Psoriasis is a chronic inflammatory disease with a strong genetic component that can be triggered by environmental factors. Disease pathogenesis is mainly driven by type 1 and type 17 cytokine-producing cells which, in healthy individuals, are modulated by regulatory T cells (Tregs). Tregs play a fundamental role in immune homeostasis and contribute to the prevention of autoimmune disease by suppressing immune responses. In psoriasis, Tregs are impaired in their suppressive function leading to an altered T-helper 17/Treg balance. Although Treg dysfunction in patients with psoriasis is associated with disease exacerbation, it is unknown how they are functionally regulated. In this review, we discuss recent insights into Tregs in the setting of psoriasis with an emphasis on the effect of current treatments on Tregs and how already available therapeutics that modulate Treg frequency or functionality could be exploited for treatment of psoriasis.

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC 50 : 1.4 nM) and interferon-α (IC 50 : 28 IU/ml, but max. 60–80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC 50 : 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.

33Japan 34 35 ABSTRACT 37 Genome-wide association studies (GWAS) have identified over 150,000 links between 38 common genetic variants and human traits or complex diseases. Over 80% of these 39 associations map to polymorphisms in non-coding DNA. Therefore, the challenge is 40 to identify disease-causing variants, the genes they affect, and the cells in which 41 these effects occur. We have developed a platform using ATAC-seq, DNaseI 42 footprints, NG Capture-C and machine learning to address this challenge. Applying 43 this approach to red blood cell traits identifies a significant proportion of known 44 causative variants and their effector genes, which we show can be validated by direct 45 in vivo modelling.Identification of the variation of the genome that determines the risk of common chronic and 48 infectious diseases informs on their primary causes, which leads to preventative or 49 therapeutic approaches and insights. Whilst genome-wide association studies (GWASs) 50 have identified thousands of chromosome regions 1 , the identification of the causal genes, 51 variants and cell types remains a major bottleneck. This is due to three major features of the 52 genome and its complex association with disease susceptibility. Trait-associated variants 53 are often tightly associated, through linkage disequilibrium (LD), with tens or hundreds of 54 other variants, mostly single-nucleotide polymorphisms (SNPs), any one or more of which 55 could be causal; the majority (>85%) the variants identified in GWAS lie within the non-56 coding genome 2 . Although non-coding regions are increasingly well annotated, many 57 variants do not correspond to known regulatory elements, and even when they do, it is rarely 58 known which genes these elements control, and in which cell types. New technical 59 approaches to link variants to the genes they control are rapidly improving but are often 60 limited by their sensitivity and resolution [3][4][5][6] ; and because so few causal variants have been 61 unequivocally linked to the genes they affect, the mechanisms by which non-coding variants 62 alter gene expression remain unknown in all but a few cases; and, third, the complexity of 63 gene regulation and cell/cell interactions means that knowing when in development, in which 64 cell type, in which activation state, and within which pathway(s) a causal variant exerts its 65 effect is usually impossible to predict. Although significant progress is being made, currently, 66 none of these problems has been adequately solved. 68Here, we have developed an integrated platform of experimental and computational 69 methods to prioritise likely causal variants, link them to the genes they regulate, and 70 determine the mechanism by which they alter gene function. To illustrate the approach we 71 have initially focussed on a single haematopoietic lineage: the development of mature red 72 blood cells (RBC), for which all stages of lineage specification and differentiation from a 73 haematopoietic stem cell to a RBC are known, and can be r...

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The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.

Group A Streptococcus (GAS) infection is associated with multiple clinical sequelae, including different subtypes of psoriasis. Such post-streptococcal disorders have been long known but are largely unexplained. CD1a is expressed at constitutively high levels by Langerhans cells and presents lipid antigens to T cells, but the potential relevance to GAS infection has not been studied. Here, we investigated whether GAS-responsive CD1a-restricted T cells contribute to the pathogenesis of psoriasis. Healthy individuals had high frequencies of circulating and cutaneous GAS-responsive CD4 + and CD8 + T cells with rapid effector functions, including the production of interleukin-22 (IL-22). Human skin and blood single-cell CITE-seq analyses of IL-22–producing T cells showed a type 17 signature with proliferative potential, whereas IFN-γ–producing T cells displayed cytotoxic T lymphocyte characteristics. Furthermore, individuals with psoriasis had significantly higher frequencies of circulating GAS-reactive T cells, enriched for markers of activation, cytolytic potential, and tissue association. In addition to responding to GAS, subsets of expanded GAS-reactive T cell clones/lines were found to be autoreactive, which included the recognition of the self-lipid antigen lysophosphatidylcholine. CD8 + T cell clones/lines produced cytolytic mediators and lysed infected CD1a-expressing cells. Furthermore, we established cutaneous models of GAS infection in a humanized CD1a transgenic mouse model and identified enhanced and prolonged local and systemic inflammation, with resolution through a psoriasis-like phenotype. Together, these findings link GAS infection to the CD1a pathway and show that GAS infection promotes the proliferation and activation of CD1a-autoreactive T cells, with relevance to post-streptococcal disease, including the pathogenesis and treatment of psoriasis.

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