RESUMEN
Mature lymphoid cells express the transcription repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells ('myeloid genes'). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2(hi) CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs.
Asunto(s)
Linfocitos B/citología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/fisiología , Células Precursoras de Linfocitos B/citología , Transactivadores/metabolismo , Animales , Linfocitos B/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linaje de la Célula , Separación Celular , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Regulación de la Expresión Génica/fisiología , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Linfopoyesis/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Precursoras de Linfocitos B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genéticaRESUMEN
Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell-specific deficiency in Uhrf1 (Uhrf1(fl/fl)Cd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1(fl/fl)Cd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.
Asunto(s)
Colitis/inmunología , Colon/inmunología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Epigénesis Genética , Proteínas Nucleares/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Proteínas Potenciadoras de Unión a CCAAT , Puntos de Control del Ciclo Celular , Proliferación Celular , Células Cultivadas , Clostridium/inmunología , Colitis/genética , Colon/microbiología , Metilación de ADN , Perfilación de la Expresión Génica , Interleucina-2 , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microbiota/inmunología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Simbiosis/inmunología , Ubiquitina-Proteína Ligasas , Regulación hacia ArribaRESUMEN
ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.
Asunto(s)
Médula Ósea , Células Madre Hematopoyéticas , Linaje de la Célula/genética , Células Madre Hematopoyéticas/metabolismo , Médula Ósea/metabolismo , Hematopoyesis/genética , ARN Mensajero/metabolismo , Diferenciación Celular/genéticaRESUMEN
Single-cell whole-transcriptome analysis is the gold standard approach to identifying molecularly defined cell phenotypes. However, this approach cannot be used for dynamics measurements such as live-cell imaging. Here, we developed a multifunctional robot, the automated live imaging and cell picking system (ALPS) and used it to perform single-cell RNA sequencing for microscopically observed cells with multiple imaging modes. Using robotically obtained data that linked cell images and the whole transcriptome, we successfully predicted transcriptome-defined cell phenotypes in a noninvasive manner using cell image-based deep learning. This noninvasive approach opens a window to determine the live-cell whole transcriptome in real time. Moreover, this work, which is based on a data-driven approach, is a proof of concept for determining the transcriptome-defined phenotypes (i.e., not relying on specific genes) of any cell from cell images using a model trained on linked datasets.
Asunto(s)
Aprendizaje Profundo , Procedimientos Quirúrgicos Robotizados , Robótica , Transcriptoma , Procesamiento de Imagen Asistido por Computador/métodos , Perfilación de la Expresión Génica , FenotipoRESUMEN
Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that â¼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.
Asunto(s)
Linfocitos B/metabolismo , Células Madre Multipotentes/metabolismo , Transcripción Genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Linaje de la Célula/genética , Células Cultivadas , Epigénesis Genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Código de Histonas , Factor 4 Similar a Kruppel , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX5/fisiología , Análisis de la Célula Individual , Transactivadores/fisiología , TranscriptomaRESUMEN
Blood cells are thought to have emerged as phagocytes in the common ancestor of animals followed by the appearance of novel blood cell lineages such as thrombocytes, erythrocytes, and lymphocytes, during evolution. However, this speculation is not based on genetic evidence and it is still possible to argue that phagocytes in different species have different origins. It also remains to be clarified how the initial blood cells evolved; whether ancient animals have solely developed de novo programs for phagocytes or they have inherited a key program from ancestral unicellular organisms. Here, we traced the evolutionary history of blood cells, and cross-species comparison of gene expression profiles revealed that phagocytes in various animal species and Capsaspora (C.) owczarzaki, a unicellular organism, are transcriptionally similar to each other. We also found that both phagocytes and C. owczarzaki share a common phagocytic program, and that CEBPα is the sole transcription factor highly expressed in both phagocytes and C. owczarzaki. We further showed that the function of CEBPα to drive phagocyte program in nonphagocytic blood cells has been conserved in tunicate, sponge, and C. owczarzaki. We finally showed that, in murine hematopoiesis, repression of CEBPα to maintain nonphagocytic lineages is commonly achieved by polycomb complexes. These findings indicate that the initial blood cells emerged inheriting a unicellular organism program driven by CEBPα and that the program has also been seamlessly inherited in phagocytes of various animal species throughout evolution.
Asunto(s)
Eucariontes , Evolución Molecular , Animales , Ratones , Filogenia , Eucariontes/genética , Regulación de la Expresión Génica , Células SanguíneasRESUMEN
In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.
Asunto(s)
Linfocitos B/citología , Transformación Celular Neoplásica/genética , Epigénesis Genética/genética , Silenciador del Gen , Proteínas del Grupo Polycomb/metabolismo , Linfocitos T/citología , Animales , Linaje de la Célula , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Ratones Endogámicos C57BL , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Complejo Represivo Polycomb 1/genética , Regiones Promotoras Genéticas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.
Asunto(s)
Asparaginasa/uso terapéutico , Aspartatoamoníaco Ligasa/genética , Variantes Farmacogenómicas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animales , Niño , Aberraciones Cromosómicas , Metilación de ADN/genética , Impresión Genómica/genética , Humanos , RatonesRESUMEN
Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.
Asunto(s)
Linfocitos T CD4-Positivos , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Evolución Clonal , Perfilación de la Expresión Génica , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/fisiología , Persona de Mediana Edad , Análisis de la Célula Individual , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CD4 and the transcription factor ThPOK are essential for the differentiation of major histocompatibility complex class II-restricted thymocytes into the helper T cell lineage; their genes (Cd4 and Zbtb7b (called 'ThPOK' here)) are repressed by transcriptional silencer elements in cytotoxic T cells. The molecular mechanisms regulating expression of these genes during helper T cell lineage differentiation remain unknown. Here we showed that inefficient upregulation of ThPOK, induced by removal of the proximal enhancer from the ThPOK locus, resulted in the transdifferentiation of helper lineage-specified cells into the cytotoxic T cell lineage. Furthermore, direct antagonism by ThPOK of the Cd4 and ThPOK silencers generated two regulatory loops that initially inhibited Cd4 downregulation and later stabilized ThPOK expression. Our results show how an initial lineage-specification signal can be amplified and stabilized during the lineage-commitment process.
Asunto(s)
Transdiferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Elementos Silenciadores Transcripcionales/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/citología , Factores de Transcripción/genética , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Citometría de Flujo , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Timo/citología , Timo/inmunología , TransfecciónRESUMEN
B-1 cells are innate-like B-cell population and produce natural antibodies that contribute to the first line of host defense. There are two subsets of B-1 cells: B-1a and B-1b. B-1a cells are the main producer of poly-reactive and autoreactive natural IgM antibodies, whereas B-1b cells can respond specifically to T-cell-independent antigens. Despite the functional significance of B-1a and B-1b cells, little information is available about what regulates the development of these two subsets. We found that Kelch-like protein 14 (KLHL14) was expressed at high levels in B cells but only at low levels in a few non-lymphoid tissues. Although mice lacking KLHL14 died right after birth, the heterozygotes developed normally with no gross abnormalities by appearance. B-cell development in the bone marrow and maturation and activation in the spleen were not affected in the heterozygous mice. However, the number of peritoneal B-1a cells was significantly reduced while B-1b cells were increased in Klhl14 heterozygous mice compared with wild-type (WT) mice. Consistently, Rag1-/- mice reconstituted with Klhl14-/- fetal liver cells had a more severe reduction of B-1a and an increase of B-1b cells in the peritoneal cavity. KLHL14 did not affect the turnover or apoptosis of B-1a and B-1b cells in vivo. Moreover, Klhl14-/- fetal liver contained a similar proportion and absolute numbers of the B-1 progenitor cells as did WT fetal liver. These results suggest that KLHL14 promotes B-1a development in mice.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Subgrupos de Linfocitos B/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficienciaRESUMEN
Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1).
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Factores de Transcripción/inmunología , Células 3T3 , Animales , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Proteínas de Unión al ADN/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/inmunología , Regiones Promotoras Genéticas/genética , Proteínas Inhibidoras de STAT Activados/inmunología , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/citología , Factores de Transcripción/genética , Ubiquitina-Proteína LigasasRESUMEN
The immune system is influenced by the vital zinc (Zn) status, and Zn deficiency triggers lymphopenia; however, the mechanisms underlying Zn-mediated lymphocyte maintenance remain elusive. Here we investigated ZIP10, a Zn transporter expressed in the early B-cell developmental process. Genetic ablation of Zip10 in early B-cell stages resulted in significant reductions in B-cell populations, and the inducible deletion of Zip10 in pro-B cells increased the caspase activity in parallel with a decrease in intracellular Zn levels. Similarly, the depletion of intracellular Zn by a chemical chelator resulted in spontaneous caspase activation leading to cell death. Collectively, these findings indicated that ZIP10-mediated Zn homeostasis is essential for early B-cell survival. Moreover, we found that ZIP10 expression was regulated by JAK-STAT pathways, and its expression was correlated with STAT activation in human B-cell lymphoma, indicating that the JAK-STAT-ZIP10-Zn signaling axis influences the B-cell homeostasis. Our results establish a role of ZIP10 in cell survival during early B-cell development, and underscore the importance of Zn homeostasis in immune system maintenance.
Asunto(s)
Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/inmunología , Zinc/metabolismo , Animales , Linfocitos B/citología , Caspasas/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Diferenciación Celular , Supervivencia Celular/inmunología , Citocinas/metabolismo , Homeostasis , Humanos , Quinasas Janus/metabolismo , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfopenia/etiología , Linfopenia/inmunología , Linfopenia/metabolismo , Ratones , Ratones Noqueados , Modelos Inmunológicos , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Zinc/deficienciaRESUMEN
The humoral immune response, also called the antibody-mediated immune response, is one of the main adaptive immune systems. The essential micronutrient zinc (Zn) is known to modulate adaptive immune responses, and dysregulated Zn homeostasis leads to immunodeficiency. However, the molecular mechanisms underlying this Zn-mediated modulation are largely unknown. Here, we show that the Zn transporter SLC39A10/ZIP10 plays an important role in B-cell antigen receptor (BCR) signal transduction. Zip10-deficiency in mature B cells attenuated both T-cell-dependent and -independent immune responses in vivo. The Zip10-deficient mature B cells proliferated poorly in response to BCR cross-linking, as a result of dysregulated BCR signaling. The perturbed signaling was found to be triggered by a reduction in CD45R phosphatase activity and consequent hyperactivation of LYN, an essential protein kinase in BCR signaling. Our data suggest that ZIP10 functions as a positive regulator of CD45R to modulate the BCR signal strength, thereby setting a threshold for BCR signaling in humoral immune responses.
Asunto(s)
Proteínas de Transporte de Catión/inmunología , Inmunidad Humoral , Receptores de Antígenos de Linfocitos B/metabolismo , Zinc/metabolismo , Inmunidad Adaptativa , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Diferenciación Celular/inmunología , Senescencia Celular/inmunología , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor, an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro. Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor, an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context-dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.
Asunto(s)
Linfopoyesis/fisiología , Modelos Inmunológicos , Complejos Multiproteicos/fisiología , Células Precursoras de Linfocitos T/fisiología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Proteínas Portadoras/metabolismo , Ciclo Celular/inmunología , Ciclo Celular/fisiología , Cartilla de ADN , Citometría de Flujo , Perfilación de la Expresión Génica , Immunoblotting , Inmunohistoquímica , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Complejos Multiproteicos/deficiencia , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteína Reguladora Asociada a mTOR , Transducción de Señal/fisiología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/deficienciaRESUMEN
Innate immune responses are important in combating various microbes during the early phases of infection. Natural killer (NK) cells are innate lymphocytes that, unlike T and B lymphocytes, do not express antigen receptors but rapidly exhibit cytotoxic activities against virus-infected cells and produce various cytokines. Here we report a new type of innate lymphocyte present in a novel lymphoid structure associated with adipose tissues in the peritoneal cavity. These cells do not express lineage (Lin) markers but do express c-Kit, Sca-1 (also known as Ly6a), IL7R and IL33R. Similar lymphoid clusters were found in both human and mouse mesentery and we term this tissue 'FALC' (fat-associated lymphoid cluster). FALC Lin(-)c-Kit(+)Sca-1(+) cells are distinct from lymphoid progenitors and lymphoid tissue inducer cells. These cells proliferate in response to IL2 and produce large amounts of T(H)2 cytokines such as IL5, IL6 and IL13. IL5 and IL6 regulate B-cell antibody production and self-renewal of B1 cells. Indeed, FALC Lin(-)c-Kit(+)Sca-1(+) cells support the self-renewal of B1 cells and enhance IgA production. IL5 and IL13 mediate allergic inflammation and protection against helminth infection. After helminth infection and in response to IL33, FALC Lin(-)c-Kit(+)Sca-1(+) cells produce large amounts of IL13, which leads to goblet cell hyperplasia-a critical step for helminth expulsion. In mice devoid of FALC Lin(-)c-Kit(+)Sca-1(+) cells, such goblet cell hyperplasia was not induced. Thus, FALC Lin(-)c-Kit(+)Sca-1(+) cells are T(H)2-type innate lymphocytes, and we propose that these cells be called 'natural helper cells'.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/inmunología , Antígenos Ly/inmunología , Citocinas/inmunología , Regulación de la Expresión Génica , Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Proteínas Proto-Oncogénicas c-kit/inmunología , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Proliferación Celular , Humanos , Proteínas de la Membrana/genética , Mesenterio/inmunología , Ratones , Ratones Endogámicos C57BL , Nematodos/fisiología , Infecciones por Nematodos/inmunología , Proteínas Proto-Oncogénicas c-kit/genética , Células Th2/inmunologíaRESUMEN
Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.
Asunto(s)
Linaje de la Célula/genética , Perfilación de la Expresión Génica , Leucemia de Células B/metabolismo , MicroARNs/metabolismo , Análisis de Varianza , Linfocitos B/metabolismo , Western Blotting , Trasplante de Médula Ósea , Línea Celular Tumoral , Linaje de la Célula/inmunología , Cartilla de ADN , Vectores Genéticos/genética , Humanos , Luciferasas , Células Progenitoras Mieloides , Oligonucleótidos/genética , Estadísticas no Paramétricas , Transactivadores/metabolismo , Factor de Transcripción 3/metabolismoRESUMEN
T, B, and NK lymphocytes are generated from pluripotent hematopoietic stem cells through a successive series of lineage restriction processes. Many regulatory components, such as transcription factors, cytokines/cytokine receptors, and signal transduction molecules orchestrate cell fate specification and determination. In particular, transcription factors play a key role in regulating lineage-associated gene programs. Recent findings suggest the involvement of epigenetic factors in the maintenance of cell fate. Here, we review the early developmental events during lymphocyte lineage determination, focusing on the transcriptional networks and epigenetic regulation. Finally, we also discuss the developmental relationship between acquired and innate lymphoid cells.
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Linfocitos B/citología , Epigénesis Genética , Linfopoyesis , Linfocitos T/citología , Animales , Linfocitos B/metabolismo , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Humanos , Linfocitos T/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Immune defect in ataxia telangiectasia patients has been attributed to either the failure of V(D)J recombination or class-switch recombination, and the chromosomal translocation in their lymphoma often involves the TCR gene. The ATM-deficient mouse exhibits fewer CD4 and CD8 single-positive T cells because of a failure to develop from the CD4(+)CD8(+) double-positive phase to the single-positive phase. Although the occurrence of chromosome 14 translocations involving TCR-δ gene in ATM-deficient lymphomas suggests that these are early events in T-cell development, a thorough analysis focusing on early T-cell development has never been performed. Here we demonstrate that ATM-deficient mouse thymocytes are perturbed in passing through the ß- or γδ-selection checkpoint, leading in part to the developmental failure of T cells. Detailed karyotype analysis using the in vitro thymocyte development system revealed that RAG-mediated TCR-α/δ locus breaks occur and are left unrepaired during the troublesome ß- or γδ-selection checkpoints. By getting through these selection checkpoints, some of the clones with random or nonrandom chromosomal translocations involving TCR-α/δ locus are selected and accumulate. Thus, our study visualized the first step of multistep evolutions toward lymphomagenesis in ATM-deficient thymocytes associated with T-lymphopenia and immunodeficiency.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Reordenamiento Génico de Linfocito T , Proteínas Serina-Treonina Quinasas/fisiología , Timocitos/inmunología , Timocitos/patología , Translocación Genética , Proteínas Supresoras de Tumor/fisiología , Recombinación V(D)J , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Trasplante de Médula Ósea , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Femenino , Citometría de Flujo , Inestabilidad Genómica , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timocitos/metabolismoRESUMEN
V(D)J recombination of Ig and TCR genes is strictly regulated in a lineage- and stage-specific manner by the accessibility of target gene chromatin to the recombinases RAG1 and RAG2. It has been shown that enforced expression of the basic helix-loop-helix protein, E2A, together with RAG1/2 in a nonlymphoid cell line BOSC23 can induce V(D)J recombination in endogenous Igκ and TCR loci by increasing chromatin accessibility of target gene segments. In this study, we demonstrate that ectopically expressed E2A proteins in BOSC23 cells have the ability to bind directly to the promoter and recombination signal sequence of Vκ genes and to recruit histone acetyltransferase CBP/p300. Overexpression of CBP/p300 in conjunction with E2A results in enhancement of E2A-induced histone acetylation, germline transcription, and Igκ rearrangement. Conversely, knockdown of endogenous CBP/p300 expression by small interfering RNA leads to a decrease in histone acetylation, germline transcription and Igκ rearrangement. Furthermore, analyses using a mouse pre-B cell line revealed that endogenous E2A proteins also bind to a distinct set of Vκ genes and regulatory regions in the mouse Igκ locus and act to increase histone acetylation by recruiting p300, confirming the similar findings observed with BOSC23 cells. These observations indicate that E2A plays critical roles in inducing Igκ rearrangement by directly binding to and increasing chromatin accessibility at target gene segments.