RESUMEN
We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.
Asunto(s)
Perfilación de la Expresión Génica , Análisis de Expresión Génica de una Sola Célula , Análisis de Secuencia de ARN , RNA-Seq , Bacterias/genética , Análisis de la Célula IndividualRESUMEN
Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.
Asunto(s)
Neoplasias Encefálicas , Melanoma , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/secundario , Linfocitos T CD8-positivos/patología , Ecosistema , Humanos , RNA-SeqRESUMEN
Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy.
Asunto(s)
Censos , Neoplasias/genética , Neoplasias/inmunología , Transcriptoma/genética , Microambiente Tumoral/inmunología , Biomarcadores de Tumor , Análisis por Conglomerados , Estudios de Cohortes , Biología Computacional/métodos , Citometría de Flujo/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/clasificación , Neoplasias/patología , RNA-Seq/métodos , San Francisco , UniversidadesRESUMEN
The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of â¼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.
Asunto(s)
Visión Ocular , Corteza Visual/citología , Corteza Visual/embriología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ácido Glutámico/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , RNA-Seq , Transcriptoma/genética , Visión Binocular/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. We interrogated the composition of BrM niches, molecularly defined the blood-tumor interface, and revealed stromal immunosuppressive states enriched with infiltrated T cells and macrophages. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our resource provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits.
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Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Adulto , Anciano , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/inmunología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Variación Genética , Humanos , Evasión Inmune , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Células Mieloides/patología , Análisis de Componente Principal , RNA-Seq , Análisis de la Célula Individual , Linfocitos T/inmunologíaRESUMEN
Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.
Asunto(s)
Quimerismo , Embrión de Mamíferos/citología , Células Madre Pluripotentes/citología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Humanos , Macaca fascicularis , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , RNA-Seq , Análisis de la Célula Individual , TranscriptomaRESUMEN
Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.
Asunto(s)
Área Hipotalámica Lateral/metabolismo , Hibridación Fluorescente in Situ , Animales , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Área Hipotalámica Lateral/citología , Imagenología Tridimensional , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuropéptidos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN/metabolismo , RNA-Seq , Análisis de la Célula Individual , Transcripción GenéticaRESUMEN
We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses distinct codes of â¼23 neuropeptide genes and â¼36 neuropeptide receptors, delineating a complex and expansive "wireless" signaling network. To demonstrate the utility of this comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression and (2) reveal adhesion proteins with potential roles in process placement and synaptic specificity. Our expression data are available at https://cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity, and function throughout the C. elegans nervous system.
Asunto(s)
Caenorhabditis elegans/metabolismo , Sistema Nervioso/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colorantes Fluorescentes/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Larva/metabolismo , Neuronas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Motivos de Nucleótidos/genética , RNA-Seq , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of â¼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Recurrencia Local de Neoplasia/patología , Análisis de la Célula Individual , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Células Mieloides/metabolismo , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/inmunología , Fenotipo , RNA-Seq , Microambiente TumoralRESUMEN
While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.
Asunto(s)
Autoinmunidad , Intestinos/inmunología , Células Madre/metabolismo , Células Th17/inmunología , Animales , Movimiento Celular , Células Clonales , Encefalomielitis Autoinmune Experimental/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Homeostasis , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Ratones Endogámicos C57BL , Especificidad de Órganos , ARN/metabolismo , RNA-Seq , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR6/metabolismo , Receptores de Interleucina/metabolismo , Reproducibilidad de los Resultados , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Análisis de la Célula Individual , Bazo/metabolismoRESUMEN
CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.
Asunto(s)
Adenocarcinoma del Pulmón/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Neoplasias Pulmonares/inmunología , Células Madre/inmunología , Secuencia de Aminoácidos , Animales , Antígeno CTLA-4/metabolismo , Epítopos , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/patología , Ratones , Péptidos/química , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , RNA-Seq , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CCR6/metabolismo , Análisis de la Célula Individual , VacunaciónRESUMEN
Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.
Asunto(s)
Pólipos del Colon/patología , Neoplasias Colorrectales/patología , Microambiente Tumoral , Inmunidad Adaptativa , Adenoma/genética , Adenoma/patología , Adulto , Anciano , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Muerte Celular , Diferenciación Celular , Pólipos del Colon/genética , Pólipos del Colon/inmunología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Heterogeneidad Genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mutación/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , RNA-Seq , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Microambiente Tumoral/inmunologíaRESUMEN
To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
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Carcinoma de Células Escamosas/metabolismo , Genómica/métodos , Neoplasias Cutáneas/metabolismo , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , RNA-Seq , Análisis de la Célula Individual , Piel/metabolismo , Neoplasias Cutáneas/patología , Transcriptoma , Trasplante HeterólogoRESUMEN
The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.
Asunto(s)
Células Endoteliales/metabolismo , Análisis de la Célula Individual , Transcriptoma , Animales , Encéfalo/citología , Sistema Cardiovascular/citología , Células Endoteliales/clasificación , Células Endoteliales/citología , Tracto Gastrointestinal/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/citología , Especificidad de Órganos , RNA-Seq , Testículo/citologíaRESUMEN
We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.
Asunto(s)
COVID-19 , Genómica , RNA-Seq , SARS-CoV-2 , Análisis de la Célula Individual , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/sangre , COVID-19/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/genética , División Celular , Senescencia Celular/genética , Secuenciación de Inmunoprecipitación de Cromatina , Cromosomas/genética , Estudios de Cohortes , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Biología Computacional , Metilación de ADN/genética , Epigenómica , Células HCT116 , Humanos , Hibridación Fluorescente in Situ , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , RNA-Seq , Análisis Espacial , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Human cerebral cortex size and complexity has increased greatly during evolution. While increased progenitor diversity and enhanced proliferative potential play important roles in human neurogenesis and gray matter expansion, the mechanisms of human oligodendrogenesis and white matter expansion remain largely unknown. Here, we identify EGFR-expressing "Pre-OPCs" that originate from outer radial glial cells (oRGs) and undergo mitotic somal translocation (MST) during division. oRG-derived Pre-OPCs provide an additional source of human cortical oligodendrocyte precursor cells (OPCs) and define a lineage trajectory. We further show that human OPCs undergo consecutive symmetric divisions to exponentially increase the progenitor pool size. Additionally, we find that the OPC-enriched gene, PCDH15, mediates daughter cell repulsion and facilitates proliferation. These findings indicate properties of OPC derivation, proliferation, and dispersion important for human white matter expansion and myelination.
Asunto(s)
Cadherinas/metabolismo , Corteza Cerebral/citología , Células Ependimogliales/metabolismo , Neurogénesis/genética , Células Precursoras de Oligodendrocitos/metabolismo , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Proliferación Celular/genética , Células Cultivadas , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Células Ependimogliales/citología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Células Precursoras de Oligodendrocitos/citología , ARN Interferente Pequeño , RNA-Seq , Análisis de la Célula Individual , Sustancia Blanca/citología , Sustancia Blanca/embriología , Sustancia Blanca/metabolismoRESUMEN
Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.
Asunto(s)
Linfocitos T CD8-positivos/citología , Antígeno CTLA-4/inmunología , Colitis/metabolismo , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Inmunoterapia/efectos adversos , Células Mieloides/metabolismo , Receptores de Quimiocina/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Antígeno CTLA-4/metabolismo , Quimiocinas/metabolismo , Colitis/tratamiento farmacológico , Colitis/genética , Colitis/inmunología , Citocinas/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/metabolismo , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Familia de Multigenes , Células Mieloides/citología , RNA-Seq , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Receptores CXCR6/genética , Receptores CXCR6/metabolismo , Receptores de Quimiocina/genética , Análisis de la Célula Individual , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.
Asunto(s)
Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Glucosa-6-Fosfato Isomerasa/metabolismo , Glucólisis/genética , Fosforilación Oxidativa , Vía de Pentosa Fosfato/fisiología , Células Th17/metabolismo , Animales , Hipoxia de la Célula/genética , Hipoxia de la Célula/inmunología , Quimera/genética , Cromatografía de Gases , Cromatografía Liquida , Infecciones por Clostridium/inmunología , Citocinas/deficiencia , Citocinas/genética , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Glucólisis/inmunología , Homeostasis/genética , Homeostasis/inmunología , Inflamación/genética , Inflamación/inmunología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiología , Vía de Pentosa Fosfato/genética , Vía de Pentosa Fosfato/inmunología , RNA-Seq , Análisis de la Célula Individual , Células Th17/inmunología , Células Th17/patologíaRESUMEN
Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.