The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an ...atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design.
This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific.
In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.
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.
Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated ...responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.
We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13−30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn’s disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction.
Circulating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40−4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4+ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls.
In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.
To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated ...variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3′ end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.
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•Capture Hi-C maps intra- and interchromosomal contacts of the immunoglobulin heavy chain•Polymer modeling shows thousands of different Igh structures that underpin diversity•The Ig loci participate in a stage-specific interchromosomal network with B lineage genes
Mielczarek et al. use Capture Hi-C to obtain a detailed map of chromosomal interactions within the immunoglobulin heavy-chain locus. Polymer modeling shows that each Igh structure is unique, enabling antibody diversity. The Ig loci associate with key B lineage genes in a developmental-stage-specific interchromosomal network.
The Human Cell Atlas Regev, Aviv; Teichmann, Sarah A; Lander, Eric S ...
eLife,
12/2017, Letnik:
6
Journal Article
Recenzirano
Odprti dostop
The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to ...identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.
γδ T cells are generally considered innate-like lymphocytes, however, an “adaptive-like” γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, ...we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αβ T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct “innate-effector” transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.
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•Vδ1+ T cells undergo transcriptional reprogramming from Tnaive to Teffector status•Vδ1+ Teffector cells are highly transcriptionally similar to CD8+ TEMRA cells•CD3 ligation of Vδ1+ Tnaive cells induces Teffector-linked transcription factors•Infections drive transition from a Vδ1+ Tnaive to Teffector transcriptional profile
Using single-cell transcriptomics, TCR repertoire analysis, ATAC-seq, and immunophenotyping, McMurray et al. show naive Vδ1+ T cells can undergo transcriptional reprogramming to an effector state extremely similar to CD8 TEMRA cells. Infections, including CMV and malaria, drive both clonotypic Vδ1+ T cell expansion and differentiation to this highly conserved effector program.
Most empirical metacommunity studies rely solely on morphological identification of taxa, precluding the species-level identification of several biotic groups, which can influence the ...characterization of metacommunities. DNA metabarcoding enables inference of species and even intraspecific diversity from community samples but has rarely been used to infer metacommunity structure. Here, we combined morphology and metabarcoding to improve the characterization of an insect metacommunity at different identification levels. We included measures of taxonomic, functional and phylogenetic richness, and we evaluated drivers affecting metacommunity structure (i.e., environmental filtering and dispersal). Communities were sampled from an area that included nine perennial, two near-perennial and two intermittent sites in a river network characterized by high hydrological variability. We identified organisms to a mixed (family to species) taxonomic level using morphology, and to operational taxonomic unit (OTU) and haplotype levels using metabarcoding of the mitochondrial cytochrome c oxidase gene. Diptera and Ephemeroptera showed the greatest increases in taxonomic and phylogenetic richness but not biological trait richness with increasing taxonomic resolution. The joint effect of environmental filtering and dispersal was more important than their individual effects in shaping metacommunity structure at all identification levels. Mixed-level and OTU-level identification were more effective than family and haplotype in characterizing the drivers of metacommunity structure. We demonstrate that the greater taxonomic resolution enabled by metabarcoding could improve understanding of metacommunities within river networks, thus enhancing our capacity to predict ecological responses in ecosystems adapting to global change.
•We explored aquatic assemblages as dry-phase indicators of health in intermittent rivers.•Diatom and plant communities differed at impacted and control sites during dry phases.•Communities at ...unimpacted sites were more diverse and supported more indicator taxa.•Diatom, plant and invertebrate indices used during wet phases varied among dry sites.•Exploring terrestrial assemblages as dry-phase biomonitors is a research priority.
Intermittent rivers and ephemeral streams (IRES) are dynamic ecosystems that shift between aquatic and terrestrial states. IRES are widespread, abundant and increasing in extent, but developing biomonitoring programmes to determine their ecological quality is challenging. To date, quality assessments have focused on the aquatic organisms present during wet phases, whereas dry-phase communities remain poorly characterized. We examined multiple biotic groups present in dry IRES channels, to compare assemblages at sites impacted and unimpacted by human activity and to evaluate the potential of each group as an ecological quality indicator. We explored existing, unpublished data for three biotic groups: an aquatic microflora (diatoms), an aquatic fauna (the invertebrate ‘seedbank’), and a mixed flora (aquatic and terrestrial plants); notably, we did not source data for terrestrial assemblages with high potential to act as indicators. Diatom and plant assemblage composition differed between impacted and unimpacted sites, and the latter assemblages were more diverse and included more indicator taxa. Invertebrate seedbank taxa richness was higher at unimpacted sites but compositional differences were not detected, probably due to the coarse taxonomic resolution to which abundant taxa were identified. Performance of standard indices of ecological quality was variable, but differences were identified between impacted and unimpacted conditions for all biotic groups. Our results can inform the enhancement of biomonitoring programmes designed to characterize IRES ecological quality in relation to legislative targets. We highlight the need to integrate wet- and dry-phase survey data in holistic quality assessments. Although we suggest diatoms, aquatic plants and the aquatic invertebrate seedbank as having the potential to inform assessment of dry-phase ecological quality, we highlight the need for research to further characterize these aquatic groups and, crucially, to explore terrestrial assemblages with high potential to act as dry-phase quality indicators.
Protecting U.S. temporary waterways Marshall, Jonathan C; Acuña, Vicenç; Allen, Daniel C ...
Science (American Association for the Advancement of Science),
08/2018, Letnik:
361, Številka:
6405
Journal Article
PDF
