RESUMO
T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eµ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-10/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/imunologia , Plicamicina/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Linfócitos T CD8-Positivos/efeitos dos fármacos , Estudos de Casos e Controles , Proliferação de Células , Modelos Animais de Doenças , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OSU-2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti-tumour activity in several haematological and solid tumour models. We have recently shown OSU-2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre-clinical activity of OSU-2S in spontaneous B-cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU-2S mediated apoptosis in canine B-cell lines and primary B-cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU-2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU-2S-mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU-2S as small molecule for treating people and dogs with lymphoma.
Assuntos
Antineoplásicos/uso terapêutico , Doenças do Cão/tratamento farmacológico , Linfoma de Células B/veterinária , Propilenoglicóis/uso terapêutico , Espécies Reativas de Oxigênio/antagonistas & inibidores , Esfingosina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Cães , Linfoma de Células B/tratamento farmacológico , Esfingosina/uso terapêuticoRESUMO
Selective cytotoxicity to cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Here we developed a tumor antigen-targeted delivery of immunonanoparticle carrying a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A (PP2A), phosphorylation and nuclear translocation of SHP1(S591) and deregulation of multiple cellular processes in chronic lymphocytic leukemia (CLL) resulting in potent cytotoxicity. To preclude OSU-2S-mediated effects on these ubiquitous phosphatases in unintended cells and avoid potential adverse effects, we developed an OSU-2S-targeted delivery of immunonanoparticles (2A2-OSU-2S-ILP), that mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 (hROR1) in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL directed immunonanoparticles and the novel transgenic (Tg) mouse model of CLL that expresses hROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia.
Assuntos
Linfócitos B/citologia , Sistemas de Liberação de Medicamentos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/terapia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Animais , Apoptose , Linfócitos B/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Cloridrato de Fingolimode , Humanos , Imunossupressores/química , Lipossomos/química , Linfoma de Célula do Manto/metabolismo , Camundongos , Camundongos Transgênicos , Nanopartículas/química , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Propilenoglicóis/química , Proteína Quinase C/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Resultado do TratamentoRESUMO
The gene encoding PTPROt (truncated isoform of protein tyrosine phosphatase receptor-type O) is methylated and suppressed in chronic lymphocytic leukemia (CLL). PTPROt exhibits in vitro tumor-suppressor characteristics through the regulation of B-cell receptor (BCR) signaling. Here we generated transgenic (Tg) mice with B-cell-specific expression of PTPROt. Although lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared with the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice, suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt-mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of BCR signaling. These results establish the in vivo tumor-suppressive function of PTPROt and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.
Assuntos
Biomarcadores Tumorais/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Inflamação/prevenção & controle , Leucemia Linfocítica Crônica de Células B/prevenção & controle , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores Tumorais/genética , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcr/genética , Proteínas Proto-Oncogênicas c-bcr/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Prognóstico , Taxa de SobrevidaRESUMO
Therapeutic regimens for chronic lymphocytic leukemia (CLL) have increasingly utilized monoclonal antibodies since the chimeric anti-CD20 antibody rituximab was introduced. Despite improved clinical outcomes, current CLL therapies are not curative. Therefore, antibodies with greater efficacy and novel targets are desirable. One promising target is CD37, a tetraspanin protein highly expressed on malignant B-cells in CLL and non-Hodgkin lymphoma. Although several novel CD37-directed therapeutics are emerging, detailed preclinical evaluation of these agents is limited by lack of appropriate animal models with spontaneous leukemia expressing the human CD37 (hCD37) target. To address this, we generated a murine CLL model that develops transplantable hCD37+ leukemia. Subsequently, we engrafted healthy mice with this leukemia to evaluate IMGN529, a novel hCD37-targeting antibody-drug conjugate. IMGN529 rapidly eliminated peripheral blood leukemia and improved overall survival. In contrast, the antibody component of IMGN529 could not alter disease course despite exhibiting substantial in vitro cytotoxicity. Furthermore, IMGN529 is directly cytotoxic to human CLL in vitro, depletes B-cells in patient whole blood and promotes killing by macrophages and natural killer cells. Our results demonstrate the utility of a novel mouse model for evaluating anti-human CD37 therapeutics and highlight the potential of IMGN529 for treatment of CLL and other CD37-positive B-cell malignancies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígenos de Neoplasias/genética , Antineoplásicos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Tetraspaninas/antagonistas & inibidores , Tetraspaninas/genética , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Imunidade Inata , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Depleção Linfocítica , Camundongos , Camundongos Transgênicos , Terapia de Alvo MolecularRESUMO
In this report, we tested the hypothesis that Ets-1 transcription factor is modulated at the mRNA level during B cell antigen receptor (BCR)-induced cell-signalling events. Quiescent B cells express high levels of Ets-1 mRNA. Stimulation through the BCR results in time-dependent inhibition of Ets-1 mRNA expression in primary splenic B cells with maximal inhibition observed by 16-h post-stimulation. Inhibition of Ets-1 expression is specific to antigen receptor but not CD40-mediated activation. Antigen receptor-induced inhibition of Ets-1 mRNA can be mimicked by phorbol myristate acetate (PMA) and/or ionomycin. PMA but not ionomycin-induced inhibition of Ets-1 expression is rescued by the inhibitors of protein kinase C and MEK. Extended time-course analysis revealed a time-dependent cyclical pattern in the re-expression of Ets-1 mRNA. While resting cells revealed maximal Ets-1 mRNA expression, activation events that induced exit from G(0) /G(1) or cells blocked in early S phase exhibited decreased Ets-1 mRNA levels. Interestingly, cells arrested at late G2 or M phase of the cell cycle failed to down modulate Ets-1 mRNA expression. Overexpression of Ets-1 in 70Z/3 B cell line caused abnormal accumulation of cells in S phase associated with increased cyclin A expression. Consistent with a requirement for Ets-1 in BCR-induced cell cycle entry, splenic B cells from mice deficient in Ets-1 showed defective antigen receptor-induced DNA synthesis and S phase entry. These results suggest a critical role for Ets-1 regulation during B cell activation and cell cycle entry.
Assuntos
Linfócitos B/metabolismo , Ciclo Celular , Regulação da Expressão Gênica , Ativação Linfocitária , Proteína Proto-Oncogênica c-ets-1/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Linfócitos B/imunologia , Cálcio/metabolismo , Linhagem Celular , Ciclina A/genética , Ciclina A/metabolismo , Feminino , Expressão Gênica , Camundongos , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos B/imunologiaAssuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD19/imunologia , Antígenos CD20/imunologia , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Glicoproteínas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adulto , Antígeno CD52 , Citometria de Fluxo , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de IgG/imunologia , Receptores de IgG/metabolismoRESUMO
Calcyon regulates activity-dependent internalization of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors and long-term depression of excitatory synapses. Elevated levels of calcyon are consistently observed in brains from schizophrenic patients, and the calcyon gene is associated with attention-deficit hyperactivity disorder. Executive function deficits are common to both disorders, and at least for schizophrenia, the etiology appears to involve both heritable and neurodevelopmental factors. Here, we show with calcyon-overexpressing Cal(OE) transgenic mice that lifelong calcyon upregulation impairs executive functions including response inhibition and working memory, without producing learning and memory deficits in general. As response inhibition and working memory, as well as the underlying neural circuitry, continue to mature into early adulthood, we functionally silenced the transgene during postnatal days 28-49, a period corresponding to adolescence. Remarkably, the response inhibition and working memory deficits including perseverative behavior were absent in adult Cal(OE) mice with the transgene silenced in adolescence. Suppressing the calcyon transgene in adulthood only partially rescued the deficits, suggesting calcyon upregulation in adolescence irreversibly alters development of neural circuits supporting mature response inhibition and working memory. Brain regional immunoblots revealed a prominent downregulation of AMPA GluR1 subunits in hippocampus and GluR2/3 subunits in hippocampus and prefrontal cortex of the Cal(OE) mice. Silencing the transgene in adolescence prevented the decrease in hippocampal GluR1, further implicating altered fronto-hippocampal connectivity in the executive function deficits observed in the Cal(OE) mice. Treatments that mitigate the effects of high levels of calcyon during adolescence could preempt adult deficits in executive functions in individuals at risk for serious mental illness.
Assuntos
Inibição Psicológica , Proteínas de Membrana/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo/fisiologia , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Modelos Animais de Doenças , Doxiciclina/metabolismo , Doxiciclina/farmacologia , Extinção Psicológica/efeitos dos fármacos , Extinção Psicológica/fisiologia , Medo/efeitos dos fármacos , Medo/fisiologia , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana/genética , Transtornos da Memória/genética , Camundongos , Camundongos Transgênicos , Receptores de AMPA/metabolismo , Percepção Espacial/efeitos dos fármacos , Percepção Espacial/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
1. The effects of enzymatically hydrolysed whole Saccharomyces cerevisiae yeast (HY) and the pellets of yeast cell wall (YCW) on production traits, the microbiology and histo-morphology of the small intestine, and humoral immune responses against Newcastle disease virus (NDV), of Ross 308 broilers were investigated. 2. The control group received a maize-soyabean meal based basal diet for 42 days. In the treated groups the basal diet was supplemented with 1 g/kg of HY and YCW. There were 8 replicate pens per group (n = 12 birds/pen). 3. HY and YCW supplementation improved live weight (P = 0·006) and FCR (P = 0·003) at 42-d as compared with the control group. 4. In the small intestine, Salmonella spp and Escherichia coli numbers were higher (P = 0·01) in the mucosa and lower (P = 0·01) in the digesta of the HY and the YCW fed groups at 25 d of age. Lactobacillus in the duodenal and jejunal digesta was higher (P < 0·05) in the HY and the YCW fed groups as compared with the control. 5. Following oral challenge with Salmonella pullorum, Escherichia coli and Lactobacillus increased (P < 0·05) in the mucosa and decreased in the digesta (P < 0·05) of the HY and YCW supplemented groups, relative to the control. 6. Supplementation of HY and YCW increased villus height in the jejunum (P = 0·02), width of villus in the ileum (P = 0·034) and number of goblet cells in villi of the jejunum (P = 0·006) and ileum (P = 0·01). 7. YCW increased antibody level against NDV at 21 and 42 d of age compared with the control and the HY supplemented diets (P < 0·05). 8. It was concluded that HY and YCW improved growth and feed efficiency in broilers, and considering the improvements in production traits and humoral immune responses, yeast cell wall may be a better dietary tool than the hydrolysed whole yeast cell as a performance enhancer for broilers.
Assuntos
Parede Celular/imunologia , Galinhas/imunologia , Galinhas/microbiologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Saccharomyces cerevisiae/imunologia , Animais , Anticorpos Antivirais/sangue , Peso Corporal/imunologia , Contagem de Colônia Microbiana/veterinária , Ingestão de Alimentos/imunologia , Histocitoquímica/veterinária , Imunidade Humoral/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestino Delgado/citologia , Intestino Delgado/ultraestruturaRESUMO
Analysis of the heavy-chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV-1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1(a) (clone 2) and IgG1(b) (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216-230. The Thr224 and Thr226 of IgG1(a) were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1(b) were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three-dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro-Ala-Ser-Ser indicated a potential structure-enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O-linked glycosylation sites and increasing the susceptibility to degradation by protease-secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1(d). Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1(c) that is distinct in both sequence and structure from the known sequence variants.
Assuntos
Alelos , Anticorpos Antivirais/genética , Bovinos/imunologia , Herpesvirus Bovino 1/imunologia , Imunoglobulina G/genética , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/química , Sequência de Bases , Feminino , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G/química , Camundongos , Modelos Moleculares , Dados de Sequência MolecularRESUMO
In this report we have investigated the role of the Ets-1 transcription factor in the differentiation of the NK cell lineage in mice. Splenic NK cells express high levels of Ets-1. Ets-1-deficient mice produced by gene targeting developed mature erythrocytes, monocytes, neutrophils, and T and B lymphocytes. However, spleens from the Ets-1-deficient mice contained significantly reduced numbers of natural killer (NK) cells, and splenocytes from these mice lacked detectable cytolytic activity against NK cell targets in vitro. Moreover, unlike wild-type animals, Ets-1-deficient mice developed tumors following subcutaneous injection of NK-susceptible RMA-S cells. These NK cell defects could not be correlated with defects in the expression of IL-12, IL-15, and IL-18 or the IL-2 or IL-15 receptors. Thus, Ets-1 defines a novel transcriptional pathway that is required for the development of the NK cell lineage in mice.
Assuntos
Células Matadoras Naturais/imunologia , Proteínas Proto-Oncogênicas/imunologia , Fatores de Transcrição/imunologia , Animais , Diferenciação Celular , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Hematopoese/genética , Hematopoese/imunologia , Técnicas In Vitro , Células Matadoras Naturais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/imunologia , Fenótipo , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Receptores de Citocinas/imunologia , Baço/citologia , Baço/imunologia , Fatores de Transcrição/genéticaRESUMO
The cAMP-responsive element-binding protein (CREB) transcription factor is required for normal T cell activation following stimulation through the T cell antigen receptor (TCR). CREB is present in resting T cells in an unphosphorylated and inactive state. TCR engagement results in the rapid phosphorylation of CREB on Ser133 and its concomitant activation. In the studies described in this report, we have investigated the signaling pathway(s) that are responsible for CREB activation in normal T cells. Using pharmacological agonists, we show that protein kinase C (PKC)-, calcium/calmodulin-, and protein kinase A-dependent pathways are each capable of independently eliciting CREB phosphorylation in T cells and thymocytes. Pharmacological inhibitor studies demonstrated that the PKC-mediated signaling pathway is required for TCR-mediated activation of CREB. In contrast, inhibitors of protein kinase A and calmodulin kinases had no effect on CREB phosphorylation following TCR cross-linking. T cells lacking the p56(lck) tyrosine kinase failed to phosphorylate CREB in response to TCR engagement. Overexpression of dominant-negative mutant Ras and Raf-1 proteins in Jurkat T cells abolished TCR-mediated CREB phosphorylation, whereas overexpression of the RSK2 serine/threonine kinase significantly potentiated TCR-mediated CREB phosphorylation. Taken together, these experiments are consistent with a model in which TCR engagement leads to the rapid phosphorylation and activation of CREB via a signaling pathway involving the activation of p56(lck), PKC, Ras, Raf-1, MEK, and RSK2. Given the importance of CREB phosphorylation in normal T cell activation, this pathway may be an attractive target for the development of novel immunosuppressive agents.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Complexo CD3/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Camundongos , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Serina/metabolismoRESUMO
CD72 is a B cell-specific glycoprotein that has been shown to be important for activation of mature B cells. Previously we showed that some of the early signaling events, such as calcium mobilization and phospholipase-gamma activation, were similar in B cell Ag receptor (BCR)- and CD72-stimulated B cells and that BCR- but not CD72-mediated early signaling events were blocked by protein kinase A activation. The present report shows that CD72 ligation induces a variety of tyrosine-phosphorylated proteins, most of which were of the same molecular mass as those seen in anti-IgM-treated B cells, except for a 72-kDa protein. Further analysis showed that the tyrosine kinases lyn and blk were activated in CD72-ligated B cells. Interestingly, the non-src kinase syk was not activated in CD72-stimulated cells whereas the tec family kinase btk was activated in both CD72- and BCR-stimulated B cells. Furthermore, B cells from xid mice were unresponsive to CD72-induced proliferation, indicating an essential role for btk in CD72-induced signaling events. Surprisingly, tyrosine phosphorylation of phospholipase C-gamma2 was normal in CD72-stimulated cells in spite of a lack of activation of syk. Furthermore, B cell proliferation through CD72 was blocked by the immunosuppressive agents cyclosporin A and FK506, indicating the important role for Ca2+-regulated activation events similar to BCR-stimulated cells. We propose that btk can substitute for syk in inducing phospholipase C-gamma2 tyrosine phosphorylation and initiating calcium mobilization in CD72-stimulated B lymphocytes.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Subpopulações de Linfócitos B/enzimologia , Precursores Enzimáticos/metabolismo , Ativação Linfocitária , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Ciclosporina/farmacologia , Feminino , Tolerância Imunológica/genética , Síndromes de Imunodeficiência/genética , Peptídeos e Proteínas de Sinalização Intracelular , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Camundongos Mutantes , Fosforilação , Especificidade por Substrato/imunologia , Quinase Syk , Tacrolimo/farmacologia , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismo , Cromossomo X/imunologiaRESUMO
Spi-B is a hematopoietic-specific Ets family transcription factor closely related to PU.1. Previous gene targeting experiments have shown that PU.1 is essential for the production of both lymphocytes and monocytes. We have now generated mice with a null mutation at the Spi-B locus. Unlike PU.1 mutant mice, Spi-B-/- mice are viable, fertile and possess mature B and T lymphocytes. However, Spi-B-/- mice exhibit severe abnormalities in B cell function and selective T cell-dependent humoral immune responses. First, although Spi-B-/- splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross-linking. Secondly, Spi-B-/- mice display abnormal T-dependent antigenic responses in vivo and produce low levels of antigen-specific IgG1, IgG2a and IgG2b after immunization. Finally, Spi-B-/- mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild-type animals, germinal centers in Spi-B-/- mice are smaller and short-lived with significantly increased numbers of apoptotic B cells. Taken together, these results demonstrate that Spi-B is essential for antigen-dependent expansion of B cells, T-dependent immune responses and maturation of normal germinal centers in vivo.
Assuntos
Linfócitos B/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Fatores de Transcrição/metabolismo , Animais , Medula Óssea/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Centro Germinativo/imunologia , Imunoglobulina M/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/genética , Tecido Linfoide/crescimento & desenvolvimento , Camundongos , Camundongos Mutantes , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Linfócitos T/imunologia , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
The basic/leucine zipper (bZip) transcription factor, CREB, binds to the CRE element (TGANNTCA). The transcriptional activity of CREB requires phosphorylation of the protein on a serine residue at position 119 (ref. 6). CREs are present in a number of T-cell genes but the precise role of CREB in T-cell differentiation and function was unknown. Here we show that resting thymocytes contain predominantly unphosphorylated (inactive) CREB, which is rapidly activated by phosphorylation on Ser 119 following thymocyte activation. T-cell development is normal in transgenic mice that express a dominant-negative form of CREB (CREBA119, with alanine at position 119) under the control of the T-cell-specific CD2 promoter/enhancer. In contrast, thymocytes and T cells from these animals display a profound proliferative defect characterized by markedly decreased interleukin-2 production, G1 cell-cycle arrest and subsequent apoptotic death in response to a number of different activation signals. This proliferative defect is associated with the markedly reduced induction of c-jun, c-fos, Fra-2 and FosB following activation of the CREBA119 transgenic thymocytes. We propose that T-cell activation leads to the phosphorylation and activation of CREB, which in turn is required for normal induction of the transcription factor AP1 and subsequent interleukin-2 production and cell-cycle progression.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Interleucina-2/biossíntese , Linfócitos T/citologia , Timo/citologia , Fatores Ativadores da Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Sanguíneas/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , DNA/metabolismo , Fase G1 , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Sequências Reguladoras de Ácido Nucleico , Serina/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição/metabolismoRESUMO
The Ets-1 proto-oncogene is a member of the Ets family of eukaryotic transcription factors. Members of this family play important roles in regulating gene expression in response to multiple developmental and mitogenic signals. Ets-1 is preferentially expressed at high levels in B and T cells of adult mice and is regulated during both thymocyte development and T-cell activation. To study the role of Ets-1 in T-cell development and function we have used the RAG-2-/- complementation system and murine embryonic stem (ES) cells containing homozygous deletions in the Ets-1 gene (Ets-1-/-). Ets-1-/(-)-RAG-2-/- chimaeric mice displayed markedly decreased numbers of mature thymocytes and peripheral T cells. Ets-1-/- T cells expressed normal levels of CD3 and T-cell antigen receptor (TCR)-alpha/beta. However, they displayed a severe proliferative defect in response to multiple activational signals and demonstrated increased rates of spontaneous apoptosis in vitro. These findings demonstrate that Ets-1 is required for the normal survival and activation of murine T cells.
Assuntos
Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA , Ativação Linfocitária/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Linfócitos T/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose/fisiologia , Complexo CD3/biossíntese , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Divisão Celular/fisiologia , Linhagem Celular , Quimera , Camundongos , Proteínas/fisiologia , Proto-Oncogene Mas , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Deleção de Sequência , Baço/citologia , Células-Tronco , Linfócitos T/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
We have recently demonstrated that pharmacological agents that elevated cAMP inhibited slgM but not Lyb2 mediated activation of murine B lymphocytes. In this report we show evidence for differential regulation of prostaglandin E2 (PGE2), a physiological agent that elevated cAMP and IFN-gamma on slgM and Lyb2 mediated B cell activation. PGE2 inhibited anti-IgM but not anti-Lyb2 induced DNA synthesis in a dose-dependent manner. Interestingly, rIFN-gamma also inhibited anti-Ig but not anti-Lyb2 induced DNA synthesis. rIFN-gamma exerted its effects directly on B cells since depletion of T cells and G-10 Sephadex adherent cells did not alter effects of IFN-gamma on anti-IgM and anti-Lyb2 induced DNA synthesis. Pretreatment of B cells with IL-4 and/or IL-5 did not prevent the IFN-gamma mediated inhibition of the anti-IgM response. The inhibitory effect of IFN-gamma was observed during early stages of B cell activation. Thus IFN-gamma inhibited anti-mu induced blast transformation and subsequent progression into the G1 phase of the cell cycle. The differential effects exerted by PGE2 and rIFN-gamma appeared to be mediated by distinct mechanisms. Thus PGE2 but not rIFN-gamma, at concentrations inhibitory to the slgM response, induced elevation of intracellular cAMP levels. These results demonstrate that physiologically relevant immunomodulators such as PGE2 and IFN-gamma can differentially regulate murine B cell responses mediated through the antigen receptor and Lyb2 molecules by cAMP dependent and independent mechanisms. Relevance of this regulation for the induction of antibody synthesis by Th1 and Th2 types of helper T cells is discussed.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , AMP Cíclico/fisiologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linfócitos B/citologia , Células Cultivadas , Dinoprostona/farmacologia , Feminino , Imunoglobulina M/fisiologia , Interferon gama/farmacologia , Interleucina-4/farmacologia , Interleucina-5/farmacologia , Interfase , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBARESUMO
The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.
Assuntos
Antígenos Ly/fisiologia , Linfócitos B/imunologia , AMP Cíclico/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Receptores de Antígenos/fisiologia , Transdução de Sinais , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Cálcio/metabolismo , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Feminino , Hidrólise , Camundongos , Camundongos Endogâmicos , Proteínas Quinases/fisiologia , Receptores da Transferrina/análiseRESUMO
B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B cells responded to the auto D1.4 T cells by increasing cell surface Ia as well as size, but failed to incorporate tritiated thymidine. The responsiveness to interleukin-4 was found to be similar between B cells from young and old mice. It appeared that the B cells from old mice are specifically defective in progressing from the G0 phase of cell cycle into the S phase when stimulated with the auto D1.4 T cells.