RESUMO
The challenges in recapitulating in vivo human T cell development in laboratory models have posed a barrier to understanding human thymopoiesis. Here, we used single-cell RNA sequencing (sRNA-seq) to interrogate the rare CD34+ progenitor and the more differentiated CD34- fractions in the human postnatal thymus. CD34+ thymic progenitors were comprised of a spectrum of specification and commitment states characterized by multilineage priming followed by gradual T cell commitment. The earliest progenitors in the differentiation trajectory were CD7- and expressed a stem-cell-like transcriptional profile, but had also initiated T cell priming. Clustering analysis identified a CD34+ subpopulation primed for the plasmacytoid dendritic lineage, suggesting an intrathymic dendritic specification pathway. CD2 expression defined T cell commitment stages where loss of B cell potential preceded that of myeloid potential. These datasets delineate gene expression profiles spanning key differentiation events in human thymopoiesis and provide a resource for the further study of human T cell development.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Linfopoese/genética , Linfócitos T/metabolismo , Timócitos/metabolismo , Animais , Biomarcadores , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunofenotipagem , Camundongos , Análise de Célula Única , Linfócitos T/citologia , Timócitos/citologia , TranscriptomaRESUMO
Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas Tirosina Quinases , Camundongos , Humanos , Animais , Proteínas Tirosina Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco Neoplásicas/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Resistencia a Medicamentos AntineoplásicosRESUMO
BACKGROUND & AIMS: Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Expression of serine-threonine kinase receptor-associated protein (STRAP) is elevated in CRCs and is associated with poor outcomes. We investigated the role of STRAP in Apc mutation-induced intestinal tumor initiation and progression. METHODS: We generated Strap intestinal epithelial knockout mice (StrapΔIEC) by crossing mice containing floxed alleles of Strap (Strapfl/fl) with Villin-Cre mice. Then we generated ApcMin/+;Strapfl/fl;Vill-Cre (ApcMin/+;StrapΔIEC) mice for RNA-sequencing analyses to determine the mechanism of function of STRAP. We used human colon cancer cell lines (DLD1, SW480, and HT29) and human and mouse colon tumor-derived organoids for STRAP knockdown and knockout and overexpression experiments. RESULTS: Strap deficiency extended the average survival of ApcMin/+ mice by 80 days and decreased the formation of intestinal adenomas. Expression profiling revealed that the intestinal stem cell signature, the Wnt/ß-catenin signaling, and the MEK/ERK pathway are down-regulated in Strap-deficient adenomas and intestinal organoids. Correlation studies suggest that these STRAP-associated oncogenic signatures are conserved across murine and human colon cancer. STRAP associates with MEK1/2, promotes binding between MEK1/2 and ERK1/2, and subsequently induces the phosphorylation of ERK1/2. STRAP activated Wnt/ß-catenin signaling through MEK/ERK-induced phosphorylation of LRP6. STRAP was identified as a target of mutated Apc and Wnt/ß-catenin signaling as chromatin immunoprecipitation and luciferase assays revealed putative binding sites of the ß-catenin/TCF4 complex on the Strap promoter. CONCLUSIONS: STRAP is a target of, and is required in, Apc mutation/deletion-induced intestinal tumorigenesis through a novel feed-forward STRAP/MEK-ERK/Wnt-ß-catenin/STRAP regulatory axis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Genes APC , Mutação , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/genética , Células Tumorais Cultivadas , Via de Sinalização WntRESUMO
Several lines of research suggest that Bcl-xL-mediated anti-apoptotic effects may contribute to the pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock-in JAK2V617F mouse model and confirmed that Bcl-xL was overexpressed in erythroid progenitors. The myeloproliferative neoplasm (MPN)-induced phenotype in the peripheral blood by conditional knock-in of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status. Mx1-Cre Jak2V617W/VF /Bcl2l1f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro-apoptotic stimuli in terminally differentiated erythroid progenitors. The pan-BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo assay using Vav-Cre Jak2V617F bone marrow cells. Both ruxolitinib and obatoclax significantly reduced spleen weights in a murine Jak2V617F MPN model but did not show additive effect. The tumor burden reduction was observed with either ruxolitinib or obatoclax in terminal differentiation stage neoplastic cells but not in myeloid-erythroid precursors. Therefore, disrupting the BCL2 balance is not sufficient to treat MPN at the stem cell level, but it is certainly an additional option for controlling the critical myeloid expansion of the disease.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Transtornos Mieloproliferativos/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células Precursoras Eritroides/patologia , Humanos , Indóis/uso terapêutico , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Nitrilas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.
Assuntos
Autorrenovação Celular/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Lisina Acetiltransferase 5/genética , Transativadores/genética , Animais , Biomarcadores , Ciclo Celular , Diferenciação Celular/genética , Dano ao DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histonas/metabolismo , Lisina Acetiltransferase 5/metabolismo , Camundongos , Transporte Proteico , Transativadores/metabolismoRESUMO
To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1's role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.
Assuntos
Células-Tronco Adultas/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Células-Tronco Adultas/patologia , Animais , Proliferação de Células , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, â¼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Células Mieloides/citologia , Regiões Promotoras Genéticas , Transativadores/metabolismo , Ativação Transcricional , Sequência de Bases , Linhagem Celular , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Humanos , Células Mieloides/metabolismo , Ligação ProteicaRESUMO
Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients.
Assuntos
Linfócitos B/imunologia , Reparo do DNA por Junção de Extremidades/genética , DNA Ligases/genética , Eczema/genética , Transtornos do Crescimento/genética , Deficiência Intelectual/genética , Linfopoese/genética , Microcefalia/genética , Imunodeficiência Combinada Severa/genética , Animais , Apoptose/genética , Apoptose/imunologia , Sequência de Bases , Proliferação de Células , Células Cultivadas , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , Modelos Animais de Doenças , Fácies , Técnicas de Introdução de Genes , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteína Supressora de Tumor p53/genéticaRESUMO
Runx transcription factors contribute to hematopoiesis and are frequently implicated in hematologic malignancies. All three Runx isoforms are expressed at the earliest stages of hematopoiesis; however, their function in hematopoietic stem cells (HSCs) is not fully elucidated. Here, we show that Runx factors are essential in HSCs by driving the expression of the hematopoietic transcription factor PU.1. Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the -14kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene. Similarly, in a mouse model of AML/ETO9a leukemia, disrupting the Runx binding sites resulted in decreased PU.1 levels. Leukemia onset was delayed, and limiting dilution transplantation experiments demonstrated functional loss of leukemia-initiating cells. This is surprising, because low PU.1 levels have been considered a hallmark of AML/ETO leukemia, as indicated in mouse models and as shown here in samples from leukemic patients. Our data demonstrate that Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells.
Assuntos
Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Animais , Pareamento de Bases/genética , Sítios de Ligação , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos C57BL , Mutação/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Dendritic cells (DCs) are master regulators of the immune system, but molecular regulation of early DC differentiation has been poorly understood. Here, we report that the transcription factor C/EBPα coordinates the development of progenitor cells required for production of multiple categories of DCs. C/EBPα was needed for differentiation from stem/progenitor cells to common DC progenitors (CDPs), but not for transition of CDP to mature DCs. C/EBPα deletion in mature DCs did not affect their numbers or function, suggesting that this transcription factor is not needed for maintenance of DCs in lymphoid tissues. ChIP-seq and microarrays were used to identify candidate genes regulated by C/EBPα and required for DC formation. Genes previously shown to be critical for DC formation were bound by C/EBPα, and their expression was decreased in the earliest hematopoietic compartments in the absence of C/EBPα. These data indicate that C/EBPα is important for the earliest stages of steady-state DC differentiation.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Diferenciação Celular/genética , Células Dendríticas/fisiologia , Células-Tronco/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Análise por Conglomerados , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco/metabolismoRESUMO
Lineage-restricted cells can be reprogrammed to a pluripotent state known as induced pluripotent stem (iPS) cells through overexpression of 4 transcription factors. iPS cells are similar to human embryonic stem (hES) cells and have the same ability to generate all the cells of the human body, including blood cells. However, this process is extremely inefficient and to date has been unsuccessful at differentiating iPS into hematopoietic stem cells (HSCs). We hypothesized that iPS cells, injected into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ immunocompromised (NSG) mice could give rise to hematopoietic stem/progenitor cells (HSPCs) during teratoma formation. Here, we report a novel in vivo system in which human iPS cells differentiate within teratomas to derive functional myeloid and lymphoid cells. Similarly, HSPCs can be isolated from teratoma parenchyma and reconstitute a human immune system when transplanted into immunodeficient mice. Our data provide evidence that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications.
Assuntos
Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Teratoma/patologia , Animais , Linfócitos B/citologia , Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Queratinócitos/fisiologia , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células Mieloides/citologia , Transplante de Neoplasias , Células Estromais/citologia , Células Estromais/fisiologia , Células Estromais/transplante , Linfócitos T/citologia , Teratoma/genética , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Antígeno B7-2/genética , Antígeno B7-2/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Linfopoese/genética , Animais , Antígeno B7-2/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Linfopoese/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/metabolismo , Células Mieloides/fisiologia , FenótipoRESUMO
Technical advances have made it possible to separate hematopoietic tissues such as the bone marrow into ever smaller populations, complicating our understanding of immune system replenishment. Patterns of surface marker expression and transcription profiles as well as results obtained with reporter mice suggest that lymphopoietic cells are not closely synchronized, and there is considerable cell to cell variation. Loss of differentiation options is gradual, and ultimate fate can be established at different stages of lineage progression. For example, individual hematopoietic stem cells can be biased such that some are very poor sources of lymphocytes as contrasted to ones with balanced outputs. Still other hematopoietic stem cells are effective at generating B and T cells but are defective with respect to expansion and difficult to distinguish from early lymphoid progenitors. That diversity carries forward to later events, and similar appearing cells in the immune system can arise from alternate differentiation pathways. In fact, new categories of lymphoid progenitors are still being discovered. Heterogeneity provides adaptability as hematopoiesis can be dramatically altered during infections, influencing numbers and types of cells that are produced.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Linfopoese/fisiologia , Células-Tronco , Animais , Camundongos , Células-Tronco/fisiologiaRESUMO
Background: Understanding how cells and tissues respond to stress factors and perturbations during disease processes is crucial for developing effective prevention, diagnosis, and treatment strategies. Single-cell RNA sequencing (scRNA-seq) enables high-resolution identification of cells and exploration of cell heterogeneity, shedding light on cell differentiation/maturation and functional differences. Recent advancements in multimodal sequencing technologies have focused on improving access to cell-specific subgroups for functional genomics analysis. To facilitate the functional annotation of cell groups and characterization of molecular mechanisms underlying cell trajectories, we introduce the Pathways, Annotated Gene Lists, and Gene Signatures Electronic Repository for Single-Cell Functional Genomics Analysis (PAGER-scFGA). Results: We have developed PAGER-scFGA, which integrates cell functional annotations and gene-set enrichment analysis into popular single-cell analysis pipelines such as Scanpy. Using differentially expressed genes (DEGs) from pairwise cell clusters, PAGER-scFGA infers cell functions through the enrichment of potential cell-marker genesets. Moreover, PAGER-scFGA provides pathways, annotated gene lists, and gene signatures (PAGs) enriched in specific cell subsets with tissue compositions and continuous transitions along cell trajectories. Additionally, PAGER-scFGA enables the construction of a gene subcellular map based on DEGs and allows examination of the gene functional compartments (GFCs) underlying cell maturation/differentiation. In a real-world case study of mouse natural killer (mNK) cells, PAGER-scFGA revealed two major stages of natural killer (NK) cells and three trajectories from the precursor stage to NK T-like mature stage within blood, spleen, and bone marrow tissues. As the trajectories progress to later stages, the DEGs exhibit greater divergence and variability. However, the DEGs in different trajectories still interact within a network during NK cell maturation. Notably, PAGER-scFGA unveiled cell cytotoxicity, exocytosis, and the response to interleukin (IL) signaling pathways and associated network models during the progression from precursor NK cells to mature NK cells. Conclusion: PAGER-scFGA enables in-depth exploration of functional insights and presents a comprehensive knowledge map of gene networks and GFCs, which can be utilized for future studies and hypothesis generation. It is expected to become an indispensable tool for inferring cell functions and detecting molecular mechanisms within cell trajectories in single-cell studies. The web app (accessible at https://au-singlecell.streamlit.app/) is publicly available.
RESUMO
Here, we examine how prenatal inflammation shapes tissue function and immunity in the lung by reprogramming tissue-resident immune cells from early development. Maternal, but not fetal, type I interferon-mediated inflammation provokes expansion and hyperactivation of group 2 innate lymphoid cells (ILC2s) seeding the developing lung. Hyperactivated ILC2s produce increased IL-5 and IL-13 and are associated with acute Th2 bias, decreased Tregs, and persistent lung eosinophilia into adulthood. ILC2 hyperactivation is recapitulated by adoptive transfer of fetal liver precursors following prenatal inflammation, indicative of developmental programming at the fetal progenitor level. Reprogrammed ILC2 hyperactivation and subsequent lung immune remodeling, including persistent eosinophilia, is concomitant with worsened histopathology and increased airway dysfunction equivalent to papain exposure, indicating increased asthma susceptibility in offspring. Our data elucidate a mechanism by which early-life inflammation results in increased asthma susceptibility in the presence of hyperactivated ILC2s that drive persistent changes to lung immunity during perinatal development.
Assuntos
Imunidade Inata , Inflamação , Pulmão , Linfócitos , Animais , Pulmão/imunologia , Pulmão/patologia , Feminino , Linfócitos/imunologia , Inflamação/patologia , Inflamação/imunologia , Gravidez , Camundongos , Asma/imunologia , Asma/patologia , Camundongos Endogâmicos C57BL , Células Th2/imunologiaRESUMO
During chronic infections and tumor progression, CD8 T cells gradually lose their effector functions and become exhausted. These exhausted CD8 T cells are heterogeneous and comprised of different subsets, including self-renewing progenitors that give rise to Ly108 - CX3CR1 + effector-like cells. Generation of these effector-like cells is essential for the control of chronic infections and tumors, albeit limited. However, the precise cues and mechanisms directing the formation and maintenance of exhausted effector-like are incompletely understood. Using genetic mouse models challenged with LCMV Clone 13 or syngeneic tumors, we show that the expression of a transcriptional repressor, growth factor independent 1 (Gfi1) is dynamically regulated in exhausted CD8 T cells, which in turn regulates the formation of exhausted effector-like cells. Gfi1 deletion in T cells dysregulates the chromatin accessibility and transcriptomic programs associated with the differentiation of LCMV Clone 13-specific CD8 T cell exhaustion, preventing the formation of effector-like and terminally exhausted cells while maintaining progenitors and a newly identified Ly108 + CX3CR1 + state. These Ly108 + CX3CR1 + cells have a distinct chromatin profile and may represent an alternative target for therapeutic interventions to combat chronic infections and cancer. In sum, we show that Gfi1 is a critical regulator of the formation of exhausted effector-like cells.
RESUMO
Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss of function of SON. While patients with ZTTK syndrome live with numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifted cell fate more toward the myeloid lineage but compromised lymphoid lineage development by reducing genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency caused inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.
Assuntos
Hematopoese , Doenças Raras , Animais , Humanos , Camundongos , Perfilação da Expressão Gênica , Hematopoese/genética , MutaçãoRESUMO
Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs. SIGNIFICANCE: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Camundongos , Células Endoteliais , Microambiente Tumoral/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Comunicação Celular , Receptores ErbB/genéticaRESUMO
Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation, and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors. However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed. Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example, minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential, would be consistent with age- or TLR-related injury. In contrast, HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity.