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The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the cellular disease dynamics remains limited. In our unique COVID-19 human challenge study we used single cell genomics of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in 16 seronegative individuals challenged with preAlpha-SARS-CoV-2. Our analyses revealed rapid changes in cell type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific timepoints or infection status. We observed that the interferon response in blood precedes the nasopharynx, and that nasopharyngeal immune infiltration occurred early in transient but later in sustained infection, and thus correlated with preventing sustained infection. Ciliated cells showed an acute response phase, upregulated MHC class II while infected, and were most permissive for viral replication, whilst nasal T cells and macrophages were infected non-productively. We resolve 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our novel computational pipeline (Cell2TCR) identifies activated antigen-responding clonotype groups and motifs in any dataset. Together, we show that our detailed time series data (covid19cellatlas.org) can serve as a “Rosetta stone” for the epithelial and immune cell responses, and reveals early dynamic responses associated with protection from infection.

Assessment of single-cell gene expression (single-cell RNA sequencing) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) has been invaluable in studying lymphocyte biology. Here we introduce Dandelion, a computational pipeline for scVDJ-seq analysis. It enables the application of standard V(D)J analysis workflows to single-cell datasets, delivering improved V(D)J contig annotation and the identification of nonproductive and partially spliced contigs. We devised a strategy to create an AIR feature space that can be used for both differential V(D)J usage analysis and pseudotime trajectory inference. The application of Dandelion improved the alignment of human thymic development trajectories of double-positive T cells to mature single-positive CD4/CD8 T cells, generating predictions of factors regulating lineage commitment. Dandelion analysis of other cell compartments provided insights into the origins of human B1 cells and ILC/NK cell development, illustrating the power of our approach. Dandelion is available at https://www.github.com/zktuong/dandelion.

Supplementary Material: Figure S1: Nuclear localisation and fluorescent properties of FRET constructs Figure S2: Quantification of FRET experiment Figure S3: Relative mRNA expression of ESR1 target genes Figure S4: Characterisation of MCF-7 clones overexpressing NFIB and YBX1 Figure S5: Separated growth curves shown in Figure 4 Figure S6: Kaplan-Meier survival curves for breast cancer cases stratified by YBX1 expression Figure S7: Response of breast cancer PDX models to tamoxifen treatment Table S1: Primers used in RT-PCR Table S2: Antibodies used in Western blots

Full data set obtained in RIME experiment.

Two opposing clusters of transcription factors (TF) have been associated with the differential risks of estrogen receptor positive or negative breast cancers, but the mechanisms underlying the opposing functions of the two clusters are undefined. In this study, we identified NFIB and YBX1 as novel interactors of the estrogen receptor (ESR1). NFIB and YBX1 are both risk TF associated with progression of ESR1-negative disease. Notably, they both interacted with the ESR1-FOXA1 complex and inhibited the transactivational potential of ESR1. Moreover, signaling through FGFR2, a known risk factor in breast cancer development, augmented these interactions and further repressed ESR1 target gene expression. We therefore show that members of two opposing clusters of risk TFs associated with ESR1-positive and -negative breast cancer can physically interact. We postulate that this interaction forms a toggle between two developmental pathways affected by FGFR2 signaling, possibly offering a junction to exploit therapeutically.Significance: Binding of the transcription factors NFIB and YBX1 to the estrogen receptor can promote an estrogen-independent phenotype that can be reverted by inhibiting FGFR2 signaling. Cancer Res; 78(2); 410–21. ©2017 AACR.

Single-cell data analysis can infer dynamic changes in cell populations, for example across time, space or in response to perturbation. To compare these dynamics between two conditions, trajectory alignment via dynamic programming (DP) optimization is frequently used, but is limited by assumptions such as a definite existence of a match. Here we describeGenes2Genes, a Bayesian information-theoretic DP framework for aligning single-cell trajectories.Genes2Genesovercomes current limitations and is able to capture sequential matches and mismatches between a reference and a query at single gene resolution, highlighting distinct clusters of genes with varying patterns of expression dynamics. Across both real world and simulated datasets,Genes2Genesaccurately captured different alignment patterns, demonstrated its utility in disease cell state trajectory analysis, and revealed that T cells differentiatedin vitromatched to an immaturein vivostate while lacking the final TNFa signaling. This use case demonstrates that precise trajectory alignment can pinpoint divergence from thein vivosystem, thus guiding the optimization ofin vitroculture conditions.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hiprogenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways.Graphical Abstract

During human development, lungs develop their roles of gas exchange and barrier function. Recent single cell studies have focused on epithelial and mesenchymal cell types, but much less is known about the developing lung immune cells, although the airways are a major site of mucosal immunity after birth. An open question is whether tissue-resident immune cells play a role in shaping the tissue as it developsin utero. In order to address this, we profiled lung immune cells using scRNAseq, smFISH and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including ILCs, NK, myeloid cells and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes high in cytotoxicity genes, and mature B lymphocytes, including B1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the abundance of immune cells, we investigated their possible effect on epithelial maturation and found that IL-1β drives epithelial progenitor exit from self-renewal and differentiation to basal cellsin vitro.In vivo, IL-1β-producing myeloid cells were found adjacent to epithelial tips, suggesting that immune cells may direct the developing lung epithelium.

RationaleSevere asthma and chronic obstructive pulmonary disease (COPD) share common pathophysiological traits such as relative corticosteroid insensitivity. We recently published three transcriptome-associated clusters (TACs) using hierarchical analysis of the sputum transcriptome in asthmatics from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort comprising one Th2-high inflammatory signature (TAC1) and two Th2-low signatures (TAC2 and TAC3).ObjectiveWe examined whether gene expression signatures obtained in asthma can be used to identify the subgroup of patients with COPD with steroid sensitivity.MethodsUsing gene set variation analysis, we examined the distribution and enrichment scores (ES) of the 3 TACs in the transcriptome of bronchial biopsies from 46 patients who participated in the Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease COPD study that received 30 months of treatment with inhaled corticosteroids (ICS) with and without an added long-acting β-agonist (LABA). The identified signatures were then associated with longitudinal clinical variables after treatment. Differential gene expression and cellular convolution were used to define key regulated genes and cell types.Measurements and main resultsBronchial biopsies in patients with COPD at baseline showed a wide range of expression of the 3 TAC signatures. After ICS±LABA treatment, the ES of TAC1 was significantly reduced at 30 months, but those of TAC2 and TAC3 were unaffected. A corticosteroid-sensitive TAC1 signature was developed from the TAC1 ICS-responsive genes. This signature consisted of mast cell-specific genes identified by single-cell RNA-sequencing and positively correlated with bronchial biopsy mast cell numbers following ICS±LABA. Baseline levels of gene transcription correlated with the change in RV/TLC %predicted following 30-month ICS±LABA.ConclusionSputum-derived transcriptomic signatures from an asthma cohort can be recapitulated in bronchial biopsies of patients with COPD and identified a signature of airway mast cells as a predictor of corticosteroid responsiveness.

Assessment of single-cell gene expression (scRNA-seq) and adaptive immune receptor sequencing (scVDJ-seq) has been invaluable in studying lymphocyte biology. Here, we introduceDandelion, a computational pipeline for scVDJ-seq analysis. It enables the application of standard V(D)J analysis workflows to single-cell datasets, delivering improved V(D)J contig annotation and the identification of non-productive and partially spliced contigs. We devised a novel strategy to create an adaptive immune receptor feature space that can be used for both differential V(D)J usage analysis and pseudotime trajectory inference. The application ofDandelionimproved the alignment of human thymic development trajectories of double positive T cells to mature single-positive CD4/CD8 T cells, with important new predictions of factors regulating lineage commitment.Dandelionanalysis of other cell compartments provided novel insights into the origins of human B1 cells and ILC/NK cell development, illustrating the power of our approach.Dandelionis an open access resource (https://www.github.com/zktuong/dandelion)that will enable future discoveries.

The balance between self‐renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self‐renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self‐renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof‐of‐principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

The yolk sac (YS) represents an evolutionarily-conserved extraembryonic structure that ensures timely delivery of nutritional support and oxygen to the developing embryo. However, the YS remains ill-defined in humans. We therefore assemble a complete single cell 3D map of human YS from 3-8 post conception weeks by integrating multiomic protein and gene expression data. We reveal the YS as a site of primitive and definitive haematopoiesis including a YS-specific accelerated route to macrophage production, a source of nutritional/metabolic support and a regulator of oxygen-carrying capacity. We reconstruct the emergence of primitive haematopoietic stem and progenitor cells from YS hemogenic endothelium and their decline upon stromal support modulation as intraembryonic organs specialise to assume these functions. The YS therefore functions as ‘three organs in one’ revealing a multifaceted relay of vital organismal functions as pregnancy proceeds.One Sentence SummaryHuman yolk sac is a key staging post in a relay of vital organismal functions during human pregnancy.

Supplemental Digital Content is available in the text., Background: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. Methods: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary–venous ECs (COL15A1neg) localized to the lung parenchyma and systemic–venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic–venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusions: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Cell type-specific differential gene expression analyses based on single-cell transcriptome datasets are sensitive to the presence of cell-free mRNA in the droplets containing single cells. This so-called ambient RNA contamination may differ between samples obtained from patients and healthy controls. Current ambient RNA correction methods were not developed specifically for single-cell differential gene expression (sc-DGE) analyses and might therefore not sufficiently correct for ambient RNA-derived signals. Here, we show that ambient RNA levels are highly sample-specific. We found that without ambient RNA correction, sc-DGE analyses erroneously identify transcripts originating from ambient RNA as cell type-specific disease-associated genes. We therefore developed a computationally lean and intuitive correction method, Fast Correction for Ambient RNA (FastCAR), optimized for sc-DGE analysis of scRNA-Seq datasets generated by droplet-based methods including the 10XGenomics Chromium platform. FastCAR uses the profile of transcripts observed in libraries that likely represent empty droplets to determine the level of ambient RNA in each individual sample, and then corrects for these ambient RNA gene expression values. FastCAR can be applied as part of the data pre-processing and QC in sc-DGE workflows comparing scRNA-Seq data in a health versus disease experimental design. We compared FastCAR with two methods previously developed to remove ambient RNA, SoupX and CellBender. All three methods identified additional genes in sc-DGE analyses that were not identified in the absence of ambient RNA correction. However, we show that FastCAR performs better at correcting gene expression values attributed to ambient RNA, resulting in a lower frequency of false-positive observations. Moreover, the use of FastCAR in a sc-DGE workflow increases the cell-type specificity of sc-DGE analyses across disease conditions.

Skeletal muscle ageing increases the incidence of age-associated frailty and sarcopenia in the elderly worldwide, leading to increased morbidity and mortality. However, our understanding of the cellular and molecular mechanisms of muscle ageing is still far from complete. Here, we generate a single-cell and single-nucleus transcriptomic atlas of skeletal muscle ageing from 15 donors across the adult human lifespan, accompanied by myofiber typing using imaging. Our atlas reveals ageing mechanisms acting across different compartments of the muscle, including muscle stem cells (MuSCs), myofibers and the muscle microenvironment. Firstly, we uncover two mechanisms driving MuSC ageing, namely a decrease in ribosome biogenesis and an increase in inflammation. Secondly, we identify a set of nuclei populations explaining the preferential degeneration of the fast-twitch myofibers and suggest two mechanisms acting to compensate for their loss. Importantly, we identify a neuromuscular junction accessory population, which helps myofiber to compensate for aged-related denervation. Thirdly, we reveal multiple microenvironment cell types contributing to the inflammatory milieu of ageing muscle by producing cytokines and chemokines to attract immune cells. Finally, we provide a comparable mouse muscle ageing atlas and further investigate conserved and specific ageing hallmarks across species. In summary, we present a comprehensive human skeletal muscle ageing resource by combining different data modalities, which significantly expands our understanding of muscle biology and ageing.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

Obesity is common and associated with more severe COVID-19, proposed to be in part related to an adipokine-driven pro-inflammatory state. Here we analysed single cell transcriptomes from bronchiolar lavage in three adult cohorts, comparing obese (Ob, body mass index (BMI) >30m2) and non-obese (N-Ob, BMI 2). Surprisingly, we found that Ob subjects had attenuated lung immune/inflammatory responses in SARS-CoV-2 infection, with decreased expression of interferon (IFN)α, IFNγ and tumour necrosis factor (TNF) alpha response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Analysis of peripheral blood immune cells in an independent adult cohort showed a similar, but less marked, reduction in type I IFN and IFNγ response genes, as well as decreased serum IFNα, in Ob patients with SARS-CoV-2. Nasal immune cells from Ob children with COVID-19 also showed reduced enrichment of IFNα and IFNγ response genes. Altogether, these findings show blunted tissue immune responses in Ob COVID-19 patients, with clinical implications.

The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The Lung Biological Network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and cell–cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework programme. discovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Human Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Human Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions.

SummaryMultiple distinct cell types of the human lung and airways have been defined by single cell RNA sequencing (scRNAseq). Here we present a multi-omics spatial lung atlas to define novel cell types which we map back into the macro- and micro-anatomical tissue context to define functional tissue microenvironments. Firstly, we have generated single cell and nuclei RNA sequencing, VDJ-sequencing and Visium Spatial Transcriptomics data sets from 5 different locations of the human lung and airways. Secondly, we define additional cell types/states, as well as spatially map novel and known human airway cell types, such as adult lung chondrocytes, submucosal gland (SMG) duct cells, distinct pericyte and smooth muscle subtypes, immune-recruiting fibroblasts, peribronchial and perichondrial fibroblasts, peripheral nerve associated fibroblasts and Schwann cells. Finally, we define a survival niche for IgA-secreting plasma cells at the SMG, comprising the newly defined epithelial SMG-Duct cells, and B and T lineage immune cells. Using our transcriptomic data for cell-cell interaction analysis, we propose a signalling circuit that establishes and supports this niche. Overall, we provide a transcriptional and spatial lung atlas with multiple novel cell types that allows for the study of specific tissue microenvironments such as the newly defined gland-associated lymphoid niche (GALN).

Lung cancer is the leading cause of cancer-related death in the world. In contrast to many other cancers, a direct connection to modifiable lifestyle risk in the form of tobacco smoke has long been established. More than 50% of all smoking-related lung cancers occur in former smokers, often many years after smoking cessation. Despite extensive research, the molecular processes for persistent lung cancer risk are unclear.To examine whether risk stratification in the clinic and in the general population can be improved upon by the addition of genetic data, and to explore the mechanisms of the persisting risk in former smokers, we have analysed transcriptomic data from accessible airway tissues of 487 subjects, including healthy volunteers and clinic patients of different smoking status. We developed a model to assess smoking associated gene expression changes and their reversibility after smoking is stopped, comparing healthy subjects to clinic patients with and without lung cancer. We find persistent smoking-associated immune alterations to be a hallmark of the clinic patients. Integrating previous GWAS data using a transcriptional network approach, we demonstrate that the same immune and interferon related pathways are strongly enriched for genes linked to known genetic risk factors, demonstrating a causal relationship between immune alteration and lung cancer risk. Finally, we used accessible airway transcriptomic data to derive a non-invasive lung cancer risk classifier.Our results provide initial evidence for germline-mediated personalised smoke injury response and risk in the general population, with potential implications for managing long-term lung cancer incidence and mortality.

Tumour behaviour is dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. These dependencies were examined through 270,000 single cell transcriptomes and 100 micro-dissected whole exomes obtained from 12 patients with kidney tumours. Tissue was sampled from multiple regions of tumour core, tumour-normal interface, normal surrounding tissues, and peripheral blood. We found the principal spatial location of CD8+ T cell clonotypes largely defined exhaustion state, with clonotypic heterogeneity not explained by somatic intra-tumoural heterogeneity. De novo mutation calling from single cell RNA sequencing data allows us to lineage-trace and infer clonality of cells. We discovered six meta-programmes that distinguish tumour cell function. An epithelial-mesenchymal transition meta-programme, enriched at the tumour-normal interface appears modulated through macrophage expressed IL1B, potentially forming a therapeutic target.

T1 Figure 1ConclusionsOverall, this largest paediatric single cell COVID-19 study to date showed significant differences in response to SARS-CoV-2 between children and adults, reflecting the changes of the immune landscape over developmental time, which in children are dominated by naive and innate responses.

We present a multiomic cell atlas of human lung development that combines single cell RNA and ATAC sequencing, high throughput spatial transcriptomics and single cell imaging. Coupling single cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial and erythrocyte/leukocyte compartments from 5-22 post conception weeks. We identify new cell states in all compartments. These include developmental-specific secretory progenitors and a new subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). Finally, to illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signalling and transcription factor hierarchies which we test using organoid models.HighlightsSpatiotemporal atlas of human lung development from 5-22 post conception weeks identifies 144 cell types/states.Tracking the developmental origins of multiple cell compartments, including new progenitor states.Functional diversity of fibroblasts in distinct anatomical signalling niches.Resource applied to interrogate and experimentally test the transcription factor code controlling neuroendocrine cell heterogeneity and the origins of small cell lung cancer.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11–12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6–7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21). A single-cell atlas of human fetal bone marrow in healthy fetuses and fetuses with Down syndrome provides insight into developmental haematopoiesis in humans and the transcription and functional differences that occur in Down syndrome.

Funder: Medical Research Council, The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Motivation Dendrogram is a classical diagram for visualizing binary trees. Although efficient to represent hierarchical relations, it provides limited space for displaying information on the leaf elements, especially for large trees. Results Here, we present TreeAndLeaf, an R/Bioconductor package that implements a hybrid layout strategy to represent tree diagrams with focus on the leaves. The TreeAndLeaf package combines force-directed graph and tree layout algorithms using a single visualization system, allowing projection of multiple layers of information onto a graph-tree diagram. The Supplementary Information provides two case studies that use breast cancer data from epidemiological and experimental studies. Availability and implementation TreeAndLeaf is written in the R language, and is available from the Bioconductor project at http://bioconductor.org/packages/TreeAndLeaf/ (version≥1.4.2). Supplementary information Supplementary data are available at Bioinformatics online.

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

Lung disease accounts for every sixth death globally. Profiling the molecular state of all lung cell types in health and disease is currently revolutionizing the identification of disease mechanisms and will aid the design of novel diagnostic and personalized therapeutic regimens. Recent progress in high-throughput techniques for single-cell genomic and transcriptomic analyses has opened up new possibilities to study individual cells within a tissue, classify these into cell types, and characterize variations in their molecular profiles as a function of genetics, environment, cell-cell interactions, developmental processes, aging, or disease. Integration of these cell state definitions with spatial information allows the in-depth molecular description of cellular neighborhoods and tissue microenvironments, including the tissue resident structural and immune cells, the tissue matrix, and the microbiome. The Human Cell Atlas consortium aims to characterize all cells in the healthy human body and has prioritized lung tissue as one of the flagship projects. Here, we present the rationale, the approach, and the expected impact of a Human Lung Cell Atlas.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.

Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell (DC) subsets emerging for the first time. B-lymphocyte expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window.

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues, in contrast to those circulating in the blood, remains limited. Here, we surveyed the immune compartment of lymphoid and non-lymphoid tissues of six adult donors by single-cell RNA sequencing, including alpha beta T-cell receptor (αβ TCR), gamma delta (γδ) TCR and B-cell receptor (BCR) variable regions. To aid systematic cell type identification we developed CellTypist, a tool for automated and accurate cell type annotation. Using this approach combined with manual curation, we determined the tissue distribution of finely phenotyped immune cell types and cell states. This revealed tissue-specific features within cell subsets, such as a subtype of activated dendritic cells in the airways expressing CSF2RA, GPR157, CRLF2, ITGAD-expressing γδ T cells in spleen and liver, and ITGAX+ splenic memory B cells. Single cell paired chain TCR analysis revealed cell type-specific biases in VDJ usage, and BCR analysis revealed characteristic patterns of somatic hypermutation and isotype usage in plasma and memory B cell subsets. In summary, our multi-tissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis and antigen receptor sequencing.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprung’s disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

While a substantial proportion of adults infected with SARS-CoV-2 progress to develop severe disease, children rarely manifest respiratory complications. Therefore, understanding differences in the local and systemic response to SARS-CoV-2 infection between children and adults may provide important clues about the pathogenesis of SARS-CoV-2 infection. To address this, we first generated a healthy reference multi-omics single cell data set from children (n=30) in whom we have profiled triple matched samples: nasal and tracheal brushings and PBMCs, where we track the developmental changes for 42 airway and 31 blood cell populations from infancy, through childhood to adolescence. This has revealed the presence of naive B and T lymphocytes in neonates and infants with a unique gene expression signature bearing hallmarks of innate immunity. We then contrast the healthy reference with equivalent data from severe paediatric and adult COVID-19 patients (total n=27), from the same three types of samples: upper and lower airways and blood. We found striking differences: children with COVID-19 as opposed to adults had a higher proportion of innate lymphoid and non-clonally expanded naive T cells in peripheral blood, and a limited interferon-response signature. In the airway epithelium, we found the highest viral load in goblet and ciliated cells and describe a novel inflammatory epithelial cell population. These cells represent a transitional regenerative state between secretory and ciliated cells; they were found in healthy children and were enriched in paediatric and adult COVID-19 patients. Epithelial cells display an antiviral and neutrophil-recruiting gene signature that is weaker in severe paediatricversusadult COVID-19. Our matched blood and airway samples allowed us to study the spatial dynamics of infection. Lastly, we provide a user-friendly interface for this data1as a highly granular reference for the study of immune responses in airways and blood in children.