The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling.

BACKGROUND: The Illumina family of Infinium Methylation BeadChip microarrays has been widely used over the last 15 years for genome-wide DNA methylation profiling, including large-scale and population-based studies, due to their ease of use and cost effectiveness. Succeeding the popular HumanMethylationEPIC BeadChip (EPICv1), the recently released Infinium MethylationEPIC v2.0 BeadChip (EPICv2) claims to extend genomic coverage to more than 935,000 CpG sites. Here, we comprehensively characterise the reproducibility, reliability and annotation of the EPICv2 array, based on bioinformatic analysis of both manifest data and new EPICv2 data from diverse biological samples. RESULTS: We find a high degree of reproducibility with EPICv1, evidenced by comparable sensitivity and precision from empirical cross-platform comparison incorporating whole genome bisulphite sequencing (WGBS), and high correlation between technical sample replicates, including between samples with DNA input levels below the manufacturer's recommendation. We provide a full assessment of probe content, evaluating genomic distribution and changes from previous array versions. We characterise EPICv2's new feature of replicated probes and provide recommendations as to the superior probes. In silico analysis of probe sequences demonstrates that probe cross-hybridisation remains a significant problem in EPICv2. By mapping the off-target sites at single nucleotide resolution and comparing with WGBS we show empirical evidence for preferential off-target binding. CONCLUSIONS: Overall, we find EPICv2 a worthy successor to the previous Infinium methylation microarrays, however some technical issues remain. To support optimal EPICv2 data analysis we provide an expanded version of the EPICv2 manifest to aid researchers in understanding probe design, data processing, choosing appropriate probes for analysis and for integration with methylation datasets from previous versions of the Infinium Methylation BeadChip.

Isolation and characterisation of PR3-specific B cells and their immunoglobulin sequences.

BACKGROUND: PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. OBJECTIVES: To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. METHODS: Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. RESULTS: We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. CONCLUSIONS: Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Comprehensive methylome sequencing reveals prognostic epigenetic biomarkers for prostate cancer mortality.

BACKGROUND: Prostate cancer is a clinically heterogeneous disease with a subset of patients rapidly progressing to lethal-metastatic prostate cancer. Current clinicopathological measures are imperfect predictors of disease progression. Epigenetic changes are amongst the earliest molecular changes in tumourigenesis. To find new prognostic biomarkers to enable earlier intervention and improved outcomes, we performed methylome sequencing of DNA from patients with localised prostate cancer and long-term clinical follow-up. METHODS: We used whole-genome bisulphite sequencing (WGBS) to comprehensively map and compare DNA methylation of radical prostatectomy tissue between patients with lethal disease (n = 7) and non-lethal (n = 8) disease (median follow-up 19.5 years). Validation of differentially methylated regions (DMRs) was performed in an independent cohort (n = 185, median follow-up 15 years) using targeted multiplex bisulphite sequencing of candidate regions. Survival was assessed via univariable and multivariable analyses including clinicopathological measures (log-rank and Cox regression models). RESULTS: WGBS data analysis identified cancer-specific methylation patterns including CpG island hypermethylation, and hypomethylation of repetitive elements, with increasing disease risk. We identified 1420 DMRs associated with prostate cancer-specific mortality (PCSM), which showed enrichment for gene sets downregulated in prostate cancer and de novo methylated in cancer. Through comparison with public prostate cancer datasets, we refined the DMRs to develop an 18-gene prognostic panel. Applying this panel to an independent cohort, we found significant associations between PCSM and hypermethylation at EPHB3, PARP6, TBX1, MARCH6 and a regulatory element within CACNA2D4. Strikingly in a multivariable model, inclusion of CACNA2D4 methylation was a better predictor of PCSM versus grade alone (Harrell's C-index: 0.779 vs. 0.684). CONCLUSIONS: Our study provides detailed methylome maps of non-lethal and lethal prostate cancer and identifies novel genic regions that distinguish these patient groups. Inclusion of our DNA methylation biomarkers with existing clinicopathological measures improves prognostic models of prostate cancer mortality, and holds promise for clinical application.

STAT5B restrains human B-cell differentiation to maintain humoral immune homeostasis.

BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

A metatranscriptomic approach to explore longitudinal tissue specimens from non-healing diabetes related foot ulcers.

Cellular mechanisms and/or microbiological interactions which contribute to chronic diabetes related foot ulcers (DRFUs) were explored using serially collected tissue specimens from chronic DRFUs and control healthy foot skin. Total RNA was isolated for next-generation sequencing. We found differentially expressed genes (DEGs) and enriched hallmark gene ontology biological processes upregulated in chronic DRFUs which primarily functioned in the host immune response including: (i) Inflammatory response; (ii) TNF signalling via NFKB; (iii) IL6 JAK-STAT3 signalling; (iv) IL2 STAT5 signalling and (v) Reactive oxygen species. A temporal analysis identified RN7SL1 signal recognition protein and IGHG4 immunoglobulin protein coding genes as being the most upregulated genes after the onset of treatment. Testing relative temporal changes between healing and non-healing DRFUs identified progressive upregulation in healed wounds of CXCR5 and MS4A1 (CD20), both canonical markers of lymphocytes (follicular B cells/follicular T helper cells and B cells, respectively). Collectively, our RNA-seq data provides insights into chronic DRFU pathogenesis.

Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint.

CD21low age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21low and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21low B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

Host-microbe metatranscriptome reveals differences between acute and chronic infections in diabetes-related foot ulcers.

Virtually all diabetes-related foot ulcers (DRFUs) will become colonized by microorganisms that may increase the risk of developing an infection. The reasons why some ulcerations develop acute clinical infections (AI-DRFUs) whilst others develop chronic infection (CI-DRFUs) and the preceding host-microbe interactions in vivo remain largely unknown. Establishing that acute and chronic infections are distinct processes requires demonstrating that these are two different strategies employed by microbes when interacting with a host. In this study, dual-RNA seq was employed to differentiate the host-microbe metatranscriptome between DRFUs that had localized chronic infection or acute clinical infection. Comparison of the host metatranscriptome in AI-DRFUs relative to CI-DRFUs identified upregulated differentially expressed genes (DEGs) that functioned as regulators of vascular lymphatic inflammatory responses, T-cell signalling and olfactory receptors. Conversely, CI-DRFUs upregulated DEGs responsible for cellular homeostasis. Gene set enrichment analysis using Hallmark annotations revealed enrichment of immune and inflammatory profiles in CI-DRFUs relative to AI-DRFUs. Analysis of the microbial metatranscriptome identified the DEGs being enriched within AI-DRFUs relative to CI-DRFUs included several toxins, two-component systems, bacterial motility, secretion systems and genes encoding for energy metabolism. Functions relevant to DRFU pathology were further explored, including biofilm and bacterial pathogenesis. This identified that the expression of biofilm-associated genes was higher within CI-DRFUs compared to that of AI-DRFUs, with mucR being the most highly expressed gene. Collectively, these data provide insights into the host-microbe function in two clinically-distinct infective phenotypes that affect DRFUs. The data reveal that bacteria in acutely infected DRFUs prioritize motility over biofilm and demonstrate greater pathogenicity and mechanisms, which likely subvert host cellular and immune pathways to establish infection. Upregulation of genes for key vascular inflammatory mediators in acutely infected ulcers may contribute, in part, to the clinical picture of a red, hot, swollen foot, which differentiates an acutely infected ulcer from that of a chronic infection.

Calling differentially methylated regions from whole genome bisulphite sequencing with DMRcate.

Whole genome bisulphite sequencing (WGBS) permits the genome-wide study of single molecule methylation patterns. One of the key goals of mammalian cell-type identity studies, in both normal differentiation and disease, is to locate differential methylation patterns across the genome. We discuss the most desirable characteristics for DML (differentially methylated locus) and DMR (differentially methylated region) detection tools in a genome-wide context and choose a set of statistical methods that fully or partially satisfy these considerations to compare for benchmarking. Our data simulation strategy is both biologically informed-employing distribution parameters derived from large-scale consortium datasets-and thorough. We report DML detection ability with respect to coverage, group methylation difference, sample size, variability and covariate size, both marginally and jointly, and exhaustively with respect to parameter combination. We also benchmark these methods on FDR control and computational time. We use this result to backend and introduce an expanded version of DMRcate: an existing DMR detection tool for microarray data that we have extended to now call DMRs from WGBS data. We compare DMRcate to a set of alternative DMR callers using a similarly realistic simulation strategy. We find DMRcate and RADmeth are the best predictors of DMRs, and conclusively find DMRcate the fastest.

Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

A multiomics approach to identify host-microbe alterations associated with infection severity in diabetic foot infections: a pilot study.

Diabetic foot infections (DFIs) are a major cause of hospitalization and can lead to lower extremity amputation. In this pilot study, we used a multiomics approach to explore the host-microbe complex within DFIs. We observed minimal differences in the overall microbial composition between PEDIS infection severities, however Staphylococcus aureus and Streptococcus genera were abundant and highly active in most mild to moderate DFIs. Further, we identified the significant enrichment of several virulence factors associated with infection pathogenicity belonging to both Staphylococcus aureus and Streptococcus. In severe DFIs, patients demonstrated a greater microbial diversity and differential gene expression demonstrated the enrichment of multispecies virulence genes suggestive of a complex polymicrobial infection. The host response in patients with severe DFIs was also significantly different as compared to mild to moderate DFIs. This was attributed to the enrichment of host genes associated with inflammation, acute phase response, cell stress and broad immune-related responses, while those associated with wound healing and myogenesis were significantly depleted.

Temporal ordering of omics and multiomic events inferred from time-series data.

Temporal changes in omics events can now be routinely measured; however, current analysis methods are often inadequate, especially for multiomics experiments. We report a novel analysis method that can infer event ordering at better temporal resolution than the experiment, and integrates omic events into two concise visualizations (event maps and sparklines). Testing our method gave results well-correlated with prior knowledge and indicated it streamlines analysis of time-series data.

Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Constitutively bound CTCF sites maintain 3D chromatin architecture and long-range epigenetically regulated domains.

The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The 'persistent' CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein (KLK) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture.

Methylome and transcriptome maps of human visceral and subcutaneous adipocytes reveal key epigenetic differences at developmental genes.

Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional differences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic differences and their contribution to cell type and depot-specific function. We found that DNA methylomes were notably distinct between different adipocyte depots and were associated with differential gene expression within pathways fundamental to adipocyte function. Most striking differential methylation was found at transcription factor and developmental genes. Our findings highlight the importance of developmental origins in the function of different fat depots.

Monozygotic twins and triplets discordant for amyotrophic lateral sclerosis display differential methylation and gene expression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons. ALS exhibits high phenotypic variability including age and site of onset, and disease duration. To uncover epigenetic and transcriptomic factors that may modify an ALS phenotype, we used a cohort of Australian monozygotic twins (n = 3 pairs) and triplets (n = 1 set) that are discordant for ALS and represent sporadic ALS and the two most common types of familial ALS, linked to C9orf72 and SOD1. Illumina Infinium HumanMethylation450K BeadChip, EpiTYPER and RNA-Seq analyses in these ALS-discordant twins/triplets and control twins (n = 2 pairs), implicated genes with consistent longitudinal differential DNA methylation and/or gene expression. Two identified genes, RAD9B and C8orf46, showed significant differential methylation in an extended cohort of >1000 ALS cases and controls. Combined longitudinal methylation-transcription analysis within a single twin set implicated CCNF, DPP6, RAMP3, and CCS, which have been previously associated with ALS. Longitudinal transcriptome data showed an 8-fold enrichment of immune function genes and under-representation of transcription and protein modification genes in ALS. Examination of these changes in a large Australian sporadic ALS cohort suggest a broader role in ALS. Furthermore, we observe that increased methylation age is a signature of ALS in older patients.

DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns.

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.

Evaluation of cross-platform and interlaboratory concordance via consensus modelling of genomic measurements.

MOTIVATION: A synoptic view of the human genome benefits chiefly from the application of nucleic acid sequencing and microarray technologies. These platforms allow interrogation of patterns such as gene expression and DNA methylation at the vast majority of canonical loci, allowing granular insights and opportunities for validation of original findings. However, problems arise when validating against a "gold standard" measurement, since this immediately biases all subsequent measurements towards that particular technology or protocol. Since all genomic measurements are estimates, in the absence of a "gold standard" we instead empirically assess the measurement precision and sensitivity of a large suite of genomic technologies via a consensus modelling method called the row-linear model. This method is an application of the American Society for Testing and Materials Standard E691 for assessing interlaboratory precision and sources of variability across multiple testing sites. Both cross-platform and cross-locus comparisons can be made across all common loci, allowing identification of technology- and locus-specific tendencies. RESULTS: We assess technologies including the Infinium MethylationEPIC BeadChip, whole genome bisulfite sequencing (WGBS), two different RNA-Seq protocols (PolyA+ and Ribo-Zero) and five different gene expression array platforms. Each technology thus is characterised herein, relative to the consensus. We showcase a number of applications of the row-linear model, including correlation with known interfering traits. We demonstrate a clear effect of cross-hybridisation on the sensitivity of Infinium methylation arrays. Additionally, we perform a true interlaboratory test on a set of samples interrogated on the same platform across twenty-one separate testing laboratories. AVAILABILITY AND IMPLEMENTATION: A full implementation of the row-linear model, plus extra functions for visualisation, are found in the R package consensus at https://github.com/timpeters82/consensus. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.