Developmental dynamics of two bipotent thymic epithelial progenitor types.

T cell development in the thymus is essential for cellular immunity and depends on the organotypic thymic epithelial microenvironment. In comparison with other organs, the size and cellular composition of the thymus are unusually dynamic, as exemplified by rapid growth and high T cell output during early stages of development, followed by a gradual loss of functional thymic epithelial cells and diminished naive T cell production with age1-10. Single-cell RNA sequencing (scRNA-seq) has uncovered an unexpected heterogeneity of cell types in the thymic epithelium of young and aged adult mice11-18; however, the identities and developmental dynamics of putative pre- and postnatal epithelial progenitors have remained unresolved1,12,16,17,19-27. Here we combine scRNA-seq and a new CRISPR-Cas9-based cellular barcoding system in mice to determine qualitative and quantitative changes in the thymic epithelium over time. This dual approach enabled us to identify two principal progenitor populations: an early bipotent progenitor type biased towards cortical epithelium and a postnatal bipotent progenitor population biased towards medullary epithelium. We further demonstrate that continuous autocrine provision of Fgf7 leads to sustained expansion of thymic microenvironments without exhausting the epithelial progenitor pools, suggesting a strategy to modulate the extent of thymopoietic activity.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

Foxn1 is not essential for T-cell development in teleosts.

In mammals, T-cell development depends on the activity of the Foxn1 transcription factor in the thymic epithelium; mutations in the vertebrate-specific Foxn1 gene are associated with profound T-cell lymphopenia and fatal immunodeficiency. Here, we examined the extent of T-cell development in teleosts lacking a functional foxn1 gene. In zebrafish carrying a deleterious internal deletion of foxn1, reduced but robust lymphopoietic activity is maintained in the mutant thymus. Moreover, pseudogenization or loss of foxn1 in the genomes of deep-sea anglerfishes is independent of the presence or absence of the canonical signatures of the T-cell lineage. Thus, in contrast to the situation in mammals, the teleost thymus can support foxn1-independent lymphopoiesis, most likely through the activity of the Foxn4, an ancient metazoan paralog of Foxn1. Our results imply that during the early stages of vertebrate evolution, genetic control of thymopoiesis was functionally redundant and thus robust; in mammals, the genetic network was reorganized to become uniquely dependent on the FOXN1 transcription factor.

A survey of the adaptive immune genes of the polka-dot batfish Ogcocephalus cubifrons.

BACKGROUND: The anglerfish, belonging to the teleost order Lophiiformes, are a diverse and species-rich group of fish that are known to exhibit a number of unique morphological, reproductive and immunological adaptations. Work to date has identified the loss of specific adaptive immune components in two of the five Lophiiformes sub-orders (Lophioidei and Ceratioidei), while no anomalies have been identified to date in two other sub-orders, Antennaroidei and Chaunacoidei. The immunogenome of the fifth sub-order, Ogcocephaloidei has not yet been investigated, and we have therefore used whole genome shotgun sequencing, combined with RNA-seq, to survey the adaptive immune capabilities of the polka-dot batfish, O. cubifrons, as a representative of this as yet unexplored sub-order. RESULTS: We find that the O. cubifrons genome encodes the core genes needed to mount adaptive T and B cell responses. These genes include those necessary for rearranging and editing antigen receptors, the antigen receptors themselves; as well as the co-receptors, signalling molecules, and antigen presenting molecules (both class I and class II) needed for B cell and T cell development and activation. CONCLUSIONS: From an immune perspective, the polka-dot batfish has a canonical complement of adaptive immune genes, and does not exhibit any of the adaptive immune changes previously identified in monkfish and oceanic anglerfish.

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing.

The rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

ReadItAndKeep: rapid decontamination of SARS-CoV-2 sequencing reads.

SUMMARY: Viral sequence data from clinical samples frequently contain contaminating human reads, which must be removed prior to sharing for legal and ethical reasons. To enable host read removal for SARS-CoV-2 sequencing data on low-specification laptops, we developed ReadItAndKeep, a fast lightweight tool for Illumina and nanopore data that only keeps reads matching the SARS-CoV-2 genome. Peak RAM usage is typically below 10 MB, and runtime less than 1 min. We show that by excluding the polyA tail from the viral reference, ReadItAndKeep prevents bleed-through of human reads, whereas mapping to the human genome lets some reads escape. We believe our test approach (including all possible reads from the human genome, human samples from each of the 26 populations in the 1000 genomes data and a diverse set of SARS-CoV-2 genomes) will also be useful for others. AVAILABILITY AND IMPLEMENTATION: ReadItAndKeep is implemented in C++, released under the MIT license, and available from https://github.com/GenomePathogenAnalysisService/read-it-and-keep. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proximal Lck Promoter-Driven Cre Function Is Limited in Neonatal and Ineffective in Adult γδ T Cell Development.

During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αß and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αß T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αß T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.

Reconciling the Potentially Irreconcilable? Genotypic and Phenotypic Amoxicillin-Clavulanate Resistance in Escherichia coli.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). A total of 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity, 23% [78/339]) but improved when genetic features associated with penicillinase hyperproduction (e.g., promoter mutations and copy number estimates) were considered (sensitivity, 82% [277/339]; P < 0.0001). Most discrepancies occurred in isolates with MICs within ±1 doubling dilution of the breakpoint. We investigated two potential causes: the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

Elephant shark genome provides unique insights into gnathostome evolution.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices.

BACKGROUND: Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections but potentially useful for any microbial sequencing, and compare detection by direct-from-clinical-sample metagenomic nanopore sequencing with Illumina sequencing and standard microbiological diagnostic techniques. RESULTS: DNA was extracted from the sonication fluids of seven explanted orthopaedic devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (> 90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing. CONCLUSIONS: We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data and use of this pipeline to show initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. The high proportion of human DNA in prosthetic joint infection extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling. The nine samples sequenced in this pilot study have shown a proof of concept for sequencing and analysis that will enable us to investigate further sequencing to improve specificity and sensitivity.

Autoimmunity associated with chemically induced thymic dysplasia.

Autoimmune and inflammatory conditions are frequent complications in patients with reduced numbers of T cells. Here, we describe a mouse model of thymic stromal dysplasia resulting in peripheral T-cell lymphopenia. In Foxn1:CFP-NTR transgenic mice, the bacterial nitroreductase enzyme is expressed in thymic epithelial cells and converts the prodrug CB1954 into a cytotoxic agent. This strategy enables titratable and durable destruction of thymopoietic tissue in early embryogenesis. Our results indicate that the resulting low levels of thymic capacity for T-cell production create a predisposition for the development of a complex autoimmune syndrome, chiefly characterized by inflammatory bowel disease and lymphocytic organ infiltrations. We conclude that the Foxn1:CFP-NTR transgenic mouse strain represents a suitable animal model to optimize established clinical protocols, such as thymus transplantation, to correct various forms of thymic dysplasia and to explore novel treatment options.

Everything in its place.

In this issue of Immunity, Griffith et al. (2009) define the thymic midcortex as a functionally inert zone between subcapsular and cortico-medullary regions, and Ehrlich et al. (2009) infer that structural features of the cortex and medulla regulate migration of thymocytes.

Quantitative fat and R2* mapping in vivo to measure lipid-rich necrotic core and intraplaque hemorrhage in carotid atherosclerosis.

PURPOSE: The aim of this work was to quantify the extent of lipid-rich necrotic core (LRNC) and intraplaque hemorrhage (IPH) in atherosclerotic plaques. METHODS: Patients scheduled for carotid endarterectomy underwent four-point Dixon and T1-weighted magnetic resonance imaging (MRI) at 3 Tesla. Fat and R2* maps were generated from the Dixon sequence at the acquired spatial resolution of 0.60 × 0.60 × 0.70 mm voxel size. MRI and three-dimensional (3D) histology volumes of plaques were registered. The registration matrix was applied to segmentations denoting LRNC and IPH in 3D histology to split plaque volumes in regions with and without LRNC and IPH. RESULTS: Five patients were included. Regarding volumes of LRNC identified by 3D histology, the average fat fraction by MRI was significantly higher inside LRNC than outside: 12.64 ± 0.2737% versus 9.294 ± 0.1762% (mean ± standard error of the mean [SEM]; P < 0.001). The same was true for IPH identified by 3D histology, R2* inside versus outside IPH was: 71.81 ± 1.276 s-1 versus 56.94 ± 0.9095 s-1 (mean ± SEM; P < 0.001). There was a strong correlation between the cumulative fat and the volume of LRNC from 3D histology (R2 = 0.92) as well as between cumulative R2* and IPH (R2 = 0.94). CONCLUSION: Quantitative mapping of fat and R2* from Dixon MRI reliably quantifies the extent of LRNC and IPH. Magn Reson Med 78:285-296, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Evolution of lymphoid tissues.

Lymphoid organs are integral parts of all vertebrate adaptive immune systems. Primary lymphoid tissues exhibit a remarkable functional dichotomy: T cells develop in specialized thymopoietic tissues located in the pharynx, whereas B cells develop in distinct areas of general hematopoietic areas, such as the kidney or bone marrow. Among secondary lymphoid tissues, the spleen is present in all vertebrates, whereas lymph nodes represent an innovation particular to mammals and some birds. A comparative analysis of anatomical, functional and genomic features thus reveals the core components of adaptive immune systems. Such information has guided recent attempts at reconstructing lymphopoietic functions in vivo and in the future might inspire the development of new strategies for medical interventions restoring and modulating immune functions.

Adaptive immunity maintains occult cancer in an equilibrium state.

The capacity of immunity to control and shape cancer, that is, cancer immunoediting, is the result of three processes that function either independently or in sequence: elimination (cancer immunosurveillance, in which immunity functions as an extrinsic tumour suppressor in naive hosts); equilibrium (expansion of transformed cells is held in check by immunity); and escape (tumour cell variants with dampened immunogenicity or the capacity to attenuate immune responses grow into clinically apparent cancers). Extensive experimental support now exists for the elimination and escape processes because immunodeficient mice develop more carcinogen-induced and spontaneous cancers than wild-type mice, and tumour cells from immunodeficient mice are more immunogenic than those from immunocompetent mice. In contrast, the equilibrium process was inferred largely from clinical observations, including reports of transplantation of undetected (occult) cancer from organ donor into immunosuppressed recipients. Herein we use a mouse model of primary chemical carcinogenesis and demonstrate that equilibrium occurs, is mechanistically distinguishable from elimination and escape, and that neoplastic cells in equilibrium are transformed but proliferate poorly in vivo. We also show that tumour cells in equilibrium are unedited but become edited when they spontaneously escape immune control and grow into clinically apparent tumours. These results reveal that, in addition to destroying tumour cells and sculpting tumour immunogenicity, the immune system of a naive mouse can also restrain cancer growth for extended time periods.

Thymopoiesis in mice depends on a Foxn1-positive thymic epithelial cell lineage.

The thymus is essential for T-cell development. Here, we focus on the role of the transcription factor Foxn1 in the development and function of thymic epithelial cells (TECs) of the mouse. TECs are of endodermal origin; they initially express Foxn1 and give rise to orthotopic (thoracic) and additional (cervical) thymi. Using Foxn1-directed cytoablation, we show that during embryogenesis, cervical thymi develop a few days after the thoracic lobes, and that bipotent epithelial progenitors of cortical and medullary compartments express Foxn1. We also show that following acute selective near-total ablation during embryogenesis, complete regeneration of TECs does not occur, providing an animal model for human thymic aplasia syndromes. Finally, we address the functional role of Foxn1-negative TECs that arise postnatally in the mouse. Lineage tracing shows that such Foxn1-negative TECs are descendants of Foxn1-positive progenitors; furthermore, Foxn1-directed subacute intoxication of TECs by polyglutamine-containing EGFP proteins indicates that a presumptive Foxn1-independent lineage does not contribute to thymopoietic function of the adult thymus. Our findings therefore support the notion that Foxn1 is the essential transcription factor regulating the differentiation of TECs and that its expression marks the major functional lineage of TECs in embryonic and adult thymic tissue.

Penicillin Binding Protein Substitutions Cooccur with Fluoroquinolone Resistance in Epidemic Lineages of Multidrug-Resistant Clostridioides difficile.

Clostridioides difficile remains a key cause of healthcare-associated infection, with multidrug-resistant (MDR) lineages causing high-mortality (≥20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship is a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species, this is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins [PBPs]). Here, we investigated five C. difficile transpeptidases (PBP1 to PBP5) for recent substitutions, associated cephalosporin MICs, and co-occurrence with fluoroquinolone resistance. Previously published genome assemblies (n = 7,096) were obtained, representing 16 geographically widespread lineages, including healthcare-associated ST1(027). Recent amino acid substitutions were found within PBP1 (n = 50) and PBP3 (n = 48), ranging from 1 to 10 substitutions per genome. ß-Lactam MICs were measured for closely related pairs of wild-type and PBP-substituted isolates separated by 20 to 273 single nucleotide polymorphisms (SNPs). Recombination-corrected phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1 to 4 doubling dilutions >wild-type, up to 1,506 µg/mL. Substitution patterns varied by lineage and clade, showed geographic structure, and occurred post-1990, coincident with the gyrA and/or gyrB substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. Their co-occurrence with fluoroquinolone resistance hinders attempts to understand the relative importance of these drugs in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. IMPORTANCE Fluoroquinolone and cephalosporin use in healthcare settings has triggered outbreaks of high-mortality, multidrug-resistant C. difficile infection. Here, we identify a mechanism associated with raised cephalosporin MICs in C. difficile comprising amino acid substitutions in two cell wall transpeptidase enzymes (penicillin binding proteins). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed that substitutions associated with raised cephalosporin and fluoroquinolone MICs were co-acquired immediately before clinically important outbreak strains emerged. PBP substitutions were geographically structured within genetic lineages, suggesting adaptation to local antimicrobial prescribing. Antimicrobial stewardship of cephalosporins and fluoroquinolones is an effective means of C. difficile outbreak control. Genetic changes associated with raised MIC may impart a "fitness cost" after antibiotic withdrawal. Our study therefore identifies a mechanism that may explain the contribution of cephalosporin stewardship to resolving outbreak conditions. However, due to the co-occurrence of raised cephalosporin MICs and fluoroquinolone resistance, further work is needed to determine the relative importance of each.