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Germinal centers (GCs) are anatomic structures where B cells undergo affinity maturation, leading to production of high-affinity antibodies. The balance between T follicular helper (TFH) and regulatory (TFR) cells is critical for adequate control of GC responses. The study of human TFH and TFR cell development has been hampered because of the lack of in vitro assays reproducing in vivo biology, along with difficult access to healthy human lymphoid tissues. We used a single-cell transcriptomics approach to study the maturation of TFH and TFR cells isolated from human blood, iliac lymph nodes (LNs), and tonsils. As independent tissues have distinct proportions of follicular T cells in different maturation states, we leveraged the heterogeneity to reconstruct the maturation trajectory for human TFH and TFR cells. We found that the dominant maturation of TFR cells follows a bifurcated trajectory from precursor Treg cells, with one arm of the bifurcation leading to blood TFR cells and the other leading to the most mature GC TFR cells. Overall, our data provide a comprehensive resource for the transcriptomics of different follicular T cell populations and their dynamic relationship across different tissues.

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 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.

The advance of livestock towards the hydrographic basin of Mutum-Parana River, in the state of Rondonia contributes to the conversion of the amazon forest to pasture. This process can decrease the plant diversity, contributing to loss of endemic microorganisms and natural enemies leading to the replacement of native biota by non-native and generalist species. Thereby, we hypothesized that this conversion changes fungal composition, reduce fungal alpha diversity and community dissimilarity and increase the incidence of potential phytopathogenic genera at pasture soils compared to forest. Soil of 10 sampling points from forest and 10 from pasture were collected at the Mutum-Parana River basin. Via ITS amplicon sequencing of the total soil DNA, differences in composition and in the taxonomic diversity between the two environments were addressed. The phylum Ascomycota predominated in both forest and pasture. Basidiomycota presented lower percent in both, but was higher in pastures. Zygomycota and Glomeromycota presented opposite tendencies, the first being predominately present in forest and the second only present in pasture with low incidence. The fungal diversity was higher in pasture soils, contrasting our hypothesis. There were also significant correlations between soil physicochemical properties and fungal community. The reduction of the dissimilarity in pasture was confirmed and this signalized for a biotic homogenization. In addition, higher incidence of potential phytopathogenic fungi was observed in pasture. These results contribute to a better understanding of how fungal communities are driven by forest-to-pasture conversion.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response., We thank N. Eling, M. Fumagalli, Y. Gilad, O. Laufman, A. Marcovitz, J. Marioni, K. Meyer, M. Muffato, D. Odom, O. Stegle, A. Stern, M. Stubbington, V. Svensson and M. Ward for discussions; G. Emerton, A. Jinat, L. Mamanova, K. Polanski, A. Fullgrabe, N. George, S. Barnett, R. Boyd, S. Patel and C. Gomez for technical assistance; the Hipsci consortium for human fibroblast lines; and members of the Teichmann laboratory for support at various stages. This project was supported by ERC grants (ThDEFINE, ThSWITCH) and an EU FET-OPEN grant (MRG-GRAMMAR No 664918) and Wellcome Sanger core funding (Grant No WT206194). T.H. was supported by an HFSP Long-Term Fellowship and by EMBO Long-Term and Advanced fellowships. V.P. is funded by Fondazione Umberto Veronesi., info:eu-repo/semantics/publishedVersion

Fibroblast, epithelial and endothelial cells are more than just the scaffold of an organ — it emerges that they communicate with immune cells and are primed to launch organ-specific gene-expression programs for antiviral defence. Fibroblast, epithelial and endothelial cells aid organ-specific defence.

The genus Tulasnella often forms mycorrhizas with orchids and has worldwide distribution. Species of this genus are associated with a wide range of orchids, including endangered hosts. Initially, species identification relied mostly on morphological features and few cultures were preserved for later phylogenetic comparisons. In this study, a total of 50 Tulasnella isolates were collected from their natural sites in Minas Gerais, Brazil, cultured, and subjected to a phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Our results, based on phylogeny, integrated with nucleotide divergence and morphology, revealed the diversity of isolated Tulasnella species, which included four new species, namely, Tulasnella brigadeiroensis, Tulasnella hadrolaeliae, Tulasnella orchidis and Tulasnella zygopetali. The conservation of these species is important due to their association with endangered orchid hosts and endemic features in the Brazilian Atlantic Forest.

In recent years, several studies have been developed to understand the impact of fermentation on the final quality of coffee and have indicated that postharvest processing could be a determinant of quality. However, a trend has appeared as a scientific counterpoint, indicating that the interactions between soil, fruit, altitude, and slope exposures with respect to the Sun are important to understand the behavior of the microbiome in coffee. Studies on the microbiota of coffee have addressed its role during the fermentation process, however the knowledge of indigenous microorganisms harbored in fruits and soil of coffee trees growing in fields are essential, as they can contribute to fermentation. Therefore, the aim of this work was to evaluate the influence of topographic and edaphic factors on the bacterial and fungal communities present in the soil and in the fruits of Coffea arabica trees. Samples of fruits and soil were collected from different growing areas at different altitudes and soil conditions. The microbial DNA was extracted and sequenced. The results showed the contribution of environmental factors in the structure of bacterial and fungal communities. The richness, evenness and diversity of the mycobiome and bacteriome were higher in the soil than in the fruits, independent of altitude. In addition, coffee trees at higher altitudes tended to have more bacteria shared between the soil and fruits. The co-occurrence/co-exclusion network showed that bacteria-bacteria connections were greater in higher altitudes. On another hand, fungi-fungi and fungi-bacteria connections were higher in low altitudes. This was the first study that evaluates in deep the influence of environmental factors in the microbiota habiting fruits and soil coffee trees, which may affect the coffee beverage quality.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Summary Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation., Graphical Abstract, Highlights • scRNA-seq reveals Treg cell tissue-specific signatures • The phenotype of Treg cells shows progressive adaptation to barrier tissues • Treg cells share transcriptional dynamics of adaptation to colon and skin • Modules of peripheral Treg cell identity are conserved between mouse and human, Treg cells are known to display tissue-specific heterogeneity. Miragaia and colleagues probe Treg cells in lymphoid and barrier tissues by single-cell transcriptomics. They show that Treg cells segregate into subpopulations along a continuum of tissue adaptation and present conserved expression programs between homeostasis and disease and mouse and human.

Background The IRE1a-XBP1 pathway is a conserved adaptive mediator of the unfolded protein response. The pathway is indispensable for the development of secretory cells by facilitating protein folding and enhancing secretory capacity. In the immune system, it is known to function in dendritic cells, plasma cells, and eosinophil development and differentiation, while its role in T helper cell is unexplored. Here, we investigated the role of the IRE1a-XBP1 pathway in regulating activation and differentiation of type-2 T helper cell (Th2), a major T helper cell type involved in allergy, asthma, helminth infection, pregnancy, and tumor immunosuppression. Methods We perturbed the IRE1a-XBP1 pathway and interrogated its role in Th2 cell differentiation. We performed genome-wide transcriptomic analysis of differential gene expression to reveal IRE1a-XBP1 pathway-regulated genes and predict their biological role. To identify direct target genes of XBP1 and define XBP1’s regulatory network, we performed XBP1 ChIPmentation (ChIP-seq). We validated our predictions by flow cytometry, ELISA, and qPCR. We also used a fluorescent ubiquitin cell cycle indicator mouse to demonstrate the role of XBP1 in the cell cycle. Results We show that Th2 lymphocytes induce the IRE1a-XBP1 pathway during in vitro and in vivo activation. Genome-wide transcriptomic analysis of differential gene expression by perturbing the IRE1a-XBP1 pathway reveals XBP1-controlled genes and biological pathways. Performing XBP1 ChIPmentation (ChIP-seq) and integrating with transcriptomic data, we identify XBP1-controlled direct target genes and its transcriptional regulatory network. We observed that the IRE1a-XBP1 pathway controls cytokine secretion and the expression of two Th2 signature cytokines, IL13 and IL5. We also discovered that the IRE1a-XBP1 pathway facilitates activation-dependent Th2 cell proliferation by facilitating cell cycle progression through S and G2/M phase. Conclusions We confirm and detail the critical role of the IRE1a-XBP1 pathway during Th2 lymphocyte activation in regulating cytokine expression, secretion, and cell proliferation. Our high-quality genome-wide XBP1 ChIP and gene expression data provide a rich resource for investigating XBP1-regulated genes. We provide a browsable online database available at http://data.teichlab.org. Electronic supplementary material The online version of this article (10.1186/s13073-018-0589-3) contains supplementary material, which is available to authorized users.

The crucial capability of T cells for discrimination between self and non-self peptides is based on negative selection of developing thymocytes by medullary thymic epithelial cells (mTECs). The mTECs purge autoreactive T cells by expression of cell-type specific genes referred to as tissue-restricted antigens (TRAs). Although the autoimmune regulator (AIRE) protein is known to promote the expression of a subset of TRAs, its mechanism of action is still not fully understood. The expression of TRAs that are not under the control of AIRE also needs further characterization. Furthermore, expression patterns of TRA genes have been suggested to change over the course of mTEC development. Herein we have used single-cell RNA-sequencing to resolve patterns of TRA expression during mTEC development. Our data indicated that mTEC development consists of three distinct stages, correlating with previously described jTEC, mTEChi and mTEClo phenotypes. For each subpopulation, we have identified marker genes useful in future studies. Aire-induced TRAs were switched on during jTEC-mTEC transition and were expressed in genomic clusters, while otherwise the subsets expressed largely overlapping sets of TRAs. Moreover, population-level analysis of TRA expression frequencies suggested that such differences might not be necessary to achieve efficient thymocyte selection., RM is supported by a PhD Fellowship from the Fundação para a Ciência e Tecnologia, Portugal (SFRH/ BD/51950/2012). XZ is supported by an Advanced Postdoc Mobility Fellowship from the Swiss National Science Foundation (SNSF, grant number P300P2_151352). Part of the work was performed during XZ’s visit to the Simons Institute for the Theory of Computing. TL is supported by the Academy of Finland (Decision 311081). The authors would like to thank Bee Ling Ng and the staff of the Cytometry Core Facility, and Stephan Lorenz and the staff of the Single Cell Genomics Core Facility for their contribution. Mark Lynch is acknowledged for technical assistance with the Fluidigm C1 platform. Mike Stubbington and Kylie James are acknowledged for revising the language of the manuscript. We thank Sarah Teichmann for help and discussions regarding the manuscript., info:eu-repo/semantics/publishedVersion

A Digital Object Identifier (DOI) disponibilizada pela revista não corresponde. A troca de saberes tem grande importância para a ciência e estimula a dialética entre a razão e a emoção. Assim, surgiu o Núcleo de Estudos em Microbiologia Agrícola (NEMA) da Universidade Federal de Viçosa (UFV), com o objetivo de promover ações de ensino, pesquisa e extensão. O NEMA é subdividido em coordenações; essas despertam o voluntariado e a construção de um legado coletivo. O Dragon Dreaming tornou-se uma metodologia importante para a dinâmica do grupo, bem como os conceitos de Paulo Freire e Edgar Morin sobre conhecimento e respeito às diferenças. As atividades realizadas até o momento, como os Ciclos de Discussões em Bioinformática e Bioestatística, o Curso de Biossegurança, os Projetos "A importância da Microbiologia para a Agroecologia" e "Micro Cultural", são indicativos de avanços na integração do Departamento de Microbiologia e do nosso Programa de Pós-graduação e deste com a sociedade. Espera-se estimular o conhecimento crítico e ampliar as conexões entre ensino, pesquisa e extensão. The exchange of knowledges is very important for the science and stimulates the dialectic between reason and emotion. Therefore, the Nucleus of Studies in Agricultural Microbiology (NEMA) of the Universidade Federal de Viçosa (UFV) was created aiming to promote activities of teaching, research and extension activities. NEMA is subdivided into coordinations; arousing volunteerism and building a collective legacy. Dragon Dreaming has become an important methodology for the group dynamics, as does the concepts of Paulo Freire and Edgar Morin on the knowledge and respect for differences. Activities such as Cycles of Discussion in Bioinformatics and Biostatistics, Biosafety course, Projects "The Importance of Microbiology for Agroecology" and "Micro Cultural", are indicative of advances in the integration between members of the Department of Microbiology and our Graduation Program and also between this group and society. It is expected to stimulate critical knowledge and widen the connections between teaching, research and extension.

The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

The single-cell revolution is paving the way towards the molecular characterisation of every cell type in the human body, revealing relationships between cell types and states at high resolution. Changes in cellular phenotypes are particularly prevalent in the immune system and can be observed in its continuous remodelling up to adulthood, response to disease and development of immunological memory. In this review, we delve into the world of cellular dynamics of the immune system. We discuss current single-cell experimental and computational approaches in this area, giving insights into plasticity and commitment of cell fates. Finally, we provide an outlook on upcoming technological developments and predict how these will improve our understanding of the immune system.

An unexpected BDCA-2+CD123+CD1a+ DC subset comprises a major DC population in acute human sterile skin inflammation. scRNA-seq shows these to be activated DCs able to express markers normally associated with plasmacytoid DCs, prompting a re-evaluation of previous studies., Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) and are traditionally defined as being BDCA-2+CD123+. pDCs are not readily detectable in healthy human skin, but have been suggested to accumulate in wounds. Here, we describe a CD1a-bearing BDCA-2+CD123int DC subset that rapidly infiltrates human skin wounds and comprises a major DC population. Using single-cell RNA sequencing, we show that these cells are largely activated DCs acquiring features compatible with lymph node homing and antigen presentation, but unexpectedly express both BDCA-2 and CD123, potentially mimicking pDCs. Furthermore, a third BDCA-2–expressing population, Axl+Siglec-6+ DCs (ASDC), was also found to infiltrate human skin during wounding. These data demonstrate early skin infiltration of a previously unrecognized CD123intBDCA-2+CD1a+ DC subset during acute sterile inflammation, and prompt a re-evaluation of previously ascribed pDC involvement in skin disease.