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1.
Blood ; 117(4): 1228-38, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21079150

RESUMO

Mammalian TOR (mTOR) regulates cell growth, proliferation, and migration. Because mTOR knock-outs are embryonic lethal, we generated a viable hypomorphic mouse by neo-insertion that partially disrupts mTOR transcription and creates a potential physiologic model of mTORC1/TORC2 inhibition. Homozygous knock-in mice exhibited reductions in body, organ, and cell size. Although reductions in most organ sizes were proportional to decreased body weight, spleens were disproportionately smaller. Decreases in the total number of T cells, particularly memory cells, and reduced responses to chemokines suggested alterations in T-cell homing/homeostasis. T-cell receptor-stimulated T cells proliferated less, produced lower cytokine levels, and expressed FoxP3. Decreased neutrophil numbers were also observed in the spleen, despite normal development and migration in the bone marrow. However, B-cell effects were most pronounced, with a partial block in B-cell development in the bone marrow, altered splenic populations, and decreases in proliferation, antibody production, and migration to chemokines. Moreover, increased AKT(Ser473) phosphorylation was observed in activated B cells, reminiscent of cancers treated with rapamycin, and was reduced by a DNA-pk inhibitor. Thus, mTOR is required for the maturation and differentiation of multiple immune cell lineages. These mice provide a novel platform for studying the consequences of constitutively reduced mTORC1/TORC2 activity.


Assuntos
Formação de Anticorpos/genética , Linfócitos B/citologia , Linfócitos B/fisiologia , Serina-Treonina Quinases TOR/genética , Animais , Linfócitos B/metabolismo , Tamanho Corporal/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Tamanho Celular , Regulação para Baixo/imunologia , Regulação para Baixo/fisiologia , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Tamanho do Órgão/genética , Baço/anatomia & histologia , Baço/metabolismo
2.
J Immunol ; 185(5): 2819-27, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20668219

RESUMO

Signaling lymphocytic activation molecule-associated protein (SAP), an adaptor molecule that recruits Fyn to the signaling lymphocytic activation molecule (SLAM) family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4(+) T cells from SAP-deficient mice have defective TCR-induced and follicular Th cell IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4(+) T cells exhibit decreased protein kinase C (PKC)-theta recruitment upon TCR stimulation. We demonstrate in this paper using GST pulldowns and coimmunoprecipitation studies that SAP constitutively associates with PKC- in T cells. SAP-PKC-theta interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP's interactions with PKC-theta occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-theta recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-theta, like SAP, was required for SLAM-mediated increases in IL-4 production, and, conversely, membrane-targeted PKC-theta mutants rescued IL-4 expression in SAP(-/-) CD4(+) T cells, providing genetic evidence that PKC-theta is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Interleucina-4/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-4/deficiência , Interleucina-4/genética , Isoenzimas/deficiência , Células Jurkat , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase C/deficiência , Proteína Quinase C-theta , Transporte Proteico/genética , Transporte Proteico/imunologia , Receptores de Superfície Celular/deficiência , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Regulação para Cima/genética , Regulação para Cima/imunologia
3.
J Exp Med ; 211(3): 529-43, 2014 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-24534190

RESUMO

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.


Assuntos
Citocinas/metabolismo , Imunidade Celular/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Vetores Genéticos/genética , Immunoblotting , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Oligonucleotídeos/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptor Cross-Talk/imunologia , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismo
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