RESUMO
Innate lymphoid cells (ILCs) are tissue-resident lymphoid cells that reside mostly at barrier surfaces and participate in the initial response against pathogens. They are classified into different types based on effector programs that are based on cytokine production and transcription factor expression. They all derive from the common lymphoid precursor, but the molecular mechanisms regulating ILC subset development is not well understood. Experiments using Id2 knockout mice have previously shown that E protein activity inhibition is an absolute requirement for the development of all ILC subsets. In this study, we use a genetic approach to demonstrate that small increases in E protein activity during ILC development selectively inhibit type 2 ILC development. Type 1 ILCs are mostly unperturbed, and type 3 ILC show only a minor inhibition. This effect is first evident at the ILC2 progenitor stage and is ILC intrinsic. Therefore, our results demonstrate that modulation of E protein activity can bias cell fate decisions in developing ILCs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Imunidade Inata/imunologia , Células T Matadoras Naturais/imunologia , Fator de Transcrição 4/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Células Progenitoras Linfoides/metabolismo , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Transduction of lymphoid progenitors with retroviral or lentiviral vectors is a powerful experimental strategy to tease out the role of a gene or pathway in T cell development via gain-of-function or loss-of-function strategies. Here we discuss different approaches to use this powerful technology, and present some protocols that we use to transduce murine HSCs, thymocytes, and lymphoid cell lines with these viral vectors.
Assuntos
Vetores Genéticos/genética , Células Precursoras de Linfócitos T/metabolismo , Retroviridae/genética , Linfócitos T/metabolismo , Transdução Genética , Animais , Linhagem Celular , Humanos , CamundongosRESUMO
E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core/biossíntese , Interferon gama/biossíntese , Subunidade beta de Receptor de Interleucina-2/biossíntese , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA não Traduzido/genética , Transdução de Sinais/imunologia , Proteínas com Domínio T/biossínteseRESUMO
iNKT cells derive from CD4(+)CD8(+) DP thymocytes, and are selected by thymocyte-thymocyte interactions through signals from their invariant Vα14-Jα18 TCR and from the costimulatory molecules SLAMF1 and SLAMF6. Genetic studies have demonstrated the contribution of different signaling pathways to this process. Surprisingly, current models imply that the Ras/MAPK pathway, one of the critical mediators of conventional αß T cell positive selection, is not necessary for iNKT cell development. Using mice defective at different levels of this pathway our results refute this paradigm, and demonstrate that Ras, and its downstream effectors Egr-1 and Egr-2 are required for positive selection of iNKT cells. Interestingly our results also show that there are differences in the contributions of several of these molecules to the development of iNKT and conventional αß T cells.
Assuntos
Sistema de Sinalização das MAP Quinases , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/enzimologia , Proteínas ras/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos CD1d/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Integrases/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Superfície Celular/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Timo/citologiaRESUMO
GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Fator de Transcrição GATA3/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Calcineurina/fisiologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/fisiologia , Fator de Transcrição GATA3/biossíntese , Fator de Transcrição GATA3/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myb/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fatores de Transcrição/fisiologia , Proteínas ras/fisiologiaRESUMO
Type I invariant NKT cells (iNKT cells) are a subset of alphabeta T cells characterized by the expression of an invariant alpha-chain variable region 14-alpha-chain joining region 18 (V(alpha)14J(alpha)18) T cell antigen receptor (TCR) alpha-chain. The iNKT cells derive from CD4(+)CD8(+) double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow V(alpha)14-J(alpha)18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM-adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.
Assuntos
Antígenos CD1d/metabolismo , Células T Matadoras Naturais/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteína bcl-X/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Transplante de Medula Óssea , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Fator de Transcrição GATA3/genética , Rearranjo Gênico do Linfócito T/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Células Precursoras de Linfócitos T/citologia , Células Precursoras de Linfócitos T/imunologia , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/imunologia , Quimera por Radiação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Saposinas/genética , Saposinas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Timo/citologia , Proteína bcl-X/genética , Proteína bcl-X/imunologiaRESUMO
During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.
