PD-1 regulates ILC3-driven intestinal immunity and homeostasis.

Interleukin-(IL) 22 production by intestinal group 3 innate lymphoid cells (ILC3) is critical to maintain gut homeostasis. However, IL-22 needs to be tightly controlled; reduced IL-22 expression is associated with intestinal epithelial barrier defect while its overexpression promotes tumor development. Here, using a single-cell ribonucleic acid sequencing approach, we identified a core set of genes associated with increased IL-22 production by ILC3. Among these genes, programmed cell death 1 (PD-1), extensively studied in the context of cancer and chronic infection, was constitutively expressed on a subset of ILC3. These cells, found in the crypt of the small intestine and colon, displayed superior capacity to produce IL-22. PD-1 expression on ILC3 was dependent on the microbiota and was induced during inflammation in response to IL-23 but, conversely, was reduced in the presence of Notch ligand. PD-1+ ILC3 exhibited distinct metabolic activity with increased glycolytic, lipid, and polyamine synthesis associated with augmented proliferation compared with their PD-1- counterparts. Further, PD-1+ ILC3 showed increased expression of mitochondrial antioxidant proteins which enable the cells to maintain their levels of reactive oxygen species. Loss of PD-1 signaling in ILC3 led to reduced IL-22 production in a cell-intrinsic manner. During inflammation, PD-1 expression was increased on natural cytotoxicity receptor (NCR)- ILC3 while deficiency in PD-1 expression resulted in increased susceptibility to experimental colitis and failure to maintain gut barrier integrity. Collectively, our findings uncover a new function of the PD-1 and highlight the role of PD-1 signaling in the maintenance of gut homeostasis mediated by ILC3 in mice.

A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

Benchmarking single-cell hashtag oligo demultiplexing methods.

Sample multiplexing is often used to reduce cost and limit batch effects in single-cell RNA sequencing (scRNA-seq) experiments. A commonly used multiplexing technique involves tagging cells prior to pooling with a hashtag oligo (HTO) that can be sequenced along with the cells' RNA to determine their sample of origin. Several tools have been developed to demultiplex HTO sequencing data and assign cells to samples. In this study, we critically assess the performance of seven HTO demultiplexing tools: hashedDrops, HTODemux, GMM-Demux, demuxmix, deMULTIplex, BFF (bimodal flexible fitting) and HashSolo. The comparison uses data sets where each sample has also been demultiplexed using genetic variants from the RNA, enabling comparison of HTO demultiplexing techniques against complementary data from the genetic 'ground truth'. We find that all methods perform similarly where HTO labelling is of high quality, but methods that assume a bimodal count distribution perform poorly on lower quality data. We also suggest heuristic approaches for assessing the quality of HTO counts in an scRNA-seq experiment.

Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification.

Hematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain-containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells.

A three-stage developmental pathway for human Vγ9Vδ2 T cells within the postnatal thymus.

Vγ9Vδ2 T cells are the largest population of γδ T cells in adults and can play important roles in providing effective immunity against cancer and infection. Many studies have suggested that peripheral Vγ9Vδ2 T cells are derived from the fetal liver and thymus and that the postnatal thymus plays little role in the development of these cells. More recent evidence suggested that these cells may also develop postnatally in the thymus. Here, we used high-dimensional flow cytometry, transcriptomic analysis, functional assays, and precursor-product experiments to define the development pathway of Vγ9Vδ2 T cells in the postnatal thymus. We identify three distinct stages of development for Vγ9Vδ2 T cells in the postnatal thymus that are defined by the progressive acquisition of functional potential and major changes in the expression of transcription factors, chemokines, and other surface markers. Furthermore, our analysis of donor-matched thymus and blood revealed that the molecular requirements for the development of functional Vγ9Vδ2 T cells are delivered predominantly by the postnatal thymus and not in the periphery. Tbet and Eomes, which are required for IFN-γ and TNFα expression, are up-regulated as Vγ9Vδ2 T cells mature in the thymus, and mature thymic Vγ9Vδ2 T cells rapidly express high levels of these cytokines after stimulation. Similarly, the postnatal thymus programs Vγ9Vδ2 T cells to express the cytolytic molecules, perforin, granzyme A, and granzyme K. This study provides a greater understanding of how Vγ9Vδ2 T cells develop in humans and may lead to opportunities to manipulate these cells to treat human diseases.

Modeling group heteroscedasticity in single-cell RNA-seq pseudo-bulk data.

Group heteroscedasticity is commonly observed in pseudo-bulk single-cell RNA-seq datasets and its presence can hamper the detection of differentially expressed genes. Since most bulk RNA-seq methods assume equal group variances, we introduce two new approaches that account for heteroscedastic groups, namely voomByGroup and voomWithQualityWeights using a blocked design (voomQWB). Compared to current gold-standard methods that do not account for group heteroscedasticity, we show results from simulations and various experiments that demonstrate the superior performance of voomByGroup and voomQWB in terms of error control and power when group variances in pseudo-bulk single-cell RNA-seq data are unequal.

A method for stabilising the XX karyotype in female mESC cultures.

Female mouse embryonic stem cells (mESCs) present differently from male mESCs in several fundamental ways; however, complications with their in vitro culture have resulted in an under-representation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female and male mESCs apart. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state that is near indistinguishable from male mESCs. If female pluripotency is to be studied effectively in this system, it is crucial that high-quality cultures of XX mESCs are available. Here, we report a method for better maintaining XX female mESCs in culture that also stabilises the male karyotype and makes study of female-specific pluripotency more feasible.

Early introduction of high-intensity eccentric loading into hamstring strain injury rehabilitation.

OBJECTIVES: This study aimed to investigate the number of days following hamstring strain injury (HSI) taken to introduce high-intensity eccentric loading (HIEL) into rehabilitation based on exercise-specific progression criteria, and whether pain resolution during isometric knee flexion strength testing occurred before or after this milestone. DESIGN: Cohort study. METHODS: We included 42 men (mean ±â€¯sd; age = 26 ±â€¯5 years; height = 181 ±â€¯8 cm; mass = 86 ±â€¯12 kg) with HSIs, who performed fully supervised rehabilitation twice per week until they met return to play clearance criteria. Isometric knee flexion strength testing was completed before every rehabilitation session and HIEL was introduced via the Nordic hamstring exercise and unilateral slider once participants could perform a bilateral slider through full eccentric knee flexion range of motion. We reported the median (IQR) number of days following HSI taken to introduce HIEL, along with participant's pain rating during isometric knee flexion strength testing before that rehabilitation session. We also reported the median (IQR) number of days following HSI taken for participants to achieve pain resolution during isometric knee flexion. RESULTS: HIEL was introduced 5 (2-8) days following HSI, despite 35/42 participants reporting pain during isometric knee flexion strength testing immediately prior to that rehabilitation session, which was rated as 3.5 (3-5) on a 0-10 numeric rating scale. Pain resolution during isometric knee flexion strength testing was achieved 11 (9-13) days following HSI. CONCLUSION: HIEL can be safely introduced into early HSI rehabilitation based on exercise-specific progression criteria, without needing to wait for pain resolution during isometric knee flexion strength testing before doing so.

Epigenetic modifier SMCHD1 maintains a normal pool of long-term hematopoietic stem cells.

SMCHD1 (structural maintenance of chromosomes hinge domain containing 1) is a noncanonical SMC protein that mediates long-range repressive chromatin structures. SMCHD1 is required for X chromosome inactivation in female cells and repression of imprinted and clustered autosomal genes, with SMCHD1 mutations linked to human diseases facioscapulohumeral muscular dystrophy (FSHD) and bosma arhinia and micropthalmia syndrome (BAMS). We used a conditional mouse model to investigate SMCHD1 in hematopoiesis. Smchd1-deleted mice maintained steady-state hematopoiesis despite showing an impaired reconstitution capacity in competitive bone marrow transplantations and age-related hematopoietic stem cell (HSC) loss. This phenotype was more pronounced in Smchd1-deleted females, which showed a loss of quiescent HSCs and fewer B cells. Gene expression profiling of Smchd1-deficient HSCs and B cells revealed known and cell-type-specific SMCHD1-sensitive genes and significant disruption to X-linked gene expression in female cells. These data show SMCHD1 is a regulator of HSCs whose effects are more profound in females.

BAF complex-mediated chromatin relaxation is required for establishment of X chromosome inactivation.

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.

A family study implicates GBE1 in the etiology of autism spectrum disorder.

Autism spectrum disorders (ASD) are neurodevelopmental disorders with an estimated heritability of >60%. Family-based genetic studies of ASD have generally focused on multiple small kindreds, searching for de novo variants of major effect. We hypothesized that molecular genetic analysis of large multiplex families would enable the identification of variants of milder effects. We studied a large multigenerational family of European ancestry with multiple family members affected with ASD or the broader autism phenotype (BAP). We identified a rare heterozygous variant in the gene encoding 1,4-ɑ-glucan branching enzyme 1 (GBE1) that was present in seven of seven individuals with ASD, nine of ten individuals with the BAP, and none of four tested unaffected individuals. We genotyped a community-acquired cohort of 389 individuals with ASD and identified three additional probands. Cascade analysis demonstrated that the variant was present in 11 of 13 individuals with familial ASD/BAP and neither of the two tested unaffected individuals in these three families, also of European ancestry. The variant was not enriched in the combined UK10K ASD cohorts of European ancestry but heterozygous GBE1 deletion was overrepresented in large ASD cohorts, collectively suggesting an association between GBE1 and ASD.

Benchmarking UMI-based single-cell RNA-seq preprocessing workflows.

BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) technologies and associated analysis methods have rapidly developed in recent years. This includes preprocessing methods, which assign sequencing reads to genes to create count matrices for downstream analysis. While several packaged preprocessing workflows have been developed to provide users with convenient tools for handling this process, how they compare to one another and how they influence downstream analysis have not been well studied. RESULTS: Here, we systematically benchmark the performance of 10 end-to-end preprocessing workflows (Cell Ranger, Optimus, salmon alevin, alevin-fry, kallisto bustools, dropSeqPipe, scPipe, zUMIs, celseq2, and scruff) using datasets yielding different biological complexity levels generated by CEL-Seq2 and 10x Chromium platforms. We compare these workflows in terms of their quantification properties directly and their impact on normalization and clustering by evaluating the performance of different method combinations. While the scRNA-seq preprocessing workflows compared vary in their detection and quantification of genes across datasets, after downstream analysis with performant normalization and clustering methods, almost all combinations produce clustering results that agree well with the known cell type labels that provided the ground truth in our analysis. CONCLUSIONS: In summary, the choice of preprocessing method was found to be less important than other steps in the scRNA-seq analysis process. Our study comprehensively compares common scRNA-seq preprocessing workflows and summarizes their characteristics to guide workflow users.

NanoMethViz: An R/Bioconductor package for visualizing long-read methylation data.

A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. The lack of R/Bioconductor tools for the effective visualization of nanopore methylation profiles between samples from different experimental groups led us to develop the NanoMethViz R package. Our software can handle methylation output generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the sample-level, we use dimensionality reduction to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across individual reads along the genome using the spaghetti plot and heatmaps, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at https://bioconductor.org/packages/NanoMethViz.

Zinc Supplementation with or without Additional Micronutrients Does Not Affect Peripheral Blood Gene Expression or Serum Cytokine Level in Bangladeshi Children.

Preventive zinc supplementation provided as a stand-alone dispersible tablet, or via home fortification as multiple micronutrient powders (MNPs), has been considered a potential strategy to prevent zinc deficiency and improve health (including immune) outcomes among children in low- and middle-income countries. However, the impact of zinc supplementation on immune profiles has not been well characterized. We sought to define the effect of zinc supplementation on peripheral blood gene expression and cytokine levels among young children in Dhaka, Bangladesh. In a sub-study of a large randomized, controlled, community-based efficacy trial where children 9-11 months of age received one of the following interventions on a daily basis for 24 weeks: (1) MNPs containing 10 mg of zinc; (2) dispersible tablet containing 10 mg zinc; or (3) placebo powder, we used RNA sequencing to profile the peripheral blood gene expression, as well as highly sensitive multiplex assays to detect cytokine profiles. We profiled samples from 100 children enrolled in the parent trial (zinc MNPs 28, zinc tablets 39, placebo 33). We did not detect an effect from either zinc intervention on differential peripheral blood gene expression at the end of the intervention, or an effect from the intervention on changes in gene expression from baseline. We also did not detect an effect from either intervention on cytokine concentrations. Exploratory analysis did not identify an association between undernutrition (defined as stunting, underweight or wasting) and peripheral blood gene expression. Zinc interventions in children did not produce a gene expression or cytokine signature in the peripheral blood. However, this study demonstrates a proof of principle that sensitive multi-omic techniques can be applied to samples collected in field studies.

Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design.

BACKGROUND: Pooling cells from multiple biological samples prior to library preparation within the same single-cell RNA sequencing experiment provides several advantages, including lower library preparation costs and reduced unwanted technological variation, such as batch effects. Computational demultiplexing tools based on natural genetic variation between individuals provide a simple approach to demultiplex samples, which does not require complex additional experimental procedures. However, to our knowledge these tools have not been evaluated in cancer, where somatic variants, which could differ between cells from the same sample, may obscure the signal in natural genetic variation. RESULTS: Here, we performed in silico benchmark evaluations by combining raw sequencing reads from multiple single-cell samples in high-grade serous ovarian cancer, which has a high copy number burden, and lung adenocarcinoma, which has a high tumor mutational burden. Our results confirm that genetic demultiplexing tools can be effectively deployed on cancer tissue using a pooled experimental design, although high proportions of ambient RNA from cell debris reduce performance. CONCLUSIONS: This strategy provides significant cost savings through pooled library preparation. To facilitate similar analyses at the experimental design phase, we provide freely accessible code and a reproducible Snakemake workflow built around the best-performing tools found in our in silico benchmark evaluations, available at https://github.com/lmweber/snp-dmx-cancer.

The histone acetyltransferase HBO1 promotes efficient tip cell sprouting during angiogenesis.

Blood vessel growth and remodelling are essential during embryonic development and disease pathogenesis. The diversity of endothelial cells (ECs) is transcriptionally evident and ECs undergo dynamic changes in gene expression during vessel growth and remodelling. Here, we investigated the role of the histone acetyltransferase HBO1 (KAT7), which is important for activating genes during development and for histone H3 lysine 14 acetylation (H3K14ac). Loss of HBO1 and H3K14ac impaired developmental sprouting angiogenesis and reduced pathological EC overgrowth in the retinal endothelium. Single-cell RNA sequencing of retinal ECs revealed an increased abundance of tip cells in Hbo1-deficient retinas, which led to EC overcrowding in the retinal sprouting front and prevented efficient tip cell migration. We found that H3K14ac was highly abundant in the endothelial genome in both intra- and intergenic regions, suggesting that HBO1 acts as a genome organiser that promotes efficient tip cell behaviour necessary for sprouting angiogenesis. This article has an associated 'The people behind the papers' interview.

Human brain region-specific variably methylated regions are enriched for heritability of distinct neuropsychiatric traits.

BACKGROUND: DNA methylation dynamics in the brain are associated with normal development and neuropsychiatric disease and differ across functionally distinct brain regions. Previous studies of genome-wide methylation differences among human brain regions focus on limited numbers of individuals and one to two brain regions. RESULTS: Using GTEx samples, we generate a resource of DNA methylation in purified neuronal nuclei from 8 brain regions as well as lung and thyroid tissues from 12 to 23 donors. We identify differentially methylated regions between brain regions among neuronal nuclei in both CpG (181,146) and non-CpG (264,868) contexts, few of which were unique to a single pairwise comparison. This significantly expands the knowledge of differential methylation across the brain by 10-fold. In addition, we present the first differential methylation analysis among neuronal nuclei from basal ganglia tissues and identify unique CpG differentially methylated regions, many associated with ion transport. We also identify 81,130 regions of variably CpG methylated regions, i.e., variable methylation among individuals in the same brain region, which are enriched in regulatory regions and in CpG differentially methylated regions. Many variably methylated regions are unique to a specific brain region, with only 202 common across all brain regions, as well as lung and thyroid. Variably methylated regions identified in the amygdala, anterior cingulate cortex, and hippocampus are enriched for heritability of schizophrenia. CONCLUSIONS: These data suggest that epigenetic variation in these particular human brain regions could be associated with the risk for this neuropsychiatric disorder.

Repeatability and sensitivity to change of non-invasive end points in PAH: the RESPIRE study.

End points that are repeatable and sensitive to change are important in pulmonary arterial hypertension (PAH) for clinical practice and trials of new therapies. In 42 patients with PAH, test-retest repeatability was assessed using the intraclass correlation coefficient and treatment effect size using Cohen's d statistic. Intraclass correlation coefficients demonstrated excellent repeatability for MRI, 6 min walk test and log to base 10 N-terminal pro-brain natriuretic peptide (log10NT-proBNP). The treatment effect size for MRI-derived right ventricular ejection fraction was large (Cohen's d 0.81), whereas the effect size for the 6 min walk test (Cohen's d 0.22) and log10NT-proBNP (Cohen's d 0.20) were fair. This study supports further evaluation of MRI as a non-invasive end point for clinical assessment and PAH therapy trials.Trial registration number NCT03841344.