Immune Network

Immune Network - Overview

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19.

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity

We performed single-cell RNA-sequencing to create a cell census of the human thymus during development, early childhood and adult life. We sampled 15 embryonic and fetal thymi spanning thymic developmental stages between 7 post-conception weeks (PCW) to 17 PCW, and 9 postnatal thymi from paediatric and adult samples. We compared the cellular composition and T cell differentiation within human and mouse thymus using newly generated and repository mouse datasets. Finally, we investigated the bias in the recombination and selection of human versus mouse TCR repertoires.

A cell atlas of human thymic development defines T cell repertoire formation

Hematopoietic Stem/Progenitor cells (HSPCs) are endowed with the role of maintaining a diverse pool of blood cells throughout the human life. Despite recent efforts, the nature of the early cell fate decisions remains contentious. Using single-cell RNA-Seq, we show that existing approaches to stratify bone marrow CD34+ cells reveal a hierarchically-structured transcriptional landscape of hematopoietic differentiation. Still, this landscape misses important early fate decisions. We here provide a broader transcriptional profiling of bone marrow lineage negative hematopoietic progenitors that recovers a key missing branchpoint into basophils and expands our understanding of the underlying structure of early adult human haematopoiesis. We also show that this map has strong similarities in topology and gene expression to that found in mouse. Finally, we identify the sialomucin CD164, as a reliable marker for the earliest branches of HSPCs specification and we showed how its use can foster the design of alternative transplantation cell products.

A comprehensive single cell transcriptional landscape of human hematopoietic progenitors

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

A human cell atlas of fetal gene expression.

Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles, it has been difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution.

A molecular cell atlas of the human lung from single-cell RNA sequencing

Human hematopoiesis is essential for producing blood cells and maintaining a healthy immune system. To provide a single-cell level characterization of the hematopoietic system, we profiled cells collected from cord blood, adult peripheral blood and adult bone marrow, to produce an immune cell atlas consisting of 1,453,784 single cells. Our atlas consists of a variety of single-cell RNA-seq assay types, such as standard assay, genetic pooling and cell hashing, which makes it an ideal dataset for benchmarking and developing new demultiplexing algorithms. We anticipate that our data will play an important role in addressing key questions of the human hematopoietic system, facilitating design of novel complete blood cell count tests and shedding light on new drug development as a health reference.

A single cell immune cell atlas of human hematopoietic system

The authors developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq.

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors.

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for approximately 1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems. Overall design: Applying single cell chromatin accessibility assays to diverse human tissue types from four donors.

A single-cell atlas of chromatin accessibility in the human genome

T cells develop from circulating precursor cells, which enter the thymus and migrate through specialised sub-compartments that support their maturation and selection. In humans, this process starts in early fetal development and is highly active until thymic involution in adolescence. To map the micro-anatomical underpinnings of this process in pre- and early postnatal stages, we established a novel quantitative morphological framework for the thymus, the Cortico-Medullary Axis, and used it to perform a spatially resolved analysis. By applying this framework to a curated multimodal single-cell atlas, spatial transcriptomics, and high-resolution multiplex imaging data, we demonstrate establishment of the lobular cytokine network, canonical thymocyte trajectories and thymic epithelial cell distributions within the first trimester of fetal development. We pinpoint tissue niches of thymic epithelial cell progenitors and distinct subtypes associated with Hassall’s corpuscles and uncover divergence in the timing of medullary entry between CD4 vs. CD8 T cell lineages. These findings provide a basis for a detailed understanding of T lymphocyte development and are complemented with a holistic toolkit for cross-platform imaging data analysis, annotation, and Organ Axis construction (TissueTag), which can be applied to any tissue.

A spatial human thymus cell atlas mapped to a continuous tissue axis

Current efforts within the HCA are largely focused on defining reference human cell types using a relatively small number of samples of predominantly European origin. However, the next phase of the HCA is likely to address the topic of human diversity by comprehensively characterizing variation in cell states across age, sex, population groups, diseases and environments. To spur this next phase, and also expand the geographical scope of the HCA, we propose an HCA-Asia seed network to generate an Asian Immune Diversity Atlas (AIDA). AIDA will generate transcriptome variation datasets from 5 major Asian population groups (Chinese, Japanese, Korean, Indian, Malay), define an atlas of Asian cell types and states, and characterize their variation associated with ethnicity, environment, age, sex, sleep status and body mass index. We will also formalize a study design based on common controls that can in principle reduce technical artifacts in a broad range of comparative single cell studies. This study design will be complemented with novel algorithms designed to normalize datasets of interest against the common controls, to increase the robustness of biological inferences. In addition to providing new tools and resources to the HCA, this seed network will serve as a template for future comparative studies in the single cell field.

AIDA

The effects of HIV infection on immune cell exhaustion at the transcriptomic level was investigated by analyzing single-cell RNA sequencing of peripheral blood mononuclear cells from four healthy subjects (37,847 cells) and six HIV-infected donors (28,610 cells).

An Atlas of Immune Cell Exhaustion in HIV-Infected Individuals Revealed by Single-Cell Transcriptomics.

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in- depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project. Overall design: The samples consists of two complex tissues (human PBMC and mouse colon) and three cell lines (HEK293-RFP, NIH3T3-GFP, MDCK-Turbo650). The primary PBMC and the colon sample constitute 90% and the cell lines 10%. The sample preparation aims to be standardized for all methods to allow a comparison of library preparation performance (6% HEK293T-RFP, 3% NIH3T3-GFP, 1% MDCK-Turbo650) of the sample content.

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 11-12 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 subsets emerging for the first time. B-lymphocytes 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. 

There are no restrictions on data availability for new data presented in this study. FASTQ and raw count matrices for droplet-based scRNA-seq data for FBM from fetuses with Down syndrome and fetuses without Down syndrome have been deposited at EMBL-EBI ArrayExpress and at the European Nucleotide Archive (ENA), with accession codes as follows: E-MTAB-9389 (FBM from fetuses with Down syndrome and FBM from fetuses without Down syndrome), E-MTAB-10042 (FBM from fetuses with Down syndrome) and ERP125305 (FBM from fetuses without Down syndrome). FASTQ and raw count matrices for all other new data in this study have been deposited at EMBL-EBI ArrayExpress and at the Gene Expression Omnibus (GEO) with accession codes E-MTAB-9801 (FBM Smart-seq2 scRNA-seq); E-MTAB-9389 (BCR- or TCR-enriched VDJ FBM scRNA-seq; FASTQs only); GSE166895 (CD34+ FBM, fetal liver and cord blood CITE-seq) and GSE166895 (FBM total CITE-seq). The following data are also available to download as Scanpy h5ad objects with transformed counts through our interactive webportal: https://fbm.cellatlas.io/: (i) FBM scRNA-seq (fetuses with Down syndrome); (ii) FBM scRNA-seq (fetuses without Down syndrome); (iii) CD34+ FBM, fetal liver and cord blood CITE-seq; and (iv) FBM total CITE-seq. All source data are available in the accompanying source data file, unless manuscript or figure legend refers to a Supplementary Table. External datasets incorporated into this study include: (i) human fetal liver and yolk sac scRNA-seq data4 (E-MTAB-7407); (ii) human blood monocyte–DC scRNA-seq data10 (GSE94820); (iii) mouse BM scRNA-seq data19 (GSE122467); (iv) fetal and paediatric thymus scRNA-seq data3 (E-MTAB-8581); and (v) adult BM and cord blood scRNA-seq data from the Human Cell Atlas Data Coordination Portal ‘Census of Immune Cells’ project (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79). At the time of submission, there are no known accessibility restrictions on these external datasets. Source data are provided with the paper.

Cell count:
Total post-QC is 164,709:
- 119,971 for 10x scRNA-seq (103,228 from fetal bone marrow; 16,743 from Down syndrome fetal bone marrow) 
- 486 for Smart-seq2 scRNA-seq (from fetal bone marrow)
- 44,252 for CITE-seq (35,273 from CD34+ fetal bone marrow, fetal liver, and cord blood; 8,978 from non-FACs sorted fetal bone marrow)

Blood and immune development in human fetal bone marrow and Down syndrome

Our aims are to generate a full representation of human hematopoiesis in blood and bone marrow of humans using a multi-tier and iterative collection and analysis of 200,000 cells from ten healthy human bone

Bone marrow plasma cells from hip replacement surgeries

Clinically important broadly reactive B cells evolve during multiple infections, with B cells re-activated after secondary infection differing from B cells activated after a primary infection. Here we studied CD27highCD38high plasmablasts from patients with a primary or secondary dengue virus infection. Three transcriptionally and functionally distinct clusters were identified. The largest cluster 0/1 was plasma cell-related, with cells coding for serotype cross-reactive antibodies of the IgG1 isotype, consistent with memory B cell activation during an extrafollicular response. Cells in clusters 2 and 3 expressed low levels of antibody genes and high levels of genes associated with oxidative phosphorylation, EIF2 pathway, and mitochondrial dysfunction. Clusters 2 and 3 showed a transcriptional footprint of T cell help, in line with activation from naive B cells or memory B cells. Our results contribute to the understanding of the parallel B cell activation events that occur in humans after natural primary and secondary infection.

CD27hiCD38hi plasmablasts are activated B cells of mixed origin with distinct function

Thymic epithelium is critical for the structural integrity of the thymus and for T cell development. Within the fully formed thymus, large numbers of hematopoietic cells shape the thymic epithelium into a scaffold-like structure which bears little similarity to classical epithelial layers, such as those observed in the skin, intestine or pancreas. Here, we show that human thymic epithelial cells (TECs) possess an epithelial identity that also incorporates the expression of mesenchymal cell associated genes, whose expression levels vary between medullary and cortical TECs (m/cTECs). Using pluripotent stem cell (PSC) differentiation systems, we identified a unique population of cells that co-expressed the master TEC transcription factor FOXN1, as well as the epithelial associated marker EPCAM and the mesenchymal associated gene CD90. Using the same serum free culture conditions, we also observed co-expression of EPCAM and CD90 on cultured TECs derived from neonatal human thymus in vitro. Single cell RNA-sequencing revealed these cultured TECs possessed an immature mTEC phenotype and expressed epithelial and mesenchymal associated genes, such as EPCAM, CLDN4, CD90 and COL1A1. Importantly, flow cytometry and single cell RNA-sequencing analysis further confirmed the presence of an EPCAM+CD90+ population in the CD45- fraction of neonatal human thymic stromal cells in vivo. Using the human thymus cell atlas, we found that cTECs displayed more pronounced mesenchymal characteristics than mTECs during embryonic development. Collectively, these results suggest human TECs possess a hybrid gene expression program comprising both epithelial and mesenchymal elements, and provide a basis for the further exploration of thymus development from primary tissues and from the in vitro differentiation of PSCs. Overall design: This dataset includes RNA samples from three independent donors as biological replicates. For each donor, we sequenced mRNA isolated from four fractions of cells based on the expression of EPCAM and CD104, as well as samples representing unsorted cells.

CD90 marks a mesenchymal program in human thymic epithelial cells in vitro and in vivo

Peripheral blood NK cells were cultured into either control or cytokine-induced memory-like NK cells as described in PMID: 32614951. After differentiation, cells were labeled with hashtag antibodies and cite-seq antibodies and control and memory-like NK cells were combined and loaded onto the 10x Genomics Chromium Instrument a the McDonnell Genome Institute at Washington University School of Medicine. The following samples were analyzed to develop a classification tool for unbiased identification of memory-like NK cells in a bulk single-cell RNA-seq population. Overall design: 1 sample: control and memory-like NK cell generated from the same normal donor are hashtagged into the same files

CITE-seq of in vitro control and memory-like human NK cells

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

The authors generated single-cell atlases of 23 lung, 16 kidney, 15 liver and 18 heart COVID-19 autopsy donor tissue samples, and spatial atlases of 14 lung donors.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

Despite rapid developments in single cell sequencing technology, sample-specific batch effects, detection of cell doublets, and the cost of generating massive datasets remain outstanding challenges. Here, we introduce cell hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins are used to uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples. We demonstrate our approach by pooling eight human PBMC samples on a single run of the 10x Chromium system, substantially reducing our per-cell costs for library generation. Cell hashing is inspired by, and complementary to, elegant multiplexing strategies based on genetic variation, which we also leverage to validate our results. We therefore envision that our approach will help to generalize the benefits of single cell multiplexing to diverse samples and experimental designs.

Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics

Cis-regulatory elements (CREs) play a critical role in the development and disease-states of all human cell types. In the retina, CREs have been implicated in several inherited disorders. To better characterize human retinal CREs, we performed single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) on the developing and adult human retina and on induced pluripotent stem cell (iPSC)-derived retinal organoids. These analyses identified developmentally dynamic, cell-class-specific CREs, enriched transcription-factor-binding motifs, and putative target genes. CREs in the retina and organoids are highly correlated at the single-cell level, and this supports the use of organoids as a model for studying disease-associated CREs. As a proof of concept, we disrupted a disease-associated CRE at 5q14.3, confirming its principal target gene as the miR-9-2 primary transcript and demonstrating its role in neurogenesis and gene regulation in mature glia. This study provides a resource for characterizing human retinal CREs and showcases organoids as a model to study the function of CREs that influence development and disease.

Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids

The Human Cell Atlas project seeks to identify and functionally characterize all cell types in the human body. Single-cell RNA-sequencing (scRNA-seq) methods quantify gene expression in individual cells. In principle, highly parallelized scRNA-seq approaches enable marker-free decomposition of cells from complex tissues to identify known and unknown cell types and provide direct insights into regulatory and functional states of each cell. Different scRNA-seq technologies vary in cell capture efficiency, library preparation, and throughput. Importantly, these approaches also vary in sensitivity and accuracy of mRNA quantification. The Human Cell Atlas project will collate data generated across many labs, necessitating the evaluation of experimental replicability, and comparing different experimental procedures. It is important to develop robust sequencing protocols and benchmark their performance on select cells and tissues. This project will perform a systematic comparison of three single cell RNA sequencing technologies, viz., Drop-Seq, Fluidigm C1, and DroNC-Seq and resulting datasets. Droplet-based methods facilitate high-throughput processing of tens of thousands of cells in parallel and at low costs, making them ideal for the Human Cell Atlas project. This project proposes to compare the sensitivity, accuracy, and precision with which the three methods quantify transcript levels within individual cells. In addition, this project will directly compare these methods using a ‘synthetic tissue’ created from a mixture of multiple cell types at known ratios.

Comparison, calibration, and benchmarking of high-throughput single cell RNA-Seq techniques for unbiased cell-type classification

Single-cell RNA sequencing was used to determine the cell type composition of all major human organs and construct a basic scheme for the human cell landscape (HCL).

Construction of A Human Cell Landscape by Single-cell mRNA-seq

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. Here, we surveyed the immune compartment of 15 tissues of six deceased adult donors by single-cell RNA sequencing and paired VDJ sequencing. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of 45 finely phenotyped immune cell types and states, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. 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.

Cross-tissue immune cell analysis reveals tissue-specific features in humans

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 uses the single-cell adaptive immune receptor repertoire to explore lymphocyte developmental origins

Single-cell RNA sequencing of PBMCs and infusion products from 32 patients treated with CD19 CAR-T therapy

Differential dynamics of response at single cell resolution following CAR-T therapy in refractory B-cell lymphoma

Cellular heterogeneity can emerge from the expression of only one parental allele, however it has remained unknown to what degree patterns of random monoallelic expression of autosomal genes (aRME) are mitotically inherited (clonal) or stochastic (dynamic) in somatic cells. Here, we resolved this by applying allele-sensitive single-cell RNA-seq on primary mouse fibroblasts and in vivo human T-cells to simultaneously investigate clonal and dynamic aRME. Dynamic aRME affected a considerable portion of the transcriptome, with levels dependent on the cell’s transcriptional activity, but clonal aRME was surprisingly scarce (<1% of genes) and affected mainly lowly expressed genes. Consequently, the overwhelming portion of aRME occurs transiently and is scattered throughout somatic populations rather than, as previously hypothesized, confined to spatially restricted patches of clonally related cells.

Dissecting the clonal nature of allelic expression in somatic cells by single-cell RNA-seq

Single cell profiling is a powerful tool for studying the molecular and cellular (dys)function of activated peripheral blood mononuclear cells (PBMCs) in the context of disease. We combined CITE-seq with two levels of multiplexing (cell hashing and individuals’ genotypes) to derive a reference database of immune cell gene/protein responses to different activation conditions. PBMCs from 10 healthy adults were profiled before and after stimulating i) T cells via anti-CD3/CD28 or ii) monocytes via LPS. By using a comprehensive antibody panel (n=39) of cell type (e.g., CD16, CD14) and cell state (e.g., CD69, CD25) markers, we discovered that all lymphocytes responded to anti-CD3/CD28 stimulation, whereas LPS specifically induced inflammation in monocytes. Pseudo-temporal analyses further revealed cell- and condition-specific heterogeneity in responses to activation that were independent of individual-specific variation. Together, these data are shared within an interactive web application (https://czi-pbmc-cite-seq.jax.org/) and will serve as a resource to guide future studies of immune cell responses.


Dissecting the protein and transcriptional responses of human immune cells to T cell and monocyte specific activation

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity, but little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterisation of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady-state. We describe distinct T helper 1 cell activation and migration profiles along the colon and characterise the transcriptional adaptation trajectory of T regulatory cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from caecum to sigmoid colon, and link this to the increasing number of reactive bacterial species

Distinct microbial and immune niches of the human colon

Systemic infections, especially in patients with chronic diseases, result in sepsis: an explosive, uncoordinated immune response that can lead to multisystem organ failure with a high mortality rate. Sepsis survivors and non-survivors oftentimes have similar clinical phenotypes or sepsis biomarker expression upon diagnosis, suggesting that the dynamics of sepsis in the critical early stage may have an impact on these opposite outcomes. To investigate this, we designed a within-subject study of patients with systemic gram-negative bacterial sepsis with surviving and fatal outcomes and performed single-cell transcriptomic analyses of peripheral blood mononuclear cells (PBMC) collected during the critical period between sepsis recognition and 6 hours. We observed that the largest sepsis-induced expression changes over time in surviving versus fatal sepsis were in CD14+ monocytes, including gene signatures previously reported for sepsis outcomes. We further identify changes in the metabolic pathways of both monocytes and platelets, the emergence of erythroid precursors, and T cell exhaustion signatures, with the most extreme differences occurring between the non-sepsis control and the sepsis non-survivor. Our single-cell observations are consistent with trends from public datasets but also reveal specific effects in individual immune cell populations, which change within hours. In conclusion, this pilot study provides the first single-cell results with a repeated measures design in sepsis to analyze the temporal changes in the immune cell population behavior in surviving or fatal sepsis. These findings indicate that tracking temporal expression changes in specific cell-types could lead to more accurate predictions of sepsis outcomes. We also identify molecular pathways that could be therapeutically controlled to improve the sepsis trajectory toward better outcomes. Summary sentence Single cell transcriptomics of peripheral blood mononuclear cells in surviving and fatal sepsis reveal inflammatory and metabolic pathways that change within hours of sepsis recognition.

Dynamic changes in human single cell transcriptional signatures during fatal sepsis

Direct comparisons of human and non-human primate brain tissue have the potential to reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is largely inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. First, we systematically evaluated the fidelity of organoid models to primary human and macaque cortex, finding organoid models preserve gene regulatory networks related to cell types and developmental processes but exhibit increased metabolic stress. Second, we identified 261 genes differentially expressed in human compared to chimpanzee organoids and macaque cortex. Many of these genes overlap with human-specific segmental duplications and a subset suggest increased PI3K/AKT/mTOR activation in human outer radial glia. Together, our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution. Overall design: Single cell mRNA sequencing of iPS-derived neural and glial progenitor cells using the Fluidigm C1 system This series includes re-analysis of publicly available data in accessions: phs000989.v3, GSE99951, GSE86207, GSE75140. Sample metadata and accession IDs for the re-analyzed samples are included in the file "GSE124299_metadata_on_processed_samples.xlsx" available on the foot of this record. The following samples have no raw data due to data loss: GSM3569728, GSM3569738, GSM3571601, GSM3571606, GSM3571615, GSM3571621, GSM3571625, and GSM3571631

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution

Single-cell RNA sequencing of memory T cells (CD45RA-CD4+) isolated from the peripheral blood (PBMCs) of 5 healthy human donors. Memory T cells were activated/stimulated with PMA/Ion/GP. A population of GM-CSF-producing Th cells (ThGM) was identified that lacked expression of signature transcription factors and cytokines of established Th lineages.

GM-CSF-producing T helper cells: a distinct subset of T helper cells.

Humans show remarkable variation in susceptibility to infectious diseases as well as chronic inflammatory and autoimmune disorders. This heterogeneity arises partially from variation in the immune response, which is responsible for preventing and controlling infection. To better understand the major factors driving antiviral immune response differences, we used single-cell RNA-sequencing to measure the effects of genetic ancestry and cis-regulatory variation on the transcriptional response to influenza infection in various immune cell types in 90 European and African American individuals. We show that monocytes are the most responsive to infection but that all cell types engage a conserved, type I IFN response, which is stronger in European individuals. Further, we detect directional, polygenic differences in expression phenotypes between populations that are under cis-genetic control and show that recent positive selection has acted on putatively causal risk loci associated with common autoimmune disorders. Our findings establish genetic ancestry and common cis-regulatory variants as important determinants governing the antiviral immune response, thus improving our understanding of the factors that contribute to differences in infectious and complex disease susceptibility. Overall design: Multiplexed single-cell RNA expression profiles of control (mock-infected) and influenza A virus (IAV)-infected peripheral blood mononuclear cells (PBMCs) collected from African and European American individuals

Genetic ancestry effects on the response to viral infection are pervasive but cell type specific

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

Heterogeneity and clonal relationships of adaptive immune cells in ulcerative colitis revealed by single-cell analyses

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.

Human SARS-CoV-2 challenge resolves local and systemic response dynamics

Preeclampsia (PE) is characterized by sustained hypertension and proteinuria at a gestational age of 20 weeks or more. Immune system alterations are associated with the origin and the pathophysiology of preeclampsia, however, little is known about the landscape and heterogeneity of maternal immune system at the single-cell level. In this study, we performed single-cell RNA sequencing of 80,429 cells in PE patients and health controls, including T cells, B cells, natural killer (NK) cells, myeloid cells in peripheral blood mononuclear cells to characterize the immune cell subgroups at the pregnant and postpartum stages of PE. Our work showed that there was excessive activation of B cells, monocytes and NK cells in PE patients at the pregnant stage. Additionally, lower immune response activation was noticed in CD4+ and CD8+ T cells in PEs, especially the low-activation of memory T cells at the pregnant and postpartum stages. Moreover, there was a higher activation of B cells in pregnancy persisted postpartum in PEs, together with lower activation of memory T cells. These indicated their persistent effects on PE and its recurrence risks. Overall design: Single-cell RNA sequencing of 80,429 cells in PE patients and health controls, including T cells, B cells, natural killer (NK) cells, myeloid cells in peripheral blood mononuclear cells to characterize the immune cell subgroups at the pregnant and postpartum stages of PE.

Immune cell profiling of preeclamptic pregnant and postpartum women by single-cell RNA sequencing

Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or malignant transformation by human papillomavirus (HPV). Here, we assessed the transcriptional profiles of 131,224 single cells from peripheral and intra-tumoral CD45+ immune populations from HPV– and HPV+ HNSCC and healthy donors. A spectrum of transcriptional signatures in immune lineages ranging from similar to highly divergent was present in the tumor microenvironments (TME) in HPV– and HPV+ HNSCC. CD8+ and regulatory CD4+ T cells were similar in HPV– and HPV+ HNSCC, compared to significant differences between helper CD4+ T cells, B cells and myeloid cells. Further dissection of the major immune lineages identified unique cell states and differentiation trajectories. We contextualized our results by performing multispectral immunofluorescence and evaluating putative cell-cell communication based on spatial proximity, and found longer progression free survival in patients with enrichment of a CD4+ T follicular helper cell signature. Overall design: Total of 63 samples were analyzed by scRNAseq. Of these, 26 samples were paired peripheral blood mononuclear cells and tumor infiltrating immune cells from HNSCC patients (18 HPV- and 8 HPV+), 6 were peripheral blood mononuclear cells from healthy donors, and 5 were tissue resident immune cells from healthy donor tonsils.

Immune landscape of viral- and carcinogen-drived head and neck cancer

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

Immune signatures underlying post-acute COVID-19 lung sequelae

Although most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected individuals experience mild coronavirus disease 2019 (COVID-19), some patients suffer from severe COVID-19, which is accompanied by acute respiratory distress syndrome and systemic inflammation. To identify factors driving severe progression of COVID-19, we performed single-cell RNA sequencing using peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, patients with mild or severe COVID-19, and patients with severe influenza. Patients with COVID-19 exhibited hyperinflammatory signatures across all types of cells among PBMCs, particularly up-regulation of the tumor necrosis factor/interleukin-1β (TNF/IL-1β)–driven inflammatory response as compared with severe influenza. In classical monocytes from patients with severe COVID-19, type I interferon (IFN) response coexisted with the TNF/IL-1β–driven inflammation, and this was not seen in patients with milder COVID-19. We documented type I IFN–driven inflammatory features in patients with severe influenza as well. On the basis of this, we propose that the type I IFN response plays a pivotal role in exacerbating inflammation in severe COVID-19.

Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Integrated analysis of multimodal single-cell data

The heart, the first organ to develop, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of CHD patients survive into adulthood but often suffer premature death from heart failure (HF) and non-cardiac causes. To gain insight into poorly understood disease progression, we performed single nuclear RNA sequencing (snRNA-seq) and analyzed 157,273 nuclei from donors and CHD patients, including hypoplastic left heart syndrome (HLHS) and Tetralogy of Fallot (TOF), two common forms of cyanotic CHD lesions, as well as, dilated (DCM) and hypertrophic (HCM) cardiomyopathies. We observed CHD specific cell states in cardiomyocytes (CMs) which had evidence of insulin resistance and increased FOXO and CRIM1 expression. Cardiac fibroblasts (CFs) in HLHS had enrichment for a low HIPPO and high YAP cell state characteristic of activated CFs. Imaging Mass Cytometry (IMC) uncovered the spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD in agreement with CHD predilection to infection and cancer. Our comprehensive CHD phenotyping provides a roadmap for future personalized medicine in CHD.

Integrated multiomic characterization of congenital heart disease

During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone-marrow- derived hematopoietic progenitors that migrate through the bloodstream. The current understanding of the na- ture of thymic immigrants is largely based on data from pre-clinical models. Here, we employed single-cell RNA sequencing (scRNA-seq) to examine the immature postnatal thymocyte population in humans. Integration of bone marrow and peripheral blood precursor datasets identified two putative thymus seeding progenitors that varied in expression of CD7; CD10; and the homing receptors CCR7, CCR9, and ITGB7. Whereas both pre- cursors supported T cell development, only one contributed to intrathymic dendritic cell (DC) differentiation, predominantly of plasmacytoid dendritic cells. Trajectory inference delineated the transcriptional dynamics un- derlying early human T lineage development, enabling prediction of transcription factor (TF) modules that drive stage-specific steps of human T cell development. This comprehensive dataset defines the expression signa- ture of immature human thymocytes and provides a resource for the further study of human thymopoiesis.

Integrated scRNA-Seq Identifies Human Postnatal Thymus Seeding Progenitors and Regulatory Dynamics of Differentiating Immature Thymocytes

Single-cell RNA sequencing of cerebrospinal fluid and PBMCs from Multiple sclerosis patients.

Integrated single cell analysis of blood and cerebrospinal fluid leukocytes in multiple sclerosis.

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 450,118 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells. Overall design: We have 49 organoids in total. We include 4 donors (AS, ThF, N1, N2; 2 males, 2 females) at 4 timepoints: 1 replicate each at the iPSC (Day 0), 1 replicate for 2 donors (N1, N2), 2 for the other two (AS, ThF) at Day 7, 3 replicates each at Days 15 and 29. For one donor (AS), we include 2 experiments at D15 and D29. For one donor (ThF), we have extended culture experiments at D32 (3 replicates) and D51(1 replicate). For ThF, we generated D29 organoids (3 replicates) from a second protocol. We have 2 transplanted organoids at D29. Organoids were pooled on lanes using a randomized design to ensure that organoids replicates from an individual batch (donor, replicate, condition) were distributed across lanes.

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

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 paediatric versus adult 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 data 1 as a highly granular reference for the study of immune responses in airways and blood in children.

Local and systemic responses to SARS-CoV-2 infection in children and adults

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, the authors performed a longitudinal multi-omics study using a two-center cohort of 14 patients. They analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients.

Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

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.

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Recent advances in single cell genomics technologies have facilitated studies on the developing immune system at unprecedented scale and resolution. However, these studies have focused on one or a few organs and were thus limited in understanding the developing immune system as a distributed network across tissues. Here, we profiled prenatal haematopoietic organs, lymphoid organs and non-lymphoid tissues using a combination of single-cell RNA sequencing, paired antigen-receptor sequencing and spatial transcriptomics to reconstruct the developing human immune system. Our analysis revealed the acquisition of immune effector transcriptome profiles in macrophages, mast cells and NK cells from the second trimester, and the transcriptomic changes accompanying the late-stage maturation of developing monocytes and T cells that extended from their organ of origin to peripheral tissues. We uncovered system-wide blood and immune cell development beyond the conventional primary haematopoietic organs. We further identified, extensively characterised and functionally validated the human prenatal B1 cells. Finally, we provide evidence for thymocyte-thymocyte selection origin for αβTCR- expressing unconventional T cells based on TCR gene usage and an in vitro artificial thymic organoid culture model. Our comprehensive atlas of the developing human immune system provides both valuable data resources and biological insights that will facilitate cell engineering, regenerative medicine and disease understanding. 

One-Sentence Summary 
By performing a comprehensive single-cell RNA sequencing atlas of human developing immune system together with antigen-receptor sequencing and spatial transcriptomics, we explored the cross-gestation and cross-organ variability in immune cells, discovered system-wide blood and immune cell development, identified, characterised and functionally validated the properties of human prenatal B1 cells and the origin of unconventional T cells.

Mapping the developing human immune system across organs

In order to investigate heterogeneity and dynamics of cancer-associated immune cells, we performed single-cell sequencing on peripheral and tumor-infiltrating immune cells in three renal clear cell carcinoma patients. We chose renal clear cell carcinoma tumors based on the responsive of these tumors to immune checkpoint blockade in the context of low mutational loads, which implies a strong influence from the tumor microenvironment. Using the 10x Genomic 5 expression platform a total of 25,672 immune cells were isolated and passed filtering for quality control, with 13,433 cells from peripheral blood and 12,239 tumor-infiltrating cells. In addition, we used the Chromium Single Cell V(D)J kit to enrich for T cell receptor sequences for nearly 10,000 T cells in with accompanying expression information, allowing for the investigation of clonality and transcriptional phenotypic diversity. We generated a comprehensive immune profile using single-cell RNA-seq data from 25,000 immune cells from three renal cell carcinoma along with matched peripheral blood. In addition, we performed VDJ sequencing on the isolated single T cells. Overall design: T cell receptor sequences of the subset of T cells in the 5' RNA expression sequencing dataset.

Mapping the immune environment in clear cell renal carcinoma by single-cell genomics

The cancer microenvironment is a complex ecosystem characterized by dynamic interactions between diverse cell types, including malignant, immune and stromal cells. Here, we performed single-cell RNA sequencing on CD45+ and CD45- cells isolated from tumour and lymph nodes during a mouse model of melanoma. The transcriptional profiles of these individual cells taken at different time points coupled with assembled T cell receptor sequences, allowed us to identify distinct immune subpopulations and delineate their developmental trajectory. Our study provides insights into the complex interplay among cells within the tumour microenvironment and presents a valuable resource for future translational applications.

Melanoma infiltration of stromal and immune cells

Natural killer (NK) cells support the anti-myeloma activity of daratumumab via antibody-dependent cellular cytotoxicity (ADCC) in multiple myeloma (MM). However, the different roles of heterogeneous NK cell subpopulations have not been elucidated in MM. Here, we delineate memory-like NK cells in the bone marrow (BM) of newly diagnosed MM (NDMM) patients using single-cell RNA sequencing, and further characterize their distinct immunophenotypic features and functions by multicolor flow cytometry. Memory-like NK cells exert robust daratumumab-mediated effector functions ex vivo, including cytokine production and degranulation, compared to conventional NK cells. The composition of memory-like NK cells in BM determines the daratumumab-mediated ex vivo functional activity of BM NK cells in NDMM patients. Unlike conventional NK cells, sorted memory-like NK cells from the BM of NDMM patients exert substantial cytotoxic activity against myeloma cells in the presence of daratumumab. Our findings indicate that memory-like NK cells are an important mediator of daratumumab-dependent effector functions in MM and support direct future efforts to better predict and improve the clinical efficacy of therapeutic antibodies by selectively employing memory-like NK cells. Overall design: NK cells sorted from 3 MM patients' bone marrow, no replicates

Memory-like natural killer cells facilitate effector functions of daratumumab in multiple myeloma

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3Neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. We also found that patients with Inflammatory Bowel Disease (IBD), demonstrate a deficiency in this specific regulatory T-cell subpopulation.  scRNA-seq analysis was performed on IL-10-producing CD4+ T cells isolated from the blood (PBMCs) of two healthy donors.

Molecular and functional heterogeneity of IL-10-producing CD4+ T cells.

Severe fever with thrombocytopenia syndrome banyangvirus (SFTSV) induces immunopathogenic disease with high fatality rate. Despite serious health concern, the mechanisms underlying clinical manifestations are largely unknown. Here, we applied targeted proteomics and single-cell transcriptomics analyses to examine the differential immune landscape of SFTS patient blood. Serum immunoprofiling distinguished Low-risk and High-risk clusters of SFTS patients with differential inflammatory cytokine levels, which matched with disease severity. Single-cell transcriptomics analysis of SFTS patient blood at different infection state showed pronounced expansion of B cells with the alteration of B-cell subset populations, specifically in fatal patients. Furthermore, plasma cells were the primary reservoir of SFTSV replication with the downregulation of IFN pathway. This study identifies not only the molecular signatures of serum inflammatory profile and B cell lineage population, but also plasma cells as the viral reservoir in SFTSV-induced fatal patients, suggesting the alteration of B cell function linked to SFTS lethal infection. Overall design: PBMC from Control, Infection, Recovery and Fatal SFTS patients were collected in hospitals throughout South Korea. PBMC from each individual were processed separately through the ChromiumTM Single Cell Platform with the 10X Genomics ChromiumTM Single Cell 3’ Library and Gel Bead Kit v2 and the Chromium Single Cell A Chip Kit using the standard manufacturer’s protocol.

Molecular signatures of inflammatory profile and B-cell function in SFTS patients

This is a human single cell sequencing repository of pancreatic cancer tumor tissue and PBMCs of pancreatic cancer patients. The goal of this study was to thorough map the immune landscape of pancreatic ductal adenocarinoma patient tumors and peripheral blood.

Multimodal mapping of the tumor and peripheral blood immune landscape in human pancreatic cancer

Here, we introduce an in-silico algorithm demuxlet that harnesses naturally occurring genetic variation in a pool of cells from unrelated individuals to discover the sample identity of each cell and identify droplets containing cells from two different individuals (doublets). These two capabilities enable a simple multiplexing design that increases single cell library construction throughput by experimental design where cells from genetically diverse samples are multiplexed and captured at 2-10x over standard workflows. We further demonstrate the utility of sample multiplexing by characterizing the interindividual variability in cell type-specific responses of ~15k PBMCs to interferon-beta, a potent cytokine. Our computational tool enables sample multiplexing of droplet-based single cell RNA-seq for large-scale studies of population variation and could be extended to other single cell datasets that incorporate natural or synthetic DNA barcodes.

Multiplexing droplet-based single cell RNA-sequencing using genetic barcodes

Peripheral blood and adipose tissue were collected from eight healthy donors and eight multiple sclerosis (MS) patients. Two of the MS patients were untreated and the rest had previously received disease modifying treatments (DMTs). This study compared the transcriptomic program induced by oleic acid to that of the pro-inflammatory arachidonic acid. The role of fatty acids in regulating tissue inflammatory signals was investigated in healthy donors and MS patients by comparing single-cell transcriptomic profiles of blood and adipose tissue samples with in vitro stimulated Treg cells.

Oleic acid restores suppressive defects in tissue-resident FOXP3 Tregs from patients with multiple sclerosis'

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.

Pathogen-induced tissue-resident memory T <sub>H</sub> 17 (T <sub>RM</sub> 17) cells amplify autoimmune kidney disease

CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, heterogeneity and clonal diversity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile and clonality in humans. The single-cell differential gene expression analysis, revealed a spectrum of known transcripts, including several linked to cytotoxic and co-stimulatory function, and transcripts of unknown cytotoxicity-related function that are expressed at higher levels in the TEMRA subset, which is highly enriched for CD4-CTLs, compared to cells in the central and effector memory subsets (TCM, TEM). Simultaneous T cells antigen receptor (TCR) analysis in single-cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared to TCM and TEM cells and that the majority of CD4-TEMRA were dengue virus (DENV)-specific in subjects with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across subjects, with four distinct clusters identified by the single-cell analysis. Most importantly, we identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and thus could provide insights into the mechanisms that may be utilized to generate durable and effective CD4-CTL immunity.

Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis

Differentiation is among the most fundamental processes in cell biology. Single cell RNA-seq studies have demonstrated that differentiation is a continuous process and in particular cell states are observed to reside on largely continuous spaces. We have developed Palantir, a graph based algorithm to model continuities in cell state transitions and cell fate choices. Modeling differentiation as a Markov chain, Palantir determines probabilities of reaching terminal states from cells in each intermediate state. The entropy of these probabilities represent the differentiation potential of the cell in the corresponding state. Applied to single cell RNA-seq dataset of CD34+ hematopoietic cells from human bone marrows, Palantir accurately identified key events leading up to cell fate commitment. Integration with ATAC-seq data from bulk sorted populations helped identify key regulators that correlate with cell fate specification and commitment.

Profiling of CD34+ cells from human bone marrow to understand hematopoiesis

Here we demonstrate single-cell RNA-5′end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay at no extra cost. We showed enhancer RNAs can be detected using sc-end5-seq methods with either random or oligo(dT) priming.

Profiling of transcribed cis-regulatory elements in single cells.

Transcriptomic profiling of skin wounds in a time-course manner. Full thickness punch biopsies were performed on the flank of healthy donors to create skin wounds. This was followed by larger biopsies, to encompass the original wounds, taken between one to four days later. The skin biopsies and blood samples were subjected to single-cell RNA sequencing analysis.

Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

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.

Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.

Regenerative lineages and immune-mediated pruning in lung cancer metastasis

We profile the transcriptomes of over 100,000 human single cells, yielding molecular definitions for non-parenchymal cell types present in healthy and cirrhotic human liver. We uncover a novel scar-associated TREM2+CD9+ macrophage subpopulation, which expands in liver fibrosis, differentiates from circulating monocytes, has a corollary population in mouse liver fibrosis and is pro-fibrogenic. We also define novel ACKR1+ and PLVAP+ endothelial cells which expand in cirrhosis, are topographically scar-restricted and enhance leucocyte transmigration. Multi-lineage ligand-receptor modelling of interactions between the novel scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides the conceptual framework required to discover rational therapeutic targets in liver cirrhosis.

Resolving the fibrotic niche of human liver cirrhosis using single-cell transcriptomics

we performed single cell RNA sequencing (scRNA-seq) in both normal and failed human heart. CD45- cells in the myocardium and immune cells (CD45+ cells) were isolated from myocardium and corresponding peripheral blood were sorted for scRNA-seq. Our findings were also validated with bulk RNAseq. Overall design: Establishing a comprehensive cell atlas of immune cells in human heart failure at single cell level.

Resolving the intertwining of inflammation and fibrosis in human heart failure at single-cell level

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment

Classic dendritic cells (cDCs) play a central role in the immune system and consist of two major subsets: CD141+ cDC (cDC1) and CD1c+ cDC (cDC2). The pre-cDCs is the immediate precursors to both cDC subsets. Previous studies showed that there were two pre-committed pre-cDC subpopulations. However, the key molecular drivers of pre-commitment in human pre-cDCs were not investigated. To address this question, we performed both single cell and bulk RNA sequencing (RNA-seq) of two cDC subsets and pre-cDCs. We inferred a list of sixteen candidate master regulator transcriptional factors (TFs) that can indeed separate pre-cDCs into two sub-populations, with one close to cDC1 and the other close to cDC2. More importantly, these two pre-cDC sub-populations are correlated with ratio of IRF8 to IRF4 expression level more than their individual expression level. Our results suggest the concept that the ratio of antagonistic TFs and their competition determine cDC subset differentiation fate.

Single Cell RNA-Seq Analysis Reveals  Fate Decision of Human Pre-cDC Determined  by Transcription Factor Competition

To investigate the relationship between genetic and transcriptional heterogeneity in a context of cancer progression, we devised a computational approach called HoneyBADGER to identify copy number variation and loss-of-heterozygosity in individual cells from single-cell RNA-sequencing data. By combining allele frequency and expression magnitude deviations, HoneyBADGER is able to infer the presence of subclone-specific alterations in individual cells and reconstruct subclonal architecture. Also HoneyBADGER to analyze single cells from a progressive multiple myeloma (MM) patient to identify major genetic subclones that exhibit distinct transcriptional signatures relevant to cancer progression.

Single cell RNA sequencing of multiple myeloma II

Self-renewal of tendons is rare since the vascular formation inside is extremely poor, thus reconstructive surgery using autologous tendons has been often taken place in the case of severe injury. However, the rate of re-injury after surgery is relatively high, and collection of autologous tendons leads muscle weakness which results in prolonged rehabilitation. Here, we introduce the induced pluripotent stem cells (iPSCs)-based technology aiming at developing a new therapeutic option after tendon injury. We derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in the vertebrate embryo. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSCs-derived tenocytes. Overall design: Single cell rnaseq analysis of the developmental trajectory of iPSC-derived tenocytes

Single cell RNAseq analysis of the developmental trajectory of iPSC-derived tenocytes

In late 2019 and through 2020, the COVID-19 pandemic swept the world, presenting both scientific and medical challenges associated with understanding and treating a previously unknown disease. To help address the need for great understanding of COVID-19, the scientific community mobilized and banded together rapidly to characterize SARS-CoV-2 infection, pathogenesis and its distinct disease trajectories. The urgency of COVID-19 provided a pressing use-case for leveraging relatively new tools, technologies, and nascent collaborative networks. Single-cell biology is one such example that has emerged over the last decade as a powerful approach that provides unprecedented resolution to the cellular and molecular underpinnings of biological processes. Early foundational work within the single-cell community, including the Human Cell Atlas, utilized published and unpublished data to characterize the putative target cells of SARS-CoV-2 sampled from diverse organs based on expression of the viral receptor ACE2 and associated entry factors TMPRSS2 and CTSL (Muus et al., 2020; Sungnak et al., 2020; Ziegler et al., 2020). This initial characterization of reference data provided an important foundation for framing infection and pathology in the airway as well as other organs. However, initial community analysis was limited to samples derived from uninfected donors and other previously-sampled disease indications. This report provides an overview of a single-cell data resource derived from samples from COVID-19 patients along with initial observations and guidance on data reuse and exploration.

Single cell profiling of COVID-19 patients: an international data resource from multiple tissues

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice This study includes single cell transcriptional profiling data from peripheral blood mononuclear cells (PBMCs) from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.

Single cell transcriptional profiling of peripheral blood mononuclear cells (PBMCs) from mice flown on Rodent Research Reference Mission-2 (RRRM-2)

In the Rodent Research Reference Mission (RRRM-2), forty female C57BL/6NTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 12 week-old and twenty 29 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 55-58 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 32 days and allowed to recover for 24 days (Live Animal Return, LAR) before sacrifice. ISS-T and LAR mice were the same age at sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice per group housed in flight hardware in matched environmental conditions), basal controls (10 mice per group sacrificed 2 days before launch), and vivarium controls (10 mice per group housed within standard vivarium habitats). Thus RRRM-2 included a total of 160 mice. This study includes single cell transcriptional profiling data from the spleens from 4 young LAR flight animals, 4 old LAR flight animals, 4 young LAR ground control animals, and 4 old LAR ground control animals.

Single cell transcriptional profiling of spleens from mice flown on Rodent Research Reference Mission-2

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.

Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle towards HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression. We further validated our results using primary CD4+ T cells isolated from HIV+ individuals.

Single-Cell RNA-Seq Reveals Transcriptional Heterogeneity in Latent and Reactivated HIV-infected Cells

IgA nephropathy (IgAN) is the leading cause of kidney failure due to an incomplete understanding of its pathogenesis. We perform single-cell RNA sequencing (RNA-seq) on kidneys and CD14+ peripheral blood mononuclear cells (PBMCs) collected from IgAN and normal samples. In IgAN, upregulation of JCHAIN in mesangial cells provides insight into the trigger mechanism for the dimerization and deposition of IgA1 in situ. The pathological mesangium also demonstrates a prominent inflammatory signature and increased cell-cell communication with other renal parenchymal cells and immune cells, suggesting disease progress from the mesangium to the entire kidney. Specific gene expression of kidney-resident macrophages and CD8+ T cells further indicates abnormal regulation associated with proliferation and inflammation. A transitional cell type among intercalated cells with fibrosis signatures is identified, suggesting an adverse outcome of interstitial fibrosis. Altogether, we systematically analyze the molecular events in the onset and progression of IgAN, providing a promising landscape for disease treatment.

Single-Cell Transcriptomics Reveal Immune Mechanisms of the Onset and Progression of IgA Nephropathy

To obtain more information about the lymph node metastasis of breast cancer cells, we selected the matched positive lymph nodes (PL), and negative lymph nodes (NL) of the same patient to perform integrated analysis. The PL, NL samples were analysed with single-cell ATAC sequencing. Overall design: Single cell ATACseq was performed using the 10X Genomics platform per the manufacturers instructions.

Single-cell ATAC sequencing reveals the mechanism of human breast cancer metastasis

Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell transcriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions.

Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma

Genome-wide association studies have identified thousands of genetic variants that are associated with disease1. Most of these variants have small effect sizes, but their downstream expression effects, so-called expression quantitative trait loci (eQTLs), are often large and celltype-specific. To identify these celltype-specific eQTLs using an unbiased approach, we used single-cell RNA sequencing to generate expression profiles of ~25,000 peripheral blood mononuclear cells from 45 donors. We identified previously reported cis-eQTLs, but also identified new celltype-specific cis-eQTLs. Finally, we generated personalized co-expression networks and identified genetic variants that significantly alter co-expression relationships (which we termed ‘co-expression QTLs’). Single-cell eQTL analysis thus allows for the identification of genetic variants that impact regulatory networks.

Single-cell RNA sequencing identifies celltype-specific cis-eQTLs and co-expression QTLs

This study aimed to explore the molecular basis of peripheral immune cells and the features of adaptive immune repertoire at a single cell level. We proﬁled 36,849 peripheral blood mononuclear cells from AD patients with amyloid-positive status and normal controls with amyloid-negative status by 5' single-cell transcriptome and immune repertoire sequencing using the cell ranger standard analysis procedure. We revealed five immune cell subsets, CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and monocytes-macrophages cells, and disentangled the characteristic alterations of cell subset proportion and gene expression patterns in AD. Thirty-one cell type-specific key genes, comprising abundant human leukocyte antigen genes, and multiple immune-related pathways were identified by protein-protein interaction network and pathway enrichment analysis. We also found high-frequency amplification clonotypes in T cells and B cells and decreased diversity in T cells in AD. As clone amplification suggested the activation of an adaptive immune response against specific antigens, we speculated that the peripheral adaptive immune response, especially mediated by T cells, may have a role in the pathogenesis of AD. This finding may also contribute to further research regarding disease mechanism and the development of immune-related biomarkers or therapy. Overall design: Three patients with AD and two cognitively normal controls (NC) >Submitter states: The analysis of TCR-seq and BCR-seq has not yet been performed<

Single-cell RNA sequencing of peripheral blood reveals immune cell signatures in Alzheimer's disease

The liver plays a critical role in both immune defense and tolerance in the body. The liver-resident immune cells (LrICs) determine the immune properties, but the unique composition and heterogeneity of these cells are incompletely understood. Here, we dissect the diversity of LrICs by a comprehensive transcriptomic profiling using unbiased single-cell RNA-sequencing (scRNA-seq). Overall design: 10x genomics was used to measure CD45+ immune cells in healthy human liver and paired spleen and PBMCs from the same donors.

Single-cell RNA sequencing reveals the heterogeneity of liver-resident immune cells in human.

Here we investigated the earliest possible evidence of subclinical neuro-inflammation (SCNI) using a small cohort of monozygotic twins where one sibling had clinically definite MS and the other been clinically "healthy" but has a maximal genetic for developing MS. In contrast to subjects with radiologically isolated syndrome (RIS), our group of very early SCNI does not even fulfill the (arbitrary) MRI criteria for RIS but have more subtle MRI changes and/or evidence of neuro-inflammation in the CSF, e.g. oligoclonal bands (OCBs). For analyzing CSF samples from Twin pairs and controls in greater detail we applied single-cell whole transcriptome sequencing (scRNAseq). Our findings demonstrate that even the earliest experimentally approachable stage of MS is characterized by synergistic activation of CD8+ T cells, CD4+ T cells and B cells.

Single-cell RNA-seq in monozygotic twins discordant for multiple sclerosis

BM samples were collected from two adult healthy donors and two AA patients at the first hospital affiliated from Zhejiang Chinese University. Mononuclear cells (MNCs) were isolated using Ficoll-Hypaque gradient separation. CD34+ cells were purified from MNCs with the human anti-CD34 MicroBeads Isolation kit according to the manufactures specifications. Isolated cell was the purification of CD34+ cell. Then, MNCs mixed with CD34+ cells at 4:1 ratio and were analyzed by 10×Genomics. Overall design: a large-scale single-cell transcriptomic sequencing of 20,000 bone marrow cells from two adult healthy donors and two AA patients

Single-cell RNA-seq of bone marrow cells from aplastic anemia patient and healthy donor

NKT cells are potent immune regulators and skew immune responses toward either inflammation or tolerance through quickly secreting either T helper (Th)1-, Th2-, Th17- or regulatory T-associated cytokines upon activation. Although the thymic development and functional differentiation of NKT cells is well defined in mice, it has not yet been studied in detail in humans. Overall design: We performed single-cell RNA-sequencing (scRNA-seq) of unstimulated and PMA/ionomycin-stimulated NKT cells from human peripheral blood mononuclear cells (PBMCs).

Single-cell RNA-seq of human peripheral blood NKT cells

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon-stimulated genes (ISGs) in monocytes, reduction of naïve CD4+ T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH+ CD8+ T cells. Cell type-specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type-specific cis-expression quantitative trait loci and to link SLE-associated variants to cell type-specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE.

Single-cell RNA-seq reveals cell type-specific molecular and genetic associations to lupus

Here, we use a microfluidics-based approach to prepare single-cell RNA-Seq libraries from 164 primary human conventional dendritic cells (cDCs) as well as cord blood (452) and blood (341) pre-cDCs. We examined heterogeneity between individual cells to document potential subpopulations within human cDCs and pre-cDCs.

Single-cell RNA-seq reveals heterogeneity within human pre-cDCs

Ascending thoracic aortic aneurysm (ATAA) is caused by the progressive weakening and dilatation of the aortic wall and can lead to aortic dissection, rupture, and other life-threatening complications. To improve understanding of ATAA pathogenesis, the authors sought to comprehensively characterize the cellular composition of the ascending aortic wall and to identify molecular alterations in each cell population of human ATAA tissues using single-cell RNA sequencing technology.

Single-cell Transcriptome Analysis Reveals Dynamic Cell Populations and Differential Gene Expression Patterns in Control and Aneurysmal Human Aortic Tissue.

Clinical benefits of cytokine blockade in ileal Crohn’s disease (iCD) are limited to a subset of patients. Here we applied single cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct connectivity network that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in 4 independent iCD cohorts (n=441), and its presence at diagnosis correlated with failure to achieve corticosteroid-free durable remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities

Single-cell analysis of Crohn’s disease lesions identifies a pathogenic cellular module associated with resistance to anti-TNF therapy

Childhood acute lymphoblastic leukemia (cALL) is the most common pediatric cancer. It is characterized by bone marrow lymphoid precursors that acquire genetic alterations, resulting in disrupted maturation and uncontrollable proliferation. More than a dozen molecular subtypes of variable severity can be used to classify cALL cases. Modern therapy protocols currently cure 85-90% of cases, but other patients are refractory or will relapse and eventually succumb to their disease. To better understand these difficult cases, we investigated the nature and extent of intra-individual transcriptional heterogeneity of cALL at the cellular level by sequencing the transcriptomes of 39,375 individual cells in eight patients (six pre-B and two pre-T) and three healthy pediatric controls. We observed intra-individual transcriptional clusters in five out of the eight patients. Using pseudotime maturation trajectories of healthy B and T cells, we obtained the predicted developmental state of each leukemia cell and observed distribution shifts within patients. We showed that the predicted developmental states of these cancer cells are inversely correlated with ribosomal protein expression levels, which could be a common contributor to intra-individual heterogeneity in cALL patients.

Single-cell analysis of childhood leukemia reveals a link between developmental states and ribosomal protein expression as a source of intra-individual heterogeneity

Lack of diversity and proportionate representation in genomics datasets and databases contributes to inequity in healthcare outcomes globally. The relationships of human diversity with biological and biomedical phenotypes are pervasive, yet remain understudied, particularly in a single-cell genomics context. Here we present the Asian Immune Diversity Atlas (AIDA), a multi-national single-cell RNA-sequencing (scRNA-seq) healthy reference atlas of human immune cells. AIDA comprises 1,265,624 circulating immune cells from 619 healthy donors and 6 controls, spanning 7 population groups across 5 countries. AIDA is one of the largest healthy blood datasets in terms of number of cells, and also the most diverse in terms of number of population groups. Though population groups are frequently compared at the continental level, we identified a pervasive impact of sub-continental diversity on cellular and molecular properties of immune cells. These included cell populations and genes implicated in disease risk and pathogenesis as well as those relevant for diagnostics. We identified numerous examples where the effects of age and sex were modulated by self-reported ethnicity. We also detected age, sex, and self-reported ethnicity differences at the resolution of cell neighbourhoods, highlighting finer-grained distinctions than were apparent at cell-type level. We discovered functional genetic variants influencing cell type-specific gene expression, including context-dependent effects, which were under-represented in analyses of non-Asian population groups, and which helped contextualise disease-associated variants. We validated our findings using multiple independent datasets and cohorts. AIDA provides fundamental insights into the relationships of human diversity with immune cell phenotypes, enables analyses of multi-ancestry disease datasets, and facilitates the development of precision medicine efforts in Asia and beyond.

Single-cell analysis of human diversity in circulating immune cells (Japan cells)

Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). We confirmed LCAMhi enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAMhi lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.

Single-cell analysis of human non-small cell lung cancer lesions refines tumor classification and patient stratification

Immunotherapies have been explored in treating solid tumors, albeit with disparate clinical effects in distinct cancer types. Systematic interrogation of immune cells in the tumor microenvironment (TME) is vital to the prediction of immunotherapy response and the development of innovative immunotherapeutics. To comprehensively characterize the immune microenvironment in advanced biliary tract cancer (BTC), we utilized single-cell RNA sequencing in unselected viable cells from 16 matched samples. We identified nineteen cell subsets from a total of 45,851 cells, in which exhausted CD8+ T cells, macrophages, and dendritic cells (DCs) in BTC were shown to augment and communicate within the TME. Transcriptional profiles coupled with T cell receptor (TCR) sequences revealed that exhausted CD8+ T cells retained clonal expansion and high proliferation in the TME, and some of them highly expressed the endoplasmic reticulum stress (ER) sensor XBP1, indicating its key role in remodeling TME. Functional assays demonstrated that XBP1 and common immune checkpoints (PD1, TIGIT) were significantly upregulated in CD8+ T cells cocultured within the TME of BTC cell lines (GBC-SD, HCCC-9810). When treating the coculture groups with the specific inhibitor of IRE1α-XBP1 (4μ8C), the downregulation of TIGIT was observed in the treatment group. Collectively, comprehensive transcriptome profiling provides deeper insights into the immune atlas in advanced BTC, which might be instrumental in exploring novel immunotherapy strategies. Overall design: Five patients diagnosed with cholangiocarcinoma in the Eastern Hepatobiliary Surgery Hospital were incorporated into the current study, including two intrahepatic cholangiocarcinoma (iCCA), two gallbladder carcinoma (GBC) and one dCCA patients. Of note, none of the patients have undergone any preoperative treatments (chemotherapy, radiation or anti-tumor medicines) prior to tumor resection. Tumor primary locus and metastasis as well as lymph node were consistently harvested for each participant, if available. Meanwhile, peripheral blood samples were collected prior to surgical procedures using anticoagulant tubes.

Single-cell atlas of diverse immune populations in the advanced biliary tract cancer microenvironment

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our data yielded unique biological insights into age-associated changes and also provided a resource of exceptional depth in both raw data and in browsable form (Synapse under accession code syn49637038).

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells

The functions of innate lymphoid cells (ILCs) in immune system are increasingly appreciated, whereas the early development of ILCs in human remains elusive. In this study, we sorted humanhematopoietic stem progenitor cells, lymphoid progenitors, presumed ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 PCW,for single-cell RNA-sequencing, followed by computational analysis and functional validation. We delineated the early phase of ILC development, from hematopoietic stem progenitor cells to multipotent lymphoid progenitors and to ILC progenitors, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor alpha (IL-3RA) as the surface marker for the lymphoid progenitors with T cell, B cell and ILC potentials. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the shared proliferating characteristics of the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2-CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity. Overall design: Here, we provided an unprecedented single-cell transcriptome atlas of stepwise ILC ontogeny in early human fetus using cells from fetal hematopoietic, lymphoid and non-lymphoid tissues spanning 8 to 12 post-conception weeks (PCWs).

Single-cell deciphering of human fetal innate lymphoid cell development

The human immune system displays substantial variation between individuals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression. We subsequently show how eQTLs have dynamic allelic effects in B cells that are transitioning from naïve to memory states and demonstrate how commonly segregating alleles lead to interindividual variation in immune function. Finally, using a Mendelian randomization approach, we identify the causal route by which 305 risk loci contribute to autoimmune disease at the cellular level. This work brings together genetic epidemiology with scRNA-seq to uncover drivers of interindividual variation in the immune system.

Single-cell eQTL mapping identifies cell type-specific genetic control of autoimmune disease

The COVID-19 pandemic, caused by SARS coronavirus 2 (SARS-CoV-2), has resulted in excess morbidity and mortality as well as economic decline. To characterise the systemic host immune response to SARS-CoV-2, we performed single-cell RNA-sequencing coupled with analysis of cell surface proteins, providing molecular profiling of over 800,000 peripheral blood mononuclear cells from a cohort of 130 patients with COVID-19. Our cohort, from three UK centres, spans the spectrum of clinical presentations and disease severities ranging from asymptomatic to critical. Three control groups were included: healthy volunteers, patients suffering from a non-COVID-19 severe respiratory illness and healthy individuals administered with intravenous lipopolysaccharide to model an acute inflammatory response. Full single cell transcriptomes coupled with quantification of 188 cell surface proteins, and T and B lymphocyte antigen receptor repertoires have provided several insights into COVID-19: 1. a new non-classical monocyte state that sequesters platelets and replenishes the alveolar macrophage pool; 2. platelet activation accompanied by early priming towards megakaryopoiesis in immature haematopoietic stem/progenitor cells and expansion of megakaryocyte-primed progenitors; 3. increased clonally expanded CD8+ effector:effector memory T cells, and proliferating CD4+ and CD8+ T cells in patients with more severe disease; and 4. relative increase of IgA plasmablasts in asymptomatic stages that switches to expansion of IgG plasmablasts and plasma cells, accompanied with higher incidence of BCR sharing, as disease severity increases. All data and analysis results are available for interrogation and data mining through an intuitive web portal. Together, these data detail the cellular processes present in peripheral blood during an acute immune response to COVID-19, and serve as a template for multi-omic single cell data integration across multiple centers to rapidly build powerful resources to help combat diseases such as COVID-19.

Single-cell multi-omics analysis of the immune response in COVID-19

We performed scRNA sequencing to understand the cellular heterogeneity and immune response landscape behind COVID-19 pathophysiology. We combine this with Ab-Seq, oligo-attached antibody based quantification of cell surface proteins to better understand the cell type and state of the cells. Samples were multiplexed using BD single-cell multiplexing kit (Human) in order to measure these data simultaneously Overall design: PBMCs were isolated from Healthy (n = 4), COVID-19 (n = 16) and recovered (n = 13) individuals, using BD vaccutainers cell prepataion tubes. PBMCs were isolated as per manufacturer's recommendation, and stored for future use. Samples were multiplexed using BD single cell multiplexing kit (Human), followed by tagging with a pool of 40 oligo-attached antibody for surface markers (Ab-Seq). Around 30000 cells were loaded per cartridge, followed by cell lysis, cDNA synthesis and final library preparation using BD WTA amplification kit as per manufacturer's recommendation. A poly-A transcripts library (WTA), Ab-Seq library and library from oligos of Sampe multiplexing kit (SMK) were constructed from each batch, and were sequenced on NovaSeq 6000 platform.

Single-cell multiomics revealed the dynamics of antigen presentation, immune response and T cell activation in the COVID-19 positive and recovered individuals

Human mononuclear phagocytes comprise phenotypically and functionally overlapping subsets of dendritic cells (DC) and monocytes, but their identification remains elusive. By integrating high-dimensional single-cell protein and RNA expression data, we clearly delineated monocytes from conventional DC2 (cDC2), identifying new markers including CD88/CD89 for monocytes and HLA-DQ/FcRI for cDC2, allowing their unambiguous characterization in blood and tissues. We also show that cDC2 can be subdivided into phenotypically and functionally distinct subsets based on CD5, CD163 and CD14 expression, including a unique subset of circulating inflammatory CD5-CD163+CD14+ cells related to previously defined DC3. These inflammatory DC3 were expanded in systemic lupus erythematosus patients, correlating with disease activity. Unravelling the heterogeneity of DC sub-populations in health and disease paves the way for specific DC subset-targeting therapies.

Single-cell omics reveal human mononuclear phagocyte heterogeneity and inflammatory DC in health and disease

Clear cell renal cell carcinomas (ccRCCs) are highly immune infiltrated, but the effect of immune heterogeneity on clinical outcome in ccRCC has not been fully characterized. Here we perform paired single-cell RNA (scRNA) and T cell receptor (TCR) sequencing of 167,283 cells from multiple tumor regions, lymph node, normal kidney, and peripheral blood of two immune checkpoint blockade (ICB)-naïve and four ICB-treated patients to map the ccRCC immune landscape. We detect extensive heterogeneity within and between patients, with enrichment of CD8A+ tissue-resident T cells in a patient responsive to ICB and tumor-associated macrophages (TAMs) in a resistant patient. A TCR trajectory framework suggests distinct T cell differentiation pathways between patients responding and resistant to ICB. Finally, scRNA-derived signatures of tissue-resident T cells and TAMs are associated with response to ICB and targeted therapies across multiple independent cohorts. Our study establishes a multimodal interrogation of the cellular programs underlying therapeutic efficacy in ccRCC.

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