RESUMO
It has been shown previously that a human CD2 (hCD2) disabled locus control region (LCR) transgene is unable to establish an open chromatin configuration in all the T cells, and this leads to position effect variegation of the transgene. In this study we show that thymus-specific overexpression of human high mobility group box transcription factor 1 (HBP1), a transcription factor that binds a specific sequence within the hCD2 LCR, affects thymus cellularity as well as the number of CD8(+) thymocytes in two independent transgenic mouse lines and increases the proportion of T cells that fully activate the transgenic locus in hCD2 variegating mice in a sequence-specific dependent manner. This finding suggests that overexpression of HBP1 can affect lineage commitment and can relieve the suppressive influence of heterochromatin, allowing thymocytes to express the variegating target locus more efficiently. These effects could be the result of direct HBP1 action on LCR activity. Alternatively, the extra HBP1 molecules may sequester repressive elements away from the LCR, thus allowing transcription permissive states to form on the transgene locus.
Assuntos
Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Timo/citologia , Timo/imunologia , Transgenes , Animais , Antígenos CD2/biossíntese , Antígenos CD2/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologia , Timo/enzimologiaRESUMO
Vav1 is a signaling protein required for both positive and negative selection of CD4(+)CD8(+) double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase Cgamma1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase Cgamma1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Proteínas Son Of Sevenless/metabolismo , Animais , Ativação Enzimática , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipase C gama , Fosforilação , Proteínas Proto-Oncogênicas c-vav , Proteína SOS1/metabolismo , Timo/citologia , Fosfolipases Tipo C/metabolismo , Proteínas ras/metabolismoRESUMO
Activation of T lineage cells through the TCR by peptide-MHC complexes on APC is critically dependent on rearrangement of the actin cytoskeleton. Vav1 is a guanine nucleotide exchange factor for members of the Rho/Rac family of GTPases which is activated following TCR stimulation, suggesting that it may transduce TCR signals to the activation of some or all actin-controlled processes. We show that Vav1-deficient double-positive thymocytes are less efficient at forming conjugates with APC presenting agonist peptide than wild-type cells are. Furthermore we demonstrate that Vav1 is required for TCR-induced activation of the integrin LFA-1, which is likely to explain the defect in conjugate formation. However, once Vav1-deficient cells form a conjugate, the assembly of proteins into an immunological synapse at the conjugate interface is normal. In contrast, thymocyte polarization is defective in the absence of Vav1, as judged by the relocalization of the microtubule-organizing center. These data demonstrate that Vav1 transduces signals to only a subset of cytoskeleton-dependent events at the immunological synapse.
Assuntos
Proteínas de Ciclo Celular , Antígeno-1 Associado à Função Linfocitária/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Sinapses/imunologia , Células Apresentadoras de Antígenos/fisiologia , Polaridade Celular , Humanos , Proteínas Proto-Oncogênicas c-vav , Proteína cdc42 de Ligação ao GTP/fisiologiaRESUMO
Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes ofmice in vivo and in vitro, allowing the generation of tissue-specific conditional mutants. We have generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R-EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single-cell level. Analysis showed that the vav promoter elements were able to direct Cre-mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre-mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre-transgenic lines will be useful in generating tissue-specific gene deletions within all the cells of hematopoietic or lymphoid tissues.
Assuntos
Marcação de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Integrases/biossíntese , Tecido Linfoide/metabolismo , Proteínas Virais/biossíntese , Animais , Linfócitos B/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Bacteriófago P1/genética , Antígenos CD2/genética , Linhagem da Célula , Genes Reporter , Humanos , Integrases/genética , Região de Controle de Locus Gênico , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Proteínas Oncogênicas/genética , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-vav , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Linfócitos T/metabolismo , Testículo/metabolismo , Proteínas Virais/genéticaRESUMO
Interactions of T cells with MHC plus peptide in the peripheral lymphoid system are important for their survival. In this study we investigated further the molecular consequences of such interactions using F5 TCR transgenic mice and peptides previously shown to induce either negative or positive selection in the thymus. Following TCR ligation with the negatively selecting agonist peptide, mature CD8(+) cells proliferated and up-regulated the activation marker CD69. Interestingly, ligation of this TCR with MHC molecules loaded with high concentrations of the positively selecting peptide also resulted in the aforementioned changes, but with slower kinetics. Analysis of the biochemical changes that occur following stimulation with these peptides showed that phosphorylation of key signaling molecules, such as ZAP-70, CD3zeta, Vav, SLP-76, LAT, and ERK-1 and 2, could be detected after exposure to agonist but not antagonist peptide. Confocal microscopy, however, revealed infrequent phosphorylation 'patches' at the site of contact between T cells and APC presenting the antagonist peptide. Our data suggest that peptides capable of inducing positive selection in the thymus can be recognized by mature T cells and cause proliferation, up-regulation of CD69 and accumulation of phosphorylated proteins at the immunological synapse with low efficiency; however no phosphorylation of signaling molecules can be detected using conventional biochemical assays.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Sítios de Ligação , Linfócitos T CD8-Positivos/efeitos dos fármacos , Lectinas Tipo C , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptídeos/farmacologia , Fosforilação , Proteínas/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais , Tirosina/químicaRESUMO
Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4(+)CD8(+) double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-gamma1 (PLCgamma1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCgamma1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCgamma1 and the adaptor molecule Src homology 2 domain-containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.