CD45R/B220 is commonly used as a pan-B cell marker in the mouse, not all B220(+) cells belong to the B cell lineage. Here we report the characterization of a subpopulation of B220(+)CD19(-) cells in murine bone marrow, which failed to express markers that are present in early CD19(--) B cell precursors. Instead, these cells expressed low levels of MHC class II and CD11c, which are typically found on dendritic cells (DC). Moreover, these B220(+)CD19(-)CD11c(+) cells expressed Gr-1, indicating that they are related to the recently identified murine plasmacytoid DC or their progenitors. Therefore, we evaluated surface marker expression of the B220(+)CD19(-)CD11c(+) cells in lymphoid tissues of C57BL/6 mice, recombinase activating gene-1 deficient mice, lacking mature B and T lymphocytes, and mice with a targeted disruption of the Ig H chain mu membrane exon (mu MT), lacking mature B lymphocytes. When comparing bone marrow and spleen, we found that the surface profiles of B220(+)CD19(-)CD11c(+) cells were remarkably similar, indicating that they are in a comparable maturation or activation stage in the two lymphoid compartments. In addition, the almost complete absence of peripheral B220(+) B-lineage cells in mu MT mice allowed the anatomical localization of the B220(+)CD19(-)CD11c(+) cells to the red pulp and the T cell areas in the spleen. Taken together, our findings indicate that the mouse bone marrow contains a recirculating population of B220(+)CD19(-) CD11c(+) plasmacytoid DC, the development of which is largely independent of the presence of mature T and B cells.
Assuntos
Antígenos de Diferenciação de Linfócitos B/análise , Células da Medula Óssea/citologia , Células Dendríticas/citologia , Antígenos Comuns de Leucócito/análise , Subpopulações de Linfócitos/citologia , Baço/citologia , Animais , Antígenos CD19/análise , Subpopulações de Linfócitos B/química , Subpopulações de Linfócitos B/classificação , Células da Medula Óssea/química , Células da Medula Óssea/classificação , Diferenciação Celular , Linhagem da Célula , Células Dendríticas/química , Citometria de Fluxo , Genes RAG-1 , Proteínas de Homeodomínio/fisiologia , Imunofenotipagem , Integrina alfaXbeta2/análise , Células Matadoras Naturais/química , Células Matadoras Naturais/classificação , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de ÓrgãosRESUMO
X-linked agammaglobulinemia (XLA) is characterized by a severe B-cell deficiency, resulting from a differentiation arrest in the bone marrow (BM). Because XLA is clinically and immunologically heterogeneous, we investigated whether the B-cell differentiation arrest in BM of XLA patients is heterogeneous as well. First, we analyzed BM samples from 19 healthy children by flow cytometry. This resulted in a normal B-cell differentiation model with eight consecutive stages. Subsequently, we analyzed BM samples from nine XLA patients. Eight patients had amino acid substitutions in the Bruton's tyrosine kinase (BTK) domain or premature stop codons, resulting in the absence of functional BTK proteins. In seven of these eight patients a major differentiation arrest was observed at the transition between cytoplasmic Ig(mu-) pre-B-I cells and cytoplasmic Ig(mu+) pre-B-II cells, consistent with a role for BTK in pre-B-cell receptor signaling. However, one patient exhibited a very early arrest at the transition between pro-B cells and pre-B-I cells, which could not be explained by a different nature of the BTK mutation. We conclude that the absence of functional BTK proteins generally leads to an almost complete arrest of B-cell development at the pre-B-I to pre-B-II transition. The ninth XLA patient had a splice site mutation associated with the presence of low levels of wild-type BTK mRNA. His BM showed an almost normal composition of the precursor B-cell compartment, suggesting that low levels of BTK can rescue the pre-B-cell receptor signaling defect, but do not lead to sufficient numbers of mature B lymphocytes in the peripheral blood.