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1.
Nat Cell Biol ; 23(11): 1129-1135, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34750578

RESUMO

Massive single-cell profiling efforts have accelerated our discovery of the cellular composition of the human body while at the same time raising the need to formalize this new knowledge. Here, we discuss current efforts to harmonize and integrate different sources of annotations of cell types and states into a reference cell ontology. We illustrate with examples how a unified ontology can consolidate and advance our understanding of cell types across scientific communities and biological domains.

2.
Hepatol Commun ; 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34792289

RESUMO

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single-cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation-related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at a single-cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte, and mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

3.
Nat Methods ; 2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34824477

RESUMO

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.

4.
Gastroenterology ; 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34780721

RESUMO

BACKGROUND AND AIMS: Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis. METHODS: To build a taxonomy model we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by: (1) overlapping syndromic features; (2) response to hematopoietic stem cell transplantation; (3) bulk RNA-seq of 32 tissues; (4) single-cell RNA-seq of >50 cell subsets from the intestine of healthy individuals and IBD patients (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4). RESULTS: Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8+ T cells, neutrophils, epithelial subsets and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mTOR activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD. CONCLUSION: Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.

5.
bioRxiv ; 2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34845454

RESUMO

Genome-wide association studies (GWAS) provide a powerful means to identify loci and genes contributing to disease, but in many cases the related cell types/states through which genes confer disease risk remain unknown. Deciphering such relationships is important for identifying pathogenic processes and developing therapeutics. Here, we introduce sc-linker, a framework for integrating single-cell RNA-seq (scRNA-seq), epigenomic maps and GWAS summary statistics to infer the underlying cell types and processes by which genetic variants influence disease. We analyzed 1.6 million scRNA-seq profiles from 209 individuals spanning 11 tissue types and 6 disease conditions, and constructed gene programs capturing cell types, disease progression, and cellular processes both within and across cell types. We evaluated these gene programs for disease enrichment by transforming them to SNP annotations with tissue-specific epigenomic maps and computing enrichment scores across 60 diseases and complex traits (average N= 297K). Cell type, disease progression, and cellular process programs captured distinct heritability signals even within the same cell type, as we show in multiple complex diseases that affect the brain (Alzheimer’s disease, multiple sclerosis), colon (ulcerative colitis) and lung (asthma, idiopathic pulmonary fibrosis, severe COVID-19). The inferred disease enrichments recapitulated known biology and highlighted novel cell-disease relationships, including GABAergic neurons in major depressive disorder (MDD), a disease progression M cell program in ulcerative colitis, and a disease-specific complement cascade process in multiple sclerosis. In autoimmune disease, both healthy and disease progression immune cell type programs were associated, whereas for epithelial cells, disease progression programs were most prominent, perhaps suggesting a role in disease progression over initiation. Our framework provides a powerful approach for identifying the cell types and cellular processes by which genetic variants influence disease.

6.
Nat Biotechnol ; 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663921

RESUMO

Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets.

7.
Nat Methods ; 18(11): 1352-1362, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34711971

RESUMO

Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.

8.
Nat Biotechnol ; 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34675424

RESUMO

Multimodal measurements of single-cell profiles are proving increasingly useful for characterizing cell states and regulatory mechanisms. In the present study, we developed PHAGE-ATAC (Assay for Transposase-Accessible Chromatin), a massively parallel droplet-based method that uses phage displaying, engineered, camelid single-domain antibodies ('nanobodies') for simultaneous single-cell measurements of protein levels and chromatin accessibility profiles, and mitochondrial DNA-based clonal tracing. We use PHAGE-ATAC for multimodal analysis in primary human immune cells, sample multiplexing, intracellular protein analysis and the detection of SARS-CoV-2 spike protein in human cell populations. Finally, we construct a synthetic high-complexity phage library for selection of antigen-specific nanobodies that bind cells of particular molecular profiles, opening an avenue for protein detection, cell characterization and screening with single-cell genomics.

9.
Nature ; 598(7879): 214-219, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616064

RESUMO

The cerebellar cortex is a well-studied brain structure with diverse roles in motor learning, coordination, cognition and autonomic regulation. However,  a complete inventory of cerebellar cell types is currently lacking. Here, using recent advances in high-throughput transcriptional profiling1-3, we molecularly define cell types across individual lobules of the adult mouse cerebellum. Purkinje neurons showed considerable regional specialization, with the greatest diversity occurring in the posterior lobules. For several types of cerebellar interneuron, the molecular variation within each type was more continuous, rather than discrete. In particular, for the unipolar brush cells-an interneuron population previously subdivided into discrete populations-the continuous variation in gene expression was associated with a graded continuum of electrophysiological properties. Notably, we found that molecular layer interneurons were composed of two molecularly and functionally distinct types. Both types show a continuum of morphological variation through the thickness of the molecular layer, but electrophysiological recordings revealed marked differences between the two types in spontaneous firing, excitability and electrical coupling. Together, these findings provide a comprehensive cellular atlas of the cerebellar cortex, and outline a methodological and conceptual framework for the integration of molecular, morphological and physiological ontologies for defining brain cell types.


Assuntos
Córtex Cerebelar/citologia , Perfilação da Expressão Gênica , Transcriptoma , Adulto , Animais , Atlas como Assunto , Eletrofisiologia , Feminino , Humanos , Interneurônios/classificação , Interneurônios/citologia , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/classificação , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/classificação , Neurônios/citologia , Neurônios/metabolismo
10.
Nat Methods ; 18(10): 1204-1212, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34608310

RESUMO

Identifying gene-regulatory targets of nuclear proteins in tissues is a challenge. Here we describe intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq), a scalable method that measures multiplexed intranuclear protein levels and the transcriptome in parallel across thousands of nuclei, enabling joint analysis of transcription factor (TF) levels and gene expression in vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed genome-wide associations of each TF and recovered known gene targets. TF-associated genes were coexpressed as distinct modules that each reflected positive or negative TF levels, showing that our approach can disentangle relative putative contributions of TFs to gene expression and add interpretability to inferred gene networks. inCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.


Assuntos
Biologia Computacional/métodos , Proteínas Nucleares/metabolismo , Análise de Célula Única/métodos , Animais , Anticorpos , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Estudo de Associação Genômica Ampla , Ácido Caínico/toxicidade , Camundongos , Proteínas Nucleares/genética , RNA , Reprodutibilidade dos Testes , Convulsões/induzido quimicamente , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
11.
Nat Genet ; 53(10): 1469-1479, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34594037

RESUMO

Single-cell RNA sequencing has revealed extensive transcriptional cell state diversity in cancer, often observed independently of genetic heterogeneity, raising the central question of how malignant cell states are encoded epigenetically. To address this, here we performed multiomics single-cell profiling-integrating DNA methylation, transcriptome and genotype within the same cells-of diffuse gliomas, tumors characterized by defined transcriptional cell state diversity. Direct comparison of the epigenetic profiles of distinct cell states revealed key switches for state transitions recapitulating neurodevelopmental trajectories and highlighted dysregulated epigenetic mechanisms underlying gliomagenesis. We further developed a quantitative framework to directly measure cell state heritability and transition dynamics based on high-resolution lineage trees in human samples. We demonstrated heritability of malignant cell states, with key differences in hierarchal and plastic cell state architectures in IDH-mutant glioma versus IDH-wild-type glioblastoma, respectively. This work provides a framework anchoring transcriptional cancer cell states in their epigenetic encoding, inheritance and transition dynamics.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Plasticidade Celular/genética , Epigênese Genética , Glioma/genética , Glioma/patologia , Padrões de Herança/genética , Transcrição Genética , Linhagem Celular Tumoral , Ilhas de CpG/genética , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Humanos , Isocitrato Desidrogenase/genética , Filogenia , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Análise de Célula Única , Transcriptoma/genética
14.
Nature ; 597(7875): 196-205, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497388

RESUMO

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.


Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologia
15.
Nat Commun ; 12(1): 5506, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535642

RESUMO

Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.


Assuntos
Anticorpos Neutralizantes/imunologia , Engenharia de Proteínas , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Anticorpos Antivirais , COVID-19/tratamento farmacológico , Humanos , Ligação Proteica , Reprodutibilidade dos Testes , Anticorpos de Domínio Único/genética , Glicoproteína da Espícula de Coronavírus
16.
Cancer Discov ; 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34561242

RESUMO

SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes CCSP+ cells within the lung in a cell-type dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex - via Smarca4 - acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution.

17.
Immunity ; 54(10): 2338-2353.e6, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34534439

RESUMO

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.


Assuntos
Adenocarcinoma de Pulmão/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Linfonodos/imunologia , Fator 1 de Transcrição de Linfócitos T/imunologia , Animais , Camundongos , Subpopulações de Linfócitos T/imunologia
18.
Cell ; 184(19): 4996-5014.e26, 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34534464

RESUMO

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.

19.
Cell Stem Cell ; 28(11): 1922-1935.e5, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34529935

RESUMO

Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II-) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.


Assuntos
Antígenos de Histocompatibilidade Classe II , Intestinos , Carcinogênese , Dieta Hiperlipídica , Células Epiteliais , Humanos
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