RESUMO
Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.
Assuntos
Proteínas Correpressoras/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Linfócitos T/citologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Separação Celular , Citometria de Fluxo , Deleção de Genes , Humanos , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Timo/citologia , TransgenesRESUMO
Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Proteínas Correpressoras/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Proteínas Correpressoras/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Hematopoese/genética , Hematopoese/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/metabolismo , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Quimera por RadiaçãoRESUMO
T cell development depends on the coordinated interplay between receptor signaling and transcriptional regulation. Through a genetic complementation screen a transcriptional repressor, NKAP, was identified. NKAP associated with the histone deacetylase HDAC3 and was shown to be part of a DNA-binding complex, as demonstrated by chromatin immunoprecipitation. NKAP also associated with the Notch corepressor complex. The expression of NKAP during T cell development inversely correlated with the expression of Notch target genes, implying that NKAP may modulate Notch-mediated transcription. To examine the function of NKAP in T cell development, we ablated NKAP by Lck(cre). Loss of NKAP blocked development of alphabeta but not gammadelta T cells, and Nkap(fl/o)Lck(cre) DP T cells expressed 8- to 20-fold higher amounts of Hes1, Deltex1, and CD25 mRNA. Thus, NKAP functions as a transcriptional repressor, acting on Notch target genes, and is required for alphabeta T cell development.
Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Correpressoras , Histona Desacetilases/metabolismo , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Proteínas Repressoras/genéticaRESUMO
Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Antígeno H-Y/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Previous reports have suggested that the protooncogene c-myb participates in T cell development in the thymus and mature T cell proliferation. We have generated two T cell-specific c-myb knockout mouse models, myb/LckCre and myb/CD4Cre. We have demonstrated that c-myb is required for the development of thymocytes at the DN3 stage, for survival and proliferation of double-positive thymocytes, for differentiation of single-positive CD4 and CD8 T cells, and for the proliferative responses of mature T cells. In addition, our data show that c-myb is directly involved in the formation of double-positive CD4+CD8+CD25+, CD4+CD25+, and CD8+CD25+ T cells, developmental processes that may imply a role for c-myb in autoimmune dysfunction.