RESUMEN
Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.
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Trasplante de Médula Ósea , Factor Estimulante de Colonias de Granulocitos , Animales , Ratones , Citocinas , Interleucina-1 , Factor de Necrosis Tumoral alfa/genética , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/metabolismoRESUMEN
A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.
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Biomarcadores , Proteínas del Ojo/metabolismo , Genómica , Células Progenitoras Linfoides/metabolismo , Análisis de la Célula Individual , Animales , Células Cultivadas , Biología Computacional/métodos , Proteínas del Ojo/genética , Perfilación de la Expresión Génica , Genómica/métodos , Hematopoyesis/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteómica , Análisis de la Célula Individual/métodosRESUMEN
Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.
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Células Dendríticas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Femenino , Expresión Génica/genética , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Células Madre/metabolismoRESUMEN
Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.
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Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Animales , Antineoplásicos/farmacología , Azepinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Mutación , Nitrilos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas , Interferencia de ARN , Receptores de Citocinas/genética , Transcriptoma , Triazoles/farmacologíaRESUMEN
Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.
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Policitemia Vera , Animales , Ratones , Policitemia Vera/genética , Policitemia Vera/complicaciones , Hepcidinas/genética , Estudio de Asociación del Genoma Completo , Hierro/metabolismo , Fenotipo , HomeostasisRESUMEN
The emergence of large language models (LLMs) and assisted artificial intelligence (AI) technologies have revolutionized the way in which we interact with technology. A recent symposium at the Walter and Eliza Hall Institute explored the current practical applications of LLMs in medical research and canvassed the emerging ethical, legal and social implications for the use of AI-assisted technologies in the sciences. This paper provides an overview of the symposium's key themes and discussions delivered by diverse speakers, including early career researchers, group leaders, educators and policy-makers highlighting the opportunities and challenges that lie ahead for scientific researchers and educators as we continue to explore the potential of this cutting-edge and emerging technology.
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Inteligencia Artificial , Investigación Biomédica , TecnologíaRESUMEN
Multi-parametric flow cytometry (MFC) has a well-established role in measurable residual disease (MRD) monitoring in patients with B-lymphoblastic leukemia (B-ALL). However, the optimal time-point (TP) for early MRD testing and associated prognostic impact remain undefined in adult B-ALL patients receiving Hyper-CVAD induction chemotherapy. To evaluate the utility of MRD analysis after one cycle (TP1) in comparison to MRD analysis after two cycles (TP2) of induction treatment with Hyper-CVAD chemotherapy, we studied 49 adult B-ALL patients over a 10-year period (2010-2020) who had available bone marrow samples for morphological and MFC MRD assessments at the two separate TPs. Median times to TP1 and TP2 relative to start of treatment were 21 and 45 days, respectively. When censored at transplant, achievement of MRD negativity at TP1 was not associated with a statistically significant improvement in either event-free survival (EFS) (p = .426) or overall survival (OS) (p = .335) when compared to patients with MRD positivity. In contrast, achieving MRD negativity at TP2 was associated with a statistically significant improvement in both EFS (p = ·005) and OS (p = .047) over patients who remained MRD positive. Multivariate analysis demonstrated that KMT2A-rearrangement and MRD positivity at TP2 were the only significant predictors of outcome, correlating with worse EFS and OS. Therefore, in the absence of residual morphologic disease, MRD analysis after one cycle of Hyper-CVAD induction chemotherapy did not provide additional benefit with regard to risk stratification or correlation with survival outcomes when compared to MRD testing after two cycles of Hyper-CVAD in adult B-ALL patients.
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Quimioterapia de Inducción , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Adulto , Citometría de Flujo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Pronóstico , Neoplasia Residual/diagnósticoRESUMEN
Deregulated overexpression of MYC is implicated in the development and malignant progression of most (â¼70%) human tumors. MYC drives cell growth and proliferation, but also, at high levels, promotes apoptosis. Here, we report that the proliferative capacity of MYC-driven normal and neoplastic B lymphoid cells depends on MNT, a MYC-related transcriptional repressor. Our genetic data establish that MNT synergizes with MYC by suppressing MYC-driven apoptosis, and that it does so primarily by reducing the level of pro-apoptotic BIM. In Eµ-Myc mice, which model the MYC/IGH chromosome translocation in Burkitt's lymphoma, homozygous Mnt deletion greatly reduced lymphoma incidence by enhancing apoptosis and markedly decreasing premalignant B lymphoid cell populations. Strikingly, by inducing Mnt deletion within transplanted fully malignant Eµ-Myc lymphoma cells, we significantly extended transplant recipient survival. The dependency of lymphomas on MNT for survival suggests that drugs inhibiting MNT could significantly boost therapy of MYC-driven tumors by enhancing intrinsic MYC-driven apoptosis.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Transformación Celular Neoplásica/genética , Linfoma/genética , Linfoma/mortalidad , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Antineoplásicos/uso terapéutico , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Linfoma/tratamiento farmacológico , Linfoma/patología , Linfoma de Células B/genética , Ratones , Ratones Transgénicos , Proteínas Represoras/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus-vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4-heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically. This hypothesis is testable in cell culture and animal models, and potentially in vivo, and if proven correct has significant implications for vaccine development and our understanding of the links between the coagulation and immune systems.
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COVID-19 , Trombocitopenia , Trombosis , Vacunas , Adenoviridae , Animales , Humanos , SARS-CoV-2 , Distribución Tisular , VacunaciónRESUMEN
During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.
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Bases de Datos Genéticas , Expresión Génica/genética , Hematopoyesis/genética , Transcriptoma/genética , Animales , Biología Computacional , Células Madre Hematopoyéticas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/tendencias , Humanos , Ratones , RNA-Seq , Programas InformáticosRESUMEN
Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(1716)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(1716)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(1716)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL.
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Cromosomas Humanos Par 21/genética , Síndrome de Down/genética , Proteínas Oncogénicas/genética , Células Madre/citología , Transactivadores/genética , Factores de Transcripción/genética , Trisomía , ADP-Ribosil Ciclasa 1/metabolismo , Alelos , Animales , Antígenos CD34/metabolismo , Linaje de la Célula , Proliferación Celular , Modelos Animales de Enfermedad , Células Eritroides/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Hematopoyesis/genética , Sistema Hematopoyético/citología , Sistema Hematopoyético/metabolismo , Humanos , Megacariocitos/metabolismo , Ratones , Ratones Noqueados , Análisis por Micromatrices , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Análisis de Secuencia de ARN , Células Madre/metabolismo , Regulador Transcripcional ERG , TranscriptomaRESUMEN
Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.
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Plaquetas/metabolismo , Megacariocitos/metabolismo , Células Mieloides/citología , Receptores de Trombopoyetina/metabolismo , Trombopoyesis , Animales , Antígenos CD34/metabolismo , Plaquetas/citología , Compartimento Celular , Proliferación Celular , Células Clonales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Marcación de Gen , Sitios Genéticos/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Integrasas/metabolismo , Megacariocitos/citología , Ratones , Modelos Biológicos , Células Mieloides/metabolismo , Trombocitosis , Trombopoyetina/genética , Trombopoyetina/metabolismo , Transcripción GenéticaAsunto(s)
Osteosclerosis , Mielofibrosis Primaria , Humanos , Recuento de Leucocitos , Macrófagos , Receptores de CalcitriolRESUMEN
A collection of tributes and remembrances from esteemed colleagues, mentees, and friends on the life and work of "the father of hematopoietic cytokines".
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Hematopoyesis , Células Madre Hematopoyéticas , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Retratos como AsuntoRESUMEN
The IFN regulatory factor family member 8 (IRF8) regulates differentiation of lymphoid and myeloid lineage cells by promoting or suppressing lineage-specific genes. How IRF8 promotes hematopoietic progenitors to commit to one lineage while preventing the development of alternative lineages is not known. In this study, we report an IRF8-EGFP fusion protein reporter mouse that revealed previously unrecognized patterns of IRF8 expression. Differentiation of hematopoietic stem cells into oligopotent progenitors is associated with progressive increases in IRF8-EGFP expression. However, significant induction of IRF8-EGFP is found in granulocyte-myeloid progenitors and the common lymphoid progenitors but not the megakaryocytic-erythroid progenitors. Surprisingly, IRF8-EGFP identifies three subsets of the seemingly homogeneous granulocyte-myeloid progenitors with an intermediate level of expression of EGFP defining bipotent progenitors that differentiation into either EGFP(hi) monocytic progenitors or EGFP(lo) granulocytic progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B population with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell committed (EGFP(int)) from those that are noncommitted. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors.
Asunto(s)
Factores Reguladores del Interferón/genética , Linfopoyesis/inmunología , Mielopoyesis/inmunología , Animales , Linfocitos B/citología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Genes Reporteros , Genotipo , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granulocitos/citología , Proteínas Fluorescentes Verdes/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Factores Reguladores del Interferón/biosíntesis , Interleucina-3/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/citología , Proteínas Recombinantes de Fusión/genética , Linfocitos T/citologíaRESUMEN
Previous studies have shown that mouse bone marrow cells can produce mast cells when stimulated in vitro by stem cell factor (SCF) and interleukin-3 (IL-3). Experiments to define the marrow cells able to generate mast cells showed that the most active subpopulations were the Kit(+) Sca1(-) progenitor cell fraction and the more ancestral Kit(+) Sca1(+) blast colony-forming cell fraction. In clonal cultures, up to 64% of blast colony-forming cells were able to generate mast cells when stimulated by SCF and IL-3, and, of these, the most active were those in the CD34(-) Flt3R(-) long-term repopulating cell fraction. Basophils, identified by the monoclonal antibody mMCP-8 to mouse mast cell serine protease-8, were also produced by 50% of blast colony-forming cells with a strong concordance in the production of both cell types by individual blast colony-forming cells. Enriched populations of marrow-derived basophils were shown to generate variable numbers of mast cells after a further incubation with SCF and IL-3. The data extend the repertoire of lineage-committed cells able to be produced by multipotential hematopoietic blast colony-forming cells and show that basophils and mast cells can have common ancestral cells and that basophils can probably generate mast cells at least under defined in vitro conditions.