Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid ...Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.
Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from ...the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple ...sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.
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•Combined scRNA/TCR-seq reveals extensive heterogeneity of tissue Th17 cells•Th17 cells exhibit strong tissue-specific signatures•Stem-like SLAMF6+ and pathogenic CXCR6+ Th17 populations are induced in autoimmunity•IL-23 converts the stem-like IL-17+ to the pathogenic GM-CSF+ IFN-γ+ population
Stem-like Th17 cells that exert homeostatic effects in the intestine form a reservoir from which pathogenic Th17 cells can differentiate and contribute to neuroinflammatory pathology in experimental autoimmune encephalomyelitis.
Coordinated cell interactions within the esophagus maintain homeostasis, and disruption can lead to eosinophilic esophagitis (EoE), a chronic inflammatory disease with poorly understood pathogenesis. ...We profile 421,312 individual cells from the esophageal mucosa of 7 healthy and 15 EoE participants, revealing 60 cell subsets and functional alterations in cell states, compositions, and interactions that highlight previously unclear features of EoE. Active disease displays enrichment of ALOX15
macrophages, PRDM16
dendritic cells expressing the EoE risk gene ATP10A, and cycling mast cells, with concomitant reduction of T
17 cells. Ligand-receptor expression uncovers eosinophil recruitment programs, increased fibroblast interactions in disease, and IL-9
IL-4
IL-13
T
2 and endothelial cells as potential mast cell interactors. Resolution of inflammation-associated signatures includes mast and CD4
T
cell contraction and cell type-specific downregulation of eosinophil chemoattractant, growth, and survival factors. These cellular alterations in EoE and remission advance our understanding of eosinophilic inflammation and opportunities for therapeutic intervention.
Reciprocal interactions between host T helper cells and gut microbiota enforce local immunological tolerance and modulate extra-intestinal immunity. However, our understanding of antigen-specific ...tolerance to the microbiome is limited. Here, we developed a systematic approach to predict HLA class-II-specific epitopes using the humanized bacteria-originated T cell antigen (hBOTA) algorithm. We identified a diverse set of microbiome epitopes spanning all major taxa that are compatible with presentation by multiple HLA-II alleles. In particular, we uncovered an immunodominant epitope from the TonB-dependent receptor SusC that was universally recognized and ubiquitous among Bacteroidales. In healthy human subjects, SusC-reactive T cell responses were characterized by IL-10-dominant cytokine profiles, whereas in patients with active Crohn’s disease, responses were associated with elevated IL-17A. Our results highlight the potential of targeted antigen discovery within the microbiome to reveal principles of tolerance and functional transitions during inflammation.
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•Discovery of microbiome epitopes across all major taxa of the human gut microbiome•TonB-dependent receptor SusC contains a conserved and immunodominant epitope hotspot•SusC-specific T cell response shifts from IL-10 to IL-17A in active Crohn’s disease•SusC-specific intestinal T cells assume regulatory and effector roles in mice
The functional basis of tolerance to the gut microbiome is incompletely understood. Pedersen et al. develop an antigen-discovery approach to assess microbiota-directed T cell immunity in health and inflammation. They reveal the dynamic nature of T cell-mediated responses toward commensal Bacteroidales, highlighting inflammation-associated changes in Crohn’s disease.
Plasma cells (PCs) constitute a significant fraction of colonic mucosal cells and contribute to inflammatory infiltrates in ulcerative colitis (UC). While gut PCs secrete bacteria-targeting IgA ...antibodies, their role in UC pathogenesis is unknown. We performed single-cell V(D)J- and RNA-seq on sorted B cells from the colon of healthy individuals and patients with UC. A large fraction of B cell clones is shared between different colon regions, but inflammation in UC broadly disrupts this landscape, causing transcriptomic changes characterized by an increase in the unfolded protein response (UPR) and antigen presentation genes, clonal expansion, and isotype skewing from IgA1 and IgA2 to IgG1. We also directly expressed and assessed the specificity of 152 mAbs from expanded PC clones. These mAbs show low polyreactivity and autoreactivity and instead target both shared bacterial antigens and specific bacterial strains. Altogether, our results characterize the microbiome-specific colon PC response and how its disruption might contribute to inflammation in UC.
Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human ...melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.
Abstract
At homeostasis, most Th17 cells are found in the lamina propria of the intestine, where they contribute to tissue homeostasis by inhibiting microbiota from tissue invasion and promote ...barrier functions. Recent studies in humans and mice have implicated intestinal Th17 cells in extra-intestinal autoimmune diseases, including multiple sclerosis, but the mechanism in which intestinal Th17 cells mediate these dichotomous functions remains unknown. Here, we combined single-cell RNA- and TCR-seq (scRNA/TCR-seq) with fate mapping of tissue Th17 cells to profile over 84,000 tissue Th17 cells and characterize their heterogeneity, plasticity, and migration at homeostasis and during CNS autoimmunity. We discovered a homeostatic Th17 cell population, that is induced by the intestinal microbiota, is present in both lymphoid organs and the intestine, and expresses IL-17. Joint scRNA- and TCR-seq showed that during EAE this homeostatic population gives rise to a pathogenic Th17 cell population, that migrates specifically through the draining lymph nodes and the spleen to the CNS, and highly expresses GM-CSF and IFNγ, but expresses little or low levels of IL-17. This conversion of homeostatic Th17 cells into encephalitogenic Th17 cells depended on IL-23R signaling. Our work characterizes Th17 heterogeneity, plasticity, and migration during homeostasis and CNS autoimmune disease, identifies a mechanism by which intestinal Th17 cells influence the generation of an autoimmune disease-inducing population, and provides further evidence for a direct relationship between the IL-17-producing and GM-CSF- and IFNγ- producing pathogenic T cells.
Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol ...biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation.
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