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Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+ CD5+ clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Treatment options for patients range from historical chemoimmunotherapy (CIT) to small molecule inhibitors targeting pro-survival pathways in leukemic B cells, such as the Bruton's tyrosine kinase inhibitor ibrutinib (IBR). Using biobanked blood samples obtained pre-therapy and at standard response evaluation timepoints, we performed an in-depth evaluation of the blood innate and adaptive immune compartments between pentostatin-based CIT and IBR and looked for correlations with clinical sequelae. CD4+ conventional T cells and CD8+ cytotoxic T cells responded similarly to CIT and IBR, although exhaustion status differed. Both treatments dramatically increased the prevalence and functional status of monocyte, dendritic cell, and natural killer cell subsets. As expected, both regimens reduced clonal B cell levels however, we observed no substantial recovery of normal B cells. Although improvements in most immune subsets were observed with CIT and IBR at response evaluation, both patient groups remained susceptible to infections and secondary malignancies during the study.
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The Class III receptor tyrosine kinase Flt3 and its ligand, the Flt3-ligand (FL), play an integral role in regulating the proliferation, differentiation, and survival of multipotent hematopoietic and lymphoid progenitors from which B cell precursors derive in bone marrow (BM). More recently, essential roles for Flt3 signaling in the regulation of peripheral B cell development and affinity maturation have come to light. Experimental findings derived from a multitude of mouse models have reinforced the importance of molecular and cellular regulation of Flt3 and FL in lymphohematopoiesis and adaptive immunity. Here, we provide a comprehensive review of the current state of the knowledge regarding molecular and cellular regulation of Flt3/FL and the roles of Flt3 signaling in hematopoietic stem cell (HSC) activation, lymphoid development, BM B lymphopoiesis, and peripheral B cell development. Cumulatively, the literature has reinforced the importance of Flt3 signaling in B cell development and function. However, it has also identified gaps in the knowledge regarding Flt3-dependent developmental-stage specific gene regulatory circuits essential for steady-state B lymphopoiesis that will be the focus of future studies.
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Inmunidad Humoral , Linfopoyesis , Animales , Linfocitos B , Diferenciación Celular , Células Madre Hematopoyéticas , Ligandos , Linfopoyesis/fisiología , Ratones , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Stimulated ABCB7-deficient splenic B cells from CD23-cre mice also had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination.
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Linfocitos B/fisiología , Cambio de Clase de Inmunoglobulina , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Proliferación Celular , Daño del ADN , Femenino , Hierro/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Bazo/citología , Azufre/metabolismoRESUMEN
Transcription factor (TF) expression levels drive developmental programs, including cell fate and function, and their measurement by flow cytometry allows for robust downstream analysis. However, significant batch-to-batch variability between replicative experiments precludes direct comparison of absolute values across experimental conditions. Here, we present a flow cytometry protocol to measure the relative abundance of multiple TFs simultaneously in single cells, allowing for direct comparison across experimental conditions/time points. This protocol uses bone marrow cells but can be adapted for other cell types. For complete details on the use and execution of this protocol, please refer to Manso et al. (2021) and Manso et al. (2019).
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Citometría de Flujo/métodos , Factores de Transcripción , Células de la Médula Ósea/citología , Células Cultivadas , Humanos , Factores de Transcripción/análisis , Factores de Transcripción/metabolismoRESUMEN
TNFα is implicated in chronic lymphocytic leukemia (CLL) immunosuppression and disease progression. TNFα is constitutively produced by CLL B cells and is a negative regulator of bone marrow (BM) myelopoiesis. Here, we show that co-culture of CLL B cells with purified normal human hematopoietic stem and progenitor cells (HSPCs) directly altered protein levels of the myeloid and erythroid cell fate determinants PU.1 and GATA-2 at the single-cell level within transitional HSPC subsets, mimicking ex vivo expression patterns. Physical separation of CLL cells from control HSPCs or neutralizing TNFα abrogated upregulation of PU.1, yet restoration of GATA-2 required TNFα neutralization, suggesting both cell contact and soluble-factor-mediated regulation. We further show that CLL patient BM myeloid progenitors are diminished in frequency and function, an effect recapitulated by chronic exposure of control HSPCs to low-dose TNFα. These findings implicate CLL B-cell-derived TNFα in impaired BM myelopoiesis.
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An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The consequences of immune dysfunction in B-chronic lymphocytic leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The well-described immune abnormalities are not able to consistently explain these complications. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. Numbers of CD34+ BM hematopoietic progenitors responsive in standard colony-forming unit (CFU) assays, including CFU-GM/GEMM and CFU-E, were significantly reduced. Flow cytometry revealed corresponding reductions in frequencies of all hematopoietic stem and progenitor cell (HSPC) subsets assessed in CLL patient marrow. Consistent with the reduction in HSPCs, BM resident monocytes and natural killer cells were reduced, a deficiency recapitulated in blood. Finally, we report increases in protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients that provides new insight into the etiology of the complex immunodeficiency state in CLL.
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Células de la Médula Ósea/patología , Médula Ósea/patología , Leucemia Linfocítica Crónica de Células B/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Femenino , Citometría de Flujo/métodos , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function. Alterations in developmental pathways have been associated with declines in stem-cell function during ageing, but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing. Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFß, JAK/STAT and senescence signalling. Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.
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Senescencia Celular , Epistasis Genética , Crecimiento y Desarrollo/genética , Proteínas de Homeodominio/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Estrés Fisiológico/genética , Envejecimiento , Animales , Senescencia Celular/genética , Cromatina/genética , Cromatina/metabolismo , Femenino , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Masculino , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Regeneración/genéticaRESUMEN
BACKGROUND: The serine threonine kinase Pim-1 has documented roles in hematopoietic progenitor and B cell precursor proliferation and survival. Pim-1 is a molecular target of the transcription factor Hoxa9. Previous studies showed that Pim-1 deficiency phenocopied the hematopoietic progenitor defect in hoxa9-/- mice and forced expression of Pim-1 normalized the in vitro proliferation defect inherent to hoxa9-/- hematopoietic progenitors. Pim-1 is induced by cytokine signaling, including the early lymphoid/B lineage regulators Flt3 and IL-7, and expression levels were shown to influence the size of the B cell compartment in bone marrow (BM). RESULTS: In this study, we sought to determine if transgenic expression of Pim-1, driven by the immunoglobulin enhancer, Eµ, was sufficient to rescue the lymphoid/B cell precursor defect in hoxa9 or flt3-ligand (flt3l) deficient mice. Unexpectedly, expression of Eµ - Pim1 exacerbated lymphoid progenitor deficiencies in flt3l-/-, and to a lesser extent, hoxa9-/- mice. Furthermore, Eµ - Pim1 expression alone reduced early myeloid and lymphoid, but not erythroid, progenitors. In contrast, Pim-1 deficiency had no significant effect on early lymphoid/B cell development through the Pre-Pro-B cell stage, but caused a significant reduction in IgM(-) B cell precursors. Importantly, loss of Pim-1 did not phenocopy hoxa9- or flt3l-deficiency on the lymphoid/early B cell progenitor pools. CONCLUSIONS: These experimental findings demonstrate that Pim-1 overexpression has developmental-stage-specific effects on B lymphopoiesis and myelopoiesis. Importantly, these suggest that Pim-1 deficiency does not contribute significantly to the early lymphoid/B cell developmental deficiency in hoxa9-/- or flt3l-/- mice.
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Linfocitos B/fisiología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Células Precursoras de Linfocitos B/fisiología , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Hematopoyesis/genética , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-pim-1/genética , Transgenes/genética , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The immune system is designed to execute rapid, specific, and protective responses against foreign pathogens. To protect against the potentially harmful effects of autoreactive escapees that might arise during the course of the immune response, multiple tolerance checkpoints exist in both the primary and secondary lymphoid organs. Regardless, autoantibodies targeting neural antigens exist in multiple neurologic diseases. The goal of this introductory chapter is to provide a foundation of the major principles and components of the immune system as a framework to understanding autoimmunity and autoimmune neurologic disorders. A broad overview of: (1) innate mechanisms of immunity and their contribution in demyelinating diseases; (2) B and T lymphocytes as effector arms of the adaptive immune response and their contribution to the pathophysiology of neurologic diseases; and (3) emerging therapeutic modalities for treatment of autoimmune disease is provided.
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Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/patología , Sistema Inmunológico/fisiología , Animales , HumanosRESUMEN
B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3-ligand (FL-/-) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL-/- mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL-/- mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF-R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL-/- mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL-/- mice. A Bcl2 transgene did not rescue TS cells in FL-/- mice, uncoupling FL-deficiency to Bcl2-dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL-/- mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.
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Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86(-) CD150(+) CD48(-) HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150(HI) CD86(-) CD18(L)°CD41(+) and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation.
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Janus Quinasa 2/metabolismo , Transducción de Señal , Células Madre/citología , Animales , Citometría de Flujo , Ratones , Ratones TransgénicosRESUMEN
Flt3 signaling plays a crucial role in regulating the survival and differentiation of lymphoid progenitors into B cell precursors (BCPs) in bone marrow. To define further the role of Flt3 signaling in lymphoid progenitor survival, mice deficient in Flt3 ligand that also expressed a Bcl2 transgene (Eµ-bcl2tg flt3l(-/-)) were generated. Intracellular flow cytometry established transgene expression in primitive hematopoietic progenitors, including lineage-negative Sca-1(+) c-kit(+) (LSK(+)) CD27(-) cells enriched for functional hematopoietic stem cells. Compared with flt3l(-/-) mice, Eµ-bcl2tg flt3l(-/-) mice had significantly increased multipotential progenitors (MPPs), IL-7R(+) common lymphoid progenitors, and B cell precursors. To determine whether forced expression of Bcl2 was sufficient to restore lymphoid priming in the absence of Flt3 signaling Eµ-bcl2tg flt3l(-/-)rag1-gfp(+) mice were generated. Analysis of Eµ-bcl2tg flt3l(-/-)rag1-gfp(+) mice revealed that the Bcl2 transgene had no effect on lymphoid priming before CD19 expression. Thus, forced expression of a survival gene can bypass the requirement for threshold levels of Flt3 signaling requisite for lymphoid priming. Temporal Flt3 ligand (FL) replacement therapy in flt3l(-/-) mice revealed specific requirements for Flt3 signaling in the expansion and maintenance of Flt3(+hi) MPP and Flt3(+) all lymphoid progenitors, but not Flt3(+) B lymphoid progenitors (BLPs), the immediate precursors of BCPs. BCPs were restored after temporal in vivo FL treatment, albeit with delayed kinetics. Together, these results show that Flt3 regulates the proliferation, survival, and maintenance of developmental stage-specific hematopoietic progenitors that give rise to BCPs.
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Proliferación Celular , Células Madre Multipotentes/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Supervivencia Celular/genética , Ratones , Ratones Noqueados , Células Madre Multipotentes/citología , Células Precursoras de Linfocitos B/citología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transgenes , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
B-cell-biased lymphoid progenitors (BLPs) and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1(+)Siglec H(+) plasmacytoid dendritic cells (pDCs) that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1(+) pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP(low/neg) pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP) stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1(+) pDCs. Thus, removal of PDCA-1(+) pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220(+)CD19(-) fraction demonstrated that AA4.1(+)Ly6D(+)PDCA-1(-) Pre-pro B cells gave rise to CD19(+) B cells at high frequency, while PDCA-1(+) pDCs in this fraction did not. Interestingly, the presence of PDCA-1(+) pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells.
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Linfocitos B/inmunología , Células Dendríticas/inmunología , Células Precursoras de Linfocitos B/inmunología , Animales , Antígenos CD19/inmunología , Antígenos Ly/inmunología , Antígeno CD11c/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Proteínas Ligadas a GPI/inmunología , Antígenos Comunes de Leucocito/inmunología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Complemento/inmunología , Receptores de Interleucina-7/inmunologíaRESUMEN
Common lymphoid progenitors (CLPs) are thought to represent major intermediates in the transition of hematopoietic stem cells (HSCs) to B lineage lymphocytes. However, it has been obvious for some time that CLPs are heterogeneous, and there has been controversy concerning their differentiation potential. We have now resolved four Flt3(+) CLP subsets that are relatively homogenous and capable of forming B cells. Differentiation potential and gene expression patterns suggest Flt3(+) CLPs lacking both Ly6D and RAG-1 are the least differentiated. In addition to B cells, they generate natural killer (NK) and dendritic cells (DCs). At the other extreme is a subset of the recently described Flt3(+) Ly6D(+) CLPs that have a history of RAG-1 expression and are B lineage restricted. These relatively abundant and potent CLPs were depleted within 48 hours of acute in vivo estrogen elevation, suggesting they descend from hormone regulated progenitors. This contrasts with the hormone insensitivity of other CLP subsets that include NK lineage progenitors. This progenitor heterogeneity and differentiation complexity may add flexibility in response to environmental changes. Expression of RAG-1 and display of Ly6D are both milestone events, but they are neither synchronized nor dependent on each other.
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Antígenos Ly/metabolismo , Linaje de la Célula/inmunología , Proteínas de Homeodominio/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Linaje de la Célula/efectos de los fármacos , Estrógenos/farmacología , Proteínas Ligadas a GPI/metabolismo , Recuento de Linfocitos , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/inmunología , Linfopoyesis/efectos de los fármacos , Linfopoyesis/inmunología , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos B/efectos de los fármacos , Esteroides/farmacologíaRESUMEN
Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9(-/-)Flt3l(-/-) mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3(+) MPP in bone marrow than the single knockouts. Although LMPP and Flt3(+) CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l(-/-) mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l(-/-) mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150(+hi)CD34(-)CD41(+) myeloid-biased stem cells in Hoxa9(-/-)Flt3l(-/-) mice. In contrast, CD150(-) MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150(-)Flt3(+) cells from wild-type control, Hoxa9(-/-), and Flt3l(-/-) single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.
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Proteínas de Homeodominio/metabolismo , Células Progenitoras Linfoides/citología , Linfopoyesis , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Linaje de la Célula/inmunología , Células Dendríticas/inmunología , Células Progenitoras de Granulocitos y Macrófagos/citología , Proteínas de Homeodominio/genética , Células Asesinas Naturales/inmunología , Recuento de Linfocitos , Células Progenitoras Linfoides/inmunología , Linfopoyesis/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/inmunología , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Transcripción Genética , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Hoxa9 is a homeodomain transcription factor important for the generation of Flt3+hiIL-7R- lymphoid biased-multipotential progenitors, Flt3+IL-7R+ common lymphoid progenitors (CLPs), and B cell precursors (BCP) in bone marrow (BM). In addition to B-cell, Flt3+IL-7R+ CLPs possess NK and DC developmental potentials, although DCs arise from Flt3+IL-7R- myeloid progenitors as well. In this study, we investigated the requirement for Hoxa9, from Flt3+ or Flt3- progenitor subsets, in the development of NK and DC lineage cells in BM. Flt3+IL-7R+Ly6D- CLPs and their Flt3+IL-7R+Ly6D+ B lineage-restricted progeny (BLP) were significantly reduced in hoxa9-/- mice. Interestingly, the reduction in Flt3+IL-7R+ CLPs in hoxa9-/- mice had no impact on the generation of NK precursor (NKP) subsets, the differentiation of NKP into mature NK cells, or NK homeostasis. Similarly, percentages and numbers of common dendritic progenitors (CDP), as well as their plasmacytoid or conventional dendritic cell progeny in hoxa9-/- mice were comparable to wildtype. These findings reveal distinct requirements for Hoxa9 or Hoxa9/Flt3 molecular circuits in regulation of B versus NK and DC development in BM.
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Linfocitos B/citología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Dendríticas/citología , Proteínas de Homeodominio/metabolismo , Células Asesinas Naturales/citología , Células Madre Multipotentes/citología , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Homeostasis/inmunología , Células Asesinas Naturales/inmunología , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Células Madre Multipotentes/inmunología , Receptores de Interleucina-7/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
A unique subset of CD86(-) HSCs was previously discovered in mice that were old or chronically stimulated with lipopolysaccharide. Functionally defective HSCs were also present in those animals, and we now show that CD86(-) CD150(+) CD48(-) HSCs from normal adult mice are particularly poor at restoring the adaptive immune system. Levels of the marker are high on all progenitors with lymphopoietic potential, and progressive loss helps to establish relations between progenitors corresponding to myeloid and erythroid lineages. CD86 represents an important tool for subdividing HSCs in several circumstances, identifying those unlikely to generate a full spectrum of hematopoietic cells.
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Antígeno B7-2/genética , Antígeno B7-2/fisiología , Células Madre Hematopoyéticas/metabolismo , Linfopoyesis/genética , Animales , Antígeno B7-2/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Femenino , Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Linfopoyesis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/citología , Células Mieloides/metabolismo , Células Mieloides/fisiología , FenotipoRESUMEN
The generation of B-cell precursors (BCP) from lymphohematopoietic progenitors (LHP) in bone marrow is dependent on signals provided by the receptor tyrosine kinase Flt3 and its ligand, Flt3-ligand (FL). Mice deficient in FL exhibit striking reductions in LHP and BCP. Currently, the mechanism by which Flt3 regulates lymphoid lineage/B-cell development is unknown. Here, we show that haploinsufficiency of FL (FL(+/) (-) ) reduced the numbers of LHP, common lymphoid progenitors, and pro-B cells, suggesting that FL levels set a threshold for B lymphopoiesis. Limiting dilution analysis confirmed reduced BCP frequency in FL(+/) (-) mice. Real-time PCR of LHP from FL(+/) (-) animals showed increased transcripts of the B lineage inhibitor id1. However, targeted deletion of id1 did not restore the lymphoid/B lineage deficiencies in FL(-/-) mice, supporting Id1-independent mechanisms. BrdU incorporation studies established that FL is not essential for the proliferation of Flt3(+) multipotential progenitors. Analysis of FL(-/-) progenitors expressing low levels of Flt3 revealed decreased levels of the pro-survival factor Mcl1. Consequently, the Flt3(+) LHP progeny of Flt3(low) LSK(+) cells exhibited increased Annexin V staining. Together, these data suggest that Flt3 signaling initiates a cascade of events in Flt3(low) precursors that promote the survival of LHP from which BCP are derived.
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Apoptosis/inmunología , Células Progenitoras Linfoides/citología , Linfopoyesis/fisiología , Proteínas de la Membrana/metabolismo , Células Precursoras de Linfocitos B/citología , Transducción de Señal/inmunología , Animales , Apoptosis/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Recuento de Células , Proliferación Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Expresión Génica/genética , Haploinsuficiencia/inmunología , Heterocigoto , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteína 1 Inhibidora de la Diferenciación/genética , Células Progenitoras Linfoides/metabolismo , Linfopoyesis/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Células Precursoras de Linfocitos B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/genética , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Early B cell factor (EBF) is a transcription factor essential for specification and commitment to the B cell fate. In this study, we show downregulation of a developmentally regulated cluster of hoxa genes, notably hoxa9, coincides with induction of EBF at the Pro-B cell stage of B cell differentiation. Analysis of the hematopoietic progenitor compartment in Hoxa9(-/-) mice revealed significantly reduced frequencies and expression levels of Flt3, a cytokine receptor important for lymphoid priming and the generation of B cell precursors (BCPs). We show that Hoxa9 directly regulates the flt3 gene. Chromatin immunoprecipitation analysis revealed binding of Hoxa9 to the flt3 promoter in a lymphoid progenitor cell line. Knockdown of Hoxa9 significantly reduced Flt3 transcription and expression. Conversely, forced expression of Hoxa9 increased Flt3 transcription and expression in a Pro-B cell line that expressed low levels of Flt3. Hoxa9 inversely correlated with ebf1 in ex vivo-isolated bone marrow progenitors and BCPs, suggesting that EBF might function to silence a Hoxa9 transcriptional program. Restoration of EBF function in an EBF(-/-) cell line induced B lineage gene expression but did not directly suppress hoxa9 transcription, revealing alternate mechanisms of Hoxa9 regulation in BCPs. These data provide new insight into Hoxa9 function and regulation during lymphoid and B cell development. Furthermore, they suggest that failure to upregulate Flt3 provides a molecular basis for the lymphoid/early B cell deficiencies in Hoxa9(-/-) mice.