Immunology in the Era of Single-Cell Technologies.

Immune cells are characterized by diversity, specificity, plasticity, and adaptability-properties that enable them to contribute to homeostasis and respond specifically and dynamically to the many threats encountered by the body. Single-cell technologies, including the assessment of transcriptomics, genomics, and proteomics at the level of individual cells, are ideally suited to studying these properties of immune cells. In this review we discuss the benefits of adopting single-cell approaches in studying underappreciated qualities of immune cells and highlight examples where these technologies have been critical to advancing our understanding of the immune system in health and disease.

Navigating COVID-19: Starting a lab during the pandemic.

For new principal investigators, the first years are key to getting a laboratory off the ground and running. COVID-19 has changed the world, bringing on unforeseen difficulties and challenges at every level. We asked these investigators to share their experiences in navigating the unique environment since the start of the pandemic-what has changed in their vision for their laboratory, how they have adapted, and what advice they can share with others in a similar situation.

Spatial multiomics map of trophoblast development in early pregnancy.

The relationship between the human placenta-the extraembryonic organ made by the fetus, and the decidua-the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal-fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell-cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.

A physical wiring diagram for the human immune system.

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention.

Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Inherent mosaicism and extensive mutation of human placentas.

Placentas can exhibit chromosomal aberrations that are absent from the fetus1. The basis of this genetic segregation, which is known as confined placental mosaicism, remains unknown. Here we investigated the phylogeny of human placental cells as reconstructed from somatic mutations, using whole-genome sequencing of 86 bulk placental samples (with a median weight of 28 mg) and of 106 microdissections of placental tissue. We found that every bulk placental sample represents a clonal expansion that is genetically distinct, and exhibits a genomic landscape akin to that of childhood cancer in terms of mutation burden and mutational imprints. To our knowledge, unlike any other healthy human tissue studied so far, the placental genomes often contained changes in copy number. We reconstructed phylogenetic relationships between tissues from the same pregnancy, which revealed that developmental bottlenecks genetically isolate placental tissues by separating trophectodermal lineages from lineages derived from the inner cell mass. Notably, there were some cases with full segregation-within a few cell divisions of the zygote-of placental lineages and lineages derived from the inner cell mass. Such early embryonic bottlenecks may enable the normalization of zygotic aneuploidy. We observed direct evidence for this in a case of mosaic trisomic rescue. Our findings reveal extensive mutagenesis in placental tissues and suggest that mosaicism is a typical feature of placental development.

A roadmap for the Human Developmental Cell Atlas.

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

Mapping Human Reproduction with Single-Cell Genomics.

The trillions of cells in the human body develop as a result of the fusion of two extremely specialized cells: an oocyte and a sperm. This process is essential for the continuation of our species, as it ensures that parental genetic information is mixed and passed on from generation to generation. In addition to producing oocytes, the female reproductive system must provide the environment for the appropriate development of the fetus until birth. New genomic and computational tools offer unique opportunities to study the tight spatiotemporal regulatory mechanisms that are required for the cycle of human reproduction. This review explores how single-cell technologies have been used to build cellular atlases of the human reproductive system across the life span and how these maps have proven useful to better understand reproductive pathologies and dissect the heterogeneity of in vitro model systems.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.

Single-cell reconstruction of the early maternal-fetal interface in humans.

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.

DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes.

BACKGROUND: Inflammasomes are cytosolic multiprotein complexes in macrophages. They assemble after infection- or stress-associated stimuli, activating both caspase-1-mediated inflammatory cytokine secretion and pyroptosis. Increased inflammasome activity resulting from gene mutations is related to monogenic autoinflammatory syndromes. However, variable penetrance among patients with the same gene mutations suggests involvement of additional mechanisms associated with inflammasome gene regulation. OBJECTIVE: We sought to investigate the role of DNA demethylation in activating inflammasome genes during macrophage differentiation and monocyte activation in healthy control subjects and patients with autoinflammatory syndrome. METHODS: Inflammasome-related genes were tested for DNA methylation and mRNA levels by using bisulfite pyrosequencing and quantitative RT-PCR in monocytes in vitro differentiated to macrophages and exposed to inflammatory conditions. The contribution of Tet methylcytosine dioxygenase 2 (TET2) and nuclear factor κB to DNA demethylation was tested by using chromatin immunoprecipitation, small interfering RNA-mediated downregulation, and pharmacologic inhibition. RESULTS: We observed that inflammasome-related genes are rapidly demethylated in both monocyte-to-macrophage differentiation and on monocyte activation. Demethylation associates with increased gene expression, and both mechanisms are impaired when TET2 and nuclear factor κB are downregulated. We analyzed DNA methylation levels of inflammasome-related genes in patients with cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever, 2 archetypical monogenic autoinflammatory syndromes. Under the above conditions, monocytes from untreated patients with CAPS undergo more efficient DNA demethylation than those of healthy subjects. Interestingly, patients with CAPS treated with anti-IL-1 drugs display methylation levels similar to those of healthy control subjects. CONCLUSION: Our study is the first to demonstrate the involvement of DNA methylation-associated alterations in patients with monogenic autoinflammatory disease and opens up possibilities for novel clinical markers.

NF-κB directly mediates epigenetic deregulation of common microRNAs in Epstein-Barr virus-mediated transformation of B-cells and in lymphomas.

MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NF-kB pathway, common to all four situations. In fact, the NF-kB subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kB pathway impairs changes in miRNA expression, NF-kB binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kB dependent. Our results highlight the relevance of the NF-kB pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL.

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

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

Human uterine natural killer cells regulate differentiation of extravillous trophoblast early in pregnancy.

In humans, balanced invasion of trophoblast cells into the uterine mucosa, the decidua, is critical for successful pregnancy. Evidence suggests that this process is regulated by uterine natural killer (uNK) cells, but how they influence reproductive outcomes is unclear. Here, we used our trophoblast organoids and primary tissue samples to determine how uNK cells affect placentation. By locating potential interaction axes between trophoblast and uNK cells using single-cell transcriptomics and in vitro modeling of these interactions in organoids, we identify a uNK cell-derived cytokine signal that promotes trophoblast differentiation at the late stage of the invasive pathway. Moreover, it affects transcriptional programs involved in regulating blood flow, nutrients, and inflammatory and adaptive immune responses, as well as gene signatures associated with disorders of pregnancy such as pre-eclampsia. Our findings suggest mechanisms on how optimal immunological interactions between uNK cells and trophoblast enhance reproductive success.

Acute response to pathogens in the early human placenta at single-cell resolution.

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

The Virtual Child.

SUMMARY: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.