RESUMEN
Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
Asunto(s)
Feto/citología , Hematopoyesis , Hígado/citología , Hígado/embriología , Células Sanguíneas/citología , Microambiente Celular , Femenino , Feto/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Hígado/metabolismo , Tejido Linfoide/citología , Análisis de la Célula Individual , Células Madre/metabolismoRESUMEN
During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.
Asunto(s)
Comunicación Celular , Feto/citología , Histocompatibilidad Materno-Fetal/inmunología , Placenta/citología , Placenta/metabolismo , Embarazo/inmunología , Análisis de la Célula Individual , Comunicación Celular/inmunología , Diferenciación Celular/genética , Decidua/citología , Decidua/inmunología , Decidua/metabolismo , Femenino , Feto/inmunología , Feto/metabolismo , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ligandos , Placenta/inmunología , ARN Citoplasmático Pequeño/genética , Análisis de Secuencia de ARN , Células del Estroma/citología , Células del Estroma/metabolismo , Transcriptoma , Trofoblastos/citología , Trofoblastos/inmunología , Trofoblastos/metabolismoRESUMEN
SUMMARY: Bulk RNA sequencing studies have demonstrated that human leukocyte antigen (HLA) genes may be expressed in a cell type-specific and allele-specific fashion. Single-cell gene expression assays have the potential to further resolve these expression patterns, but currently available methods do not perform allele-specific quantification at the molecule level. Here, we present scHLAcount, a post-processing workflow for single-cell RNA-seq data that computes allele-specific molecule counts of the HLA genes based on a personalized reference constructed from the sample's HLA genotypes. AVAILABILITY AND IMPLEMENTATION: scHLAcount is available under the MIT license at https://github.com/10XGenomics/scHLAcount. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Análisis de la Célula Individual , Programas Informáticos , Alelos , Expresión Génica , Humanos , Análisis de Secuencia de ARN , Flujo de TrabajoRESUMEN
The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell-specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.
Asunto(s)
Evolución Molecular , Células Asesinas Naturales/inmunología , Receptores de IgG/genética , Transcriptoma , Proteínas de Pez Cebra/genética , Animales , Células Cultivadas , Secuencia Conservada , Humanos , Células Asesinas Naturales/citología , Ratones , Análisis de la Célula Individual , Pez Cebra/genética , Pez Cebra/inmunologíaRESUMEN
We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4(+) T cells as well as mature effector and memory cells.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Receptores de Antígenos de Linfocitos T/genética , Salmonelosis Animal/inmunología , Análisis de la Célula Individual/métodos , Programas Informáticos , Transcriptoma , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Activación de Linfocitos , Ratones , Salmonella/genética , Salmonelosis Animal/genéticaRESUMEN
BACKGROUND & AIMS: Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation. METHODS: We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13-30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn's disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor Vß genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction. RESULTS: Circulating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40-4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vß repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4+ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls. CONCLUSIONS: In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.
Asunto(s)
Bacterias/inmunología , Linfocitos T CD4-Positivos/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Microbioma Gastrointestinal/inmunología , Intestinos/inmunología , Bacterias/clasificación , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/microbiología , Estudios de Casos y Controles , Línea Celular , Colitis Ulcerosa/sangre , Colitis Ulcerosa/diagnóstico , Enfermedad de Crohn/sangre , Enfermedad de Crohn/diagnóstico , Interacciones Huésped-Patógeno , Humanos , Inmunidad Mucosa , Memoria Inmunológica , Interleucina-17/inmunología , Intestinos/microbiología , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Células Th17/inmunología , Células Th17/microbiologíaRESUMEN
The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3's function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression.
Asunto(s)
ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Linfocitos T Reguladores/fisiología , Animales , Linaje de la Célula , Núcleo Celular/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Ratones Endogámicos BALB C , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Linfocitos T Reguladores/citologíaAsunto(s)
Receptores de Antígenos de Linfocitos B/genética , Análisis de Secuencia de ARN/estadística & datos numéricos , Programas Informáticos , Animales , Linfocitos B/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Gráficos por Computador , Humanos , Ratones , Análisis de la Célula Individual/estadística & datos numéricosRESUMEN
The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees to isolate over 9,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. Among the recovered antibodies was TXG-0078, an N-terminal domain (NTD)-specific neutralizing mAb that recognizes diverse alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy-chain-dominant binding pattern seen in other NTD-supersite-specific neutralizing Abs with much narrower specificity. We also report CC24.2, a pan-sarbecovirus neutralizing antibody that targets a unique receptor-binding domain (RBD) epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 shows protection in vivo, suggesting their potential use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Epítopos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/virología , Epítopos/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Monoclonales/inmunología , Animales , Betacoronavirus/inmunología , RatonesRESUMEN
Development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. Current strategies for developing pan-coronavirus countermeasures have largely focused on the receptor binding domain (RBD) and S2 regions of the coronavirus Spike protein; it has been unclear whether the N-terminal domain (NTD) is a viable target for universal vaccines and broadly neutralizing antibodies (Abs). Additionally, many RBD-targeting Abs have proven susceptible to viral escape. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees using multiplexed panels of uniquely barcoded antigens in a high-throughput single cell workflow to isolate over 9,000 SARS-CoV-2-specific monoclonal Abs (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. We observed many instances of clonal coalescence between individuals, suggesting that Ab responses frequently converge independently on similar genetic solutions. Among the recovered antibodies was TXG-0078, a public neutralizing mAb that binds the NTD supersite region of the coronavirus Spike protein and recognizes a diverse collection of alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy chain-dominant binding pattern seen in other NTD supersite-specific neutralizing Abs with much narrower specificity. We also report the discovery of CC24.2, a pan-sarbecovirus neutralizing mAb that targets a novel RBD epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 provides protection against in vivo challenge with SARS-CoV-2, suggesting potential future use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.
RESUMEN
To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.
Asunto(s)
Linfocitos B , Inmunoglobulinas , Recombinación V(D)J/genética , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Células Precursoras de Linfocitos BRESUMEN
Links between T cell clonotypes, as defined by T cell receptor (TCR) sequences, and phenotype, as reflected in gene expression (GEX) profiles, surface protein expression and peptide:major histocompatibility complex binding, can reveal functional relationships beyond the features shared by clonally related cells. Here we present clonotype neighbor graph analysis (CoNGA), a graph theoretic approach that identifies correlations between GEX profile and TCR sequence through statistical analysis of GEX and TCR similarity graphs. Using CoNGA, we uncovered associations between TCR sequence and GEX profiles that include a previously undescribed 'natural lymphocyte' population of human circulating CD8+ T cells and a set of TCR sequence determinants of differentiation in thymocytes. These examples show that CoNGA might help elucidate complex relationships between TCR sequence and T cell phenotype in large, heterogeneous, single-cell datasets.
Asunto(s)
Linfocitos T CD8-positivos , Receptores de Antígenos de Linfocitos T alfa-beta , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.
Asunto(s)
Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T , Adulto , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Preescolar , Granzimas/metabolismo , Humanos , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα-/- bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3' VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα-/- B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.
Asunto(s)
Diversidad de Anticuerpos/genética , Linfocitos B/metabolismo , Médula Ósea/metabolismo , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Receptores de Interleucina-7/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Linaje de la Célula/genética , Cromatina/metabolismo , ADN Intergénico/genética , Feto/metabolismo , Hígado/embriología , Hígado/metabolismo , Ratones Endogámicos C57BL , Motivos de Nucleótidos/genética , Factor de Transcripción PAX5/metabolismo , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
In this chapter, we describe TraCeR and BraCeR, our computational tools for reconstruction of paired full-length antigen receptor sequences and clonality inference from single-cell RNA-seq (scRNA-seq) data. In brief, TraCeR reconstructs T-cell receptor (TCR) sequences from scRNA-seq data by extracting sequencing reads derived from TCRs by aligning the reads from each cell against synthetic TCR sequences. TCR-derived reads are then assembled into full-length recombined TCR sequences. BraCeR builds on the TraCeR pipeline and accounts for somatic hypermutations (SHM) and isotype switching. Here we discuss experimental design, use of the tools, and interpretation of the results.
Asunto(s)
ARN Citoplasmático Pequeño/genética , ARN/genética , Receptores de Antígenos/genética , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Receptores de Antígenos de Linfocitos T/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Programas InformáticosRESUMEN
The Human Cell Atlas is a large, international consortium that aims to identify and describe every cell type in the human body. The comprehensive cellular maps that arise from this ambitious effort have the potential to transform many aspects of fundamental biology and clinical practice. Here, we discuss the technical approaches that could be used today to generate such a resource and also the technical challenges that will be encountered.
Asunto(s)
Bases de Datos Factuales , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Análisis de la Célula Individual , HumanosRESUMEN
A recently published study in Genome Biology shows that cells isolated from cryopreserved tissues are a reliable source of genetic material for single-cell RNA-sequencing experiments.Please see related Method article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1171-9.
Asunto(s)
Criopreservación , Análisis de Secuencia de ARN , Análisis de la Célula Individual/métodos , Animales , Humanos , ARN/genéticaRESUMEN
The immune system varies in cell types, states, and locations. The complex networks, interactions, and responses of immune cells produce diverse cellular ecosystems composed of multiple cell types, accompanied by genetic diversity in antigen receptors. Within this ecosystem, innate and adaptive immune cells maintain and protect tissue function, integrity, and homeostasis upon changes in functional demands and diverse insults. Characterizing this inherent complexity requires studies at single-cell resolution. Recent advances such as massively parallel single-cell RNA sequencing and sophisticated computational methods are catalyzing a revolution in our understanding of immunology. Here we provide an overview of the state of single-cell genomics methods and an outlook on the use of single-cell techniques to decipher the adaptive and innate components of immunity.
Asunto(s)
Genómica/métodos , Sistema Inmunológico , Análisis de la Célula Individual/métodos , Transcriptoma , Inmunidad Adaptativa/genética , Animales , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata/genéticaRESUMEN
Noncoding transcripts originating upstream of the immunoglobulin constant region (I transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of Iε and Iγ1 transcription in response to interleukin 4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. In this study, we combine novel mouse reporters and single-cell RNA sequencing to reveal the heterogeneity in IL-4-induced I transcription. We identify an early population of cells expressing Iε but not Iγ1 and demonstrate that early Iε transcription leads to switching to IgE and occurs at lower activation levels than Iγ1. Our results reveal how probabilistic transcription with a lower activation threshold for Iε directs the early choice of IgE versus IgG1, a key physiological response against parasitic infestations and a mediator of allergy and asthma.