RESUMEN
The mechanisms underlying the ability of embryonic stem cells (ESCs) to rapidly activate lineage-specific genes during differentiation remain largely unknown. Through multiple CRISPR-activation screens, we discovered human ESCs have pre-established transcriptionally competent chromatin regions (CCRs) that support lineage-specific gene expression at levels comparable to differentiated cells. CCRs reside in the same topological domains as their target genes. They lack typical enhancer-associated histone modifications but show enriched occupancy of pluripotent transcription factors, DNA demethylation factors, and histone deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while HDAC1 family members prevent premature activation. This "push and pull" feature resembles bivalent domains at developmental gene promoters but involves distinct molecular mechanisms. Our study provides new insights into pluripotency regulation and cellular plasticity in development and disease. One sentence summary: We report a class of distal regulatory regions distinct from enhancers that confer human embryonic stem cells with the competence to rapidly activate the expression of lineage-specific genes.
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The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.
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Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/terapia , Inmunoterapia , Interleucina-15/uso terapéutico , Melanoma Experimental/terapia , Animales , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Humanos , Interleucina-15/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Regnase-1 is an emerging regulator of immune responses with essential roles in the posttranscriptional control of immune cell activation. Regnase-1 is expressed in B cells; however, its B cell-specific functions remain unknown. Here, we demonstrate that Regnase-1 prevents severe autoimmune pathology and show its essential role in maintaining B cell homeostasis. Using Cre driver mice for ablation of Regnase-1 at various stages of B cell development, we demonstrate that loss of Regnase-1 leads to aberrant B cell activation and differentiation, resulting in systemic autoimmunity and early morbidity. The basis of these findings was informed by gene expression data revealing a regulatory role for Regnase-1 in the suppression of a transcriptional program that promotes B cell activation, survival, and differentiation. Overall, our study shows that Regnase-1 exerts critical control of B cell activation, which is required for prevention of immunopathology.
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Autoinmunidad/genética , Linfocitos B/metabolismo , Homeostasis/genética , Activación de Linfocitos/genética , Ribonucleasas/genética , Animales , Diferenciación Celular/genética , Perfilación de la Expresión Génica/métodos , Ratones Noqueados , Ratones Transgénicos , RNA-Seq/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ribonucleasas/metabolismoRESUMEN
Humoral immune responses depend on the generation of high-affinity antigen-specific antibodies. Germinal center (GC) B cells are the cornerstone of this response in peripheral lymphoid organs. High purities of GC B cells, and also naive B cells, are required for accurate analysis in downstream assays to yield essential knowledge on immunity. This protocol lays out quick and easy steps to purify GC B cells from spleens of immunized mice or B cells from naive animals by negative selection using MACS. For complete details on the use and execution of this protocol, please refer to Ramezani-Rad et al. (2020).
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Separación Celular/métodos , Separación Inmunomagnética/métodos , Animales , Linfocitos B/citología , Centro Germinal/citología , Inmunidad Humoral , Inmunización , Leucosialina/inmunología , Ratones , Bazo/inmunologíaRESUMEN
Germinal center (GC) B cells surge in their proliferative capacity, which poses a direct risk for B cell malignancies. G1- to S-phase transition is dependent on the expression and stability of D-type cyclins. We show that cyclin D3 expression specifically regulates dark zone (DZ) GC B cell proliferation. B cell receptor (BCR) stimulation of GC B cells downregulates cyclin D3 but induces c-Myc, which subsequently requires cyclin D3 to exert GC expansion. Control of DZ proliferation requires degradation of cyclin D3, which is dependent on phosphorylation of residue Thr283 and can be bypassed by cyclin D3T283A hyperstabilization as observed in B cell lymphoma. Thereby, selected GC B cells in the light zone potentially require disengagement from BCR signaling to accumulate cyclin D3 and undergo clonal expansion in the DZ.
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Ciclina D3/metabolismo , Centro Germinal/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , División Celular , Proliferación Celular , Ciclina D2/metabolismo , Ciclina D3/genética , Ciclinas/metabolismo , Femenino , Centro Germinal/fisiología , Activación de Linfocitos , Linfoma de Células B/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Receptores de Antígenos de Linfocitos B/metabolismo , Ovinos , Transducción de SeñalRESUMEN
Mature naive B cells expressing BCRs of the IgM and IgD isotypes respond to Ag in secondary lymphoid organs. However, the vast majority of B cells do not undergo productive Ag encounter and have finite life spans dependent on survival signals propagated by the BCR and the BAFFR. In this study, we show that the E3 ubiquitin ligase Fbw7 is required for the maintenance of mature B cell populations in mice. BCR stimulation of B cells induced substantial apoptosis along with proliferative and growth defects upon the loss of Fbw7. Analysis of B cell proteomes revealed aberrant signaling patterns, including lower Bcl2 and diminished NF-κB signaling. Further, excessive accumulation of Fbw7 substrate c-Myc, increased Bim expression, and loss of PI3K signaling mediated apoptosis downstream of BCR signaling. In accordance, strong prosurvival signals delivered through ectopic expression of BCL2 in B cells could largely rescue apoptotic cells in the absence of Fbw7. Overall, this study reveals an unexpected role for Fbw7 in the survival and fitness of mature B cells.
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Linfocitos B/fisiología , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Animales , Apoptosis/inmunología , Proteína 11 Similar a Bcl2/metabolismo , Proliferación Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Recuento de Linfocitos , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Ubiquitinación/genética , Ubiquitinación/inmunologíaRESUMEN
The PI3K pathway is integral for the germinal center (GC) response. However, the contribution of protein kinase B (AKT) as a PI3K effector in GC B cells remains unknown. Here, we show that mice lacking the AKT1 and AKT2 isoforms in B cells failed to form GCs, which undermined affinity maturation and antibody production in response to immunization. Upon B-cell receptor stimulation, AKT1/2-deficient B cells showed poor survival, reduced proliferation, and impaired mitochondrial and metabolic fitness, which collectively halted GC development. By comparison, Foxo1 T24A mutant, which cannot be inactivated by AKT1/2 phosphorylation and is sequestered in the nucleus, significantly enhanced antibody class switch recombination via induction of activation-induced cytidine deaminase (AID) expression. By contrast, repression of FOXO1 activity by AKT1/2 promoted IRF4-driven plasma cell differentiation. Last, we show that T-cell help via CD40, but not enforced expression of Bcl2, rescued the defective GC response in AKT1/2-deficient animals by restoring proliferative expansion and energy production. Overall, our study provides mechanistic insights into the key role of AKT and downstream pathways on B cell fate decisions during the GC response.
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Linfocitos B/citología , Centro Germinal/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Formación de Anticuerpos , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Femenino , Proteína Forkhead Box O1/metabolismo , Centro Germinal/citología , Cambio de Clase de Inmunoglobulina , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas , Receptores de Antígenos de Linfocitos B/genética , Linfocitos T/inmunologíaRESUMEN
The FOXP1 transcription factor is expressed throughout B cell development until its extinction just prior to terminal differentiation. Foxp1 nulls die of cardiac defects at midgestation, but adult rescue via fetal liver transfer led to a strong pre-B cell block. To circumvent these limitations and to investigate FOXP1 function at later stages of B cell differentiation, we generated and analyzed floxed (F) Foxp1 alleles deleted at pro-B, transitional (T) 1, and mature B cell stages. Mb-1cre-mediated deletion of Foxp1F/F confirmed its requirement for pro-B to pre-B transition. Cd21- and Cd19cre deletion led to significant reduction of germinal center formation and a second block in differentiation at the T2/marginal zone precursor stage. T-dependent and -independent immunization of FOXP1 mutants led to reduction of Ag-specific IgM, whereas responses of class-switched Abs were unimpaired. Yet, unexpectedly, plasmablast and plasma cell numbers were significantly increased by in vitro BCR stimulation of Foxp1F/F splenic follicular B cells but rapidly lost, as they were highly prone to apoptosis. RNA sequencing, gene set enrichment analysis, and chromatin immunoprecipitation sequencing analyses revealed strong enrichment for signatures related to downregulation of immune responses, apoptosis, and germinal center biology, including direct activation of Bcl6 and downregulation of Aicda/AID, the primary effector of somatic hypermutation, and class-switch recombination. These observations support a role for FOXP1 as a direct transcriptional regulator at key steps underlying B cell development in the mouse.
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Linfocitos B/fisiología , Diferenciación Celular , Factores de Transcripción Forkhead/fisiología , Proteínas Represoras/fisiología , Animales , Factores de Transcripción Forkhead/genética , Ratones Noqueados , Proteínas Represoras/genéticaRESUMEN
Immune homeostasis in the gut associated lymphoid tissues (GALT) is critical to prevent the development of inadvertent pathologies. B cells as the producers of antibodies and cytokines plays an important role in maintaining the GALT homeostasis. However, the mechanism by which B cells specifically direct their responses towards non-self-antigens and become ignorant to self-antigens in the GALT is not known. Therefore, we developed a novel mouse model by expressing Duck Egg Lysozyme (DEL) in gut epithelial cells in presence of HEL reactive B cells. Notably, we observed a transient activation and rapid deletion of self-reactive B cells in Peyers Patches and Mesenteric lymph nodes upon self-antigen exposure. The survival of self-reactive B cells upon exposure to their self-antigen was partially rescued by blocking receptor editing but could be completely rescued by stronger survival signal like ectopic expression of BCL2. Importantly, rescuing the self-reactive B cells promoted production of auto-antibodies and gut inflammation. Mechanistically, we identify a specific activation of TGFß signaling in self-reactive B cells in the gut and a critical role of this pathway in maintaining peripheral tolerance. Collectively, our studies describe functional consequences and fate of self-reactive B cells in GALT and provide novel mechanistic insights governing self-tolerance of B cells in the gut.
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Linfocitos B/inmunología , Tracto Gastrointestinal/inmunología , Inflamación/prevención & control , Activación de Linfocitos , Animales , Autoantígenos/inmunología , Médula Ósea , Células Epiteliales/inmunología , Tracto Gastrointestinal/patología , Homeostasis , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Muramidasa/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Transcriptional regulatory mechanisms of lineage priming in embryonic development are largely uncharacterized because of the difficulty of isolating transient progenitor populations. Directed differentiation of human pluripotent stem cells (hPSCs) combined with gene editing provides a powerful system to define precise temporal gene requirements for progressive chromatin changes during cell fate transitions. Here, we map the dynamic chromatin landscape associated with sequential stages of pancreatic differentiation from hPSCs. Our analysis of chromatin accessibility dynamics led us to uncover a requirement for FOXA2, known as a pioneer factor, in human pancreas specification not previously shown from mouse knockout studies. FOXA2 knockout hPSCs formed reduced numbers of pancreatic progenitors accompanied by impaired recruitment of GATA6 to pancreatic enhancers. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming. This work highlights the power of combining hPSC differentiation, genome editing, and computational genomics for discovering transcriptional mechanisms during development.
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Factor Nuclear 3-beta del Hepatocito/fisiología , Páncreas/fisiología , Diferenciación Celular/fisiología , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Masculino , Páncreas/citología , Páncreas/metabolismo , TranscriptomaRESUMEN
MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos.
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Apoptosis , Autorrenovación de las Células , ARN Helicasas DEAD-box/metabolismo , Células Madre Embrionarias Humanas/citología , Ribonucleasa III/metabolismo , Secuencia de Bases , Línea Celular , ARN Helicasas DEAD-box/genética , Eliminación de Gen , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células Madre Embrionarias Humanas/metabolismo , Humanos , MicroARNs/genética , Ribonucleasa III/genéticaRESUMEN
B cell activation is accompanied by metabolic adaptations to meet the increased energetic demands of proliferation. The metabolic composition of the microenvironment is known to change during a germinal center response, in inflamed tissue and to vary significantly between different organs. To sustain cellular homeostasis B cells need to be able to dynamically adapt to changes in their environment. An inability to take up and process available nutrients can result in impaired B cell growth and a diminished humoral immune response. Furthermore, the metabolic microenvironment can affect B cell signaling and provide a means to avoid aberrant proliferation or modulate B cell function. Thus, a better understanding of the intricate interplay between cell signaling and metabolism could provide novel insight into how B cell function is regulated and have implications for the development of vaccines or treatment of autoimmune disorders and B cell derived malignancies.
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Linfocitos B/fisiología , Proliferación Celular , Homeostasis , Transducción de Señal , Estrés Fisiológico , AnimalesRESUMEN
B cells contribute critically to an effective immune response by producing Ag-specific Abs. During the immune response to so-called "thymus-dependent Ags," activated B cells seek T cell help and form germinal centers. In contrast, thymus-independent Ags generally do not induce germinal center formation. In the germinal center, B cells undergo somatic hypermutation, affinity-based clonal expansion, and differentiation to produce plasma cells and memory B cells. Valuable insight into these processes has been gained by using model hapten-carrier complexes or SRBCs. SRBCs induce robust germinal center formation in mice. Therefore, this Ag is commonly used to study germinal center responses. In contrast to haptenated Ags, thus far it has been difficult to measure the titer of Ag-specific Abs or the expansion of Ag-specific B cells after immunization with SRBCs. We have developed new, simple methods to access these parameters, thus providing new tools to study germinal center and Ab responses.
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Linfocitos B/fisiología , Transfusión de Eritrocitos/métodos , Eritrocitos/inmunología , Centro Germinal/inmunología , Inmunidad Humoral , Técnicas Inmunológicas/métodos , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Bovinos , Diferenciación Celular , Células Cultivadas , Femenino , Inmunización , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Hipermutación Somática de InmunoglobulinaRESUMEN
Productive humoral responses require that naive B cells and their differentiated progeny move among distinct micro-environments. In this review, we discuss how studies are beginning to address the nature of these niches as well as the interplay between cellular signaling, metabolic programming, and adaptation to the locale. Recent work adds evidence to the expectation that B cells at distinct stages of development or functional subsets are influenced by the altered profiles of nutrients and metabolic by-products that distinguish these sites. Moreover, emerging findings reveal a cross-talk among the external milieu, signal transduction pathways, and transcription factors that direct B cell fate in the periphery.
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Metabolismo Energético , Inmunidad Humoral , Inmunidad Adaptativa , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Ciclo Celular , Microambiente Celular/genética , Microambiente Celular/inmunología , Selección Clonal Mediada por Antígenos , Regulación de la Expresión Génica , Centro Germinal/inmunología , Centro Germinal/metabolismo , Homeostasis , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Inmunidad Innata , Activación de Linfocitos/genética , Activación de Linfocitos/inmunologíaRESUMEN
The germinal center (GC) reaction is an adaptive immune response to select B cells bearing high-affinity B cell receptors (BCRs) to undergo further differentiation into antibody-producing cells or memory B cells. To drive affinity maturation, (GC) B cells undergo rounds of hypermutation and rapid proliferation, which can enhance susceptibility to malignant transformation. Lymphomas frequently originate from GC B cells, but the etiology for most lymphoma subtypes is unknown. Work in the past decade has more fully documented the mutational landscape in lymphomas, but the impact of these genomic lesions is often difficult to ascertain. In addition, while mutations affecting BCR signaling are well studied, the impact of extrinsic microenvironmental factors has not been widely addressed. Murine models are useful tools to study lymphomagenesis and disease progression, as well as potential treatment in a pre-clinical setting. Herein we discuss advances in murine models of lymphoma and how they inform on key characteristics of human lymphomas.
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Centro Germinal/inmunología , Linfoma/inmunología , Neoplasias Experimentales/inmunología , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Centro Germinal/patología , Humanos , Linfoma/patología , Ratones , Neoplasias Experimentales/patología , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.
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Linfocitos B/fisiología , Centro Germinal/inmunología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Animales , Antígenos CD19/genética , Antígenos CD19/metabolismo , Apoptosis/genética , Ligando de CD40/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Glucógeno Sintasa Quinasa 3 beta/genética , Glucólisis , Interleucina-4/metabolismo , Ratones , Ratones Noqueados , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.
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Linfocitos B/fisiología , Caspasas/fisiología , Proteínas de Neoplasias/fisiología , Animales , Apoptosis , Linfocitos B/citología , Diferenciación Celular , Supervivencia Celular , Centro Germinal/fisiología , Activación de Linfocitos , Ratones , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Receptores de Antígenos de Linfocitos B/fisiología , Proteína bcl-X/análisisRESUMEN
B cell growth and proliferation is tightly regulated by signaling through the B cell receptor and by other membrane bound receptors responding to different cytokines. The PI3K signaling pathway has been shown to play a crucial role in B cell activation, differentiation and survival. Activated B cells undergo metabolic reprograming in response to changing energetic and biosynthetic demands. B cells also need to be able to coordinate metabolic activity and proliferation with nutrient availability. The PI3K signaling network has been implicated in regulating nutrient acquisition, utilization and biosynthesis, thus integrating receptor-mediated signaling with cell metabolism. In this review, we discuss the current knowledge about metabolic changes induced in activated B cells, strategies to adapt to metabolic stress and the role of PI3K signaling in these processes.
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Linfocitos B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Animales , Autofagia , Linfocitos B/citología , Diferenciación Celular , Proliferación Celular , Humanos , Redes y Vías Metabólicas , Mitocondrias/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Survivin is a member of the inhibitor of apoptosis family of proteins and a biomarker of poor prognosis in aggressive B cell non-Hodgkin's lymphoma. In addition to its role in inhibition of apoptosis, survivin also regulates mitosis. In this article, we show that deletion of survivin during early B cell development results in a complete block at the cycling pre-B stage. In the periphery, B cell homeostasis is not affected, but survivin-deficient B cells are unable to mount humoral responses. Correspondingly, we show that survivin is required for cell division in response to mitogenic stimulation. Thus, survivin is essential for proliferation of B cell progenitors and activated mature B cells, but is dispensable for B cell survival. Moreover, a small-molecule inhibitor of survivin strongly impaired the growth of representative B lymphoma lines in vitro, supporting the validity of survivin as an attractive therapeutic target for high-grade B cell non-Hodgkin's lymphoma.
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Linfocitos B/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Células Precursoras de Linfocitos B/metabolismo , Proteínas Represoras/genética , Alelos , Animales , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Apoptosis/genética , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Biomarcadores , Diferenciación Celular/genética , Supervivencia Celular/genética , Daño del ADN , Expresión Génica , Genotipo , Inmunidad Humoral/genética , Inmunidad Humoral/inmunología , Inmunofenotipificación , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/deficiencia , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/deficiencia , SurvivinRESUMEN
Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5'- and 3'-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.