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The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific facilities. Pre-prints by Institute authors can be viewed on the Institute's bioRxiv channel. We believe that free and open access to the outputs of publicly‐funded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.
 

Contursi A, Schiavone S, Dovizio M, Hinz C, Fullone R, Tacconelli S, Tyrrell VJ, Grande R, Lanuti P, Marchisio M, Zucchelli M, Ballerini P, Lanas A, O'Donnell VB, Patrignani P Signalling

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.

+view abstract Journal of lipid research , PMID: 34428433

Hall BS, Dos Santos SJ, Hsieh LT, Manifava M, Ruf MT, Pluschke G, Ktistakis N, Simmonds RE Signalling

The exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease. ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

+view abstract Autophagy , PMID: 34424124

Linterman MA, Denton AE Immunology

Inhibition of ferroptosis via selenium supplementation promotes the survival of follicular helper T cells, boosting the germinal center and antibody response following vaccination in mice and people.

+view abstract Nature immunology , PMID: 34413522

Pantarelli C, Pan D, Chetwynd S, Stark AK, Hornigold K, Machin P, Crossland L, Cleary SJ, Baker MJ, Hampson E, Mandel A, Segonds-Pichon A, Walker R, van 't Veer C, Riffo-Vasquez Y, Okkenhaug K, Pitchford S, Welch HCE Signalling , Bioinformatics

Streptococcal pneumonia is a worldwide health problem that kills ∼2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein-coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophil-mediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli-induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species-dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections.

+view abstract Blood advances , PMID: 34402884

Freire-Pritchett P, Ray-Jones H, Della Rosa M, Eijsbouts CQ, Orchard WR, Wingett SW, Wallace C, Cairns J, Spivakov M, Malysheva V Bioinformatics

Capture Hi-C is widely used to obtain high-resolution profiles of chromosomal interactions involving, at least on one end, regions of interest such as gene promoters. Signal detection in Capture Hi-C data is challenging and cannot be adequately accomplished with tools developed for other chromosome conformation capture methods, including standard Hi-C. Capture Hi-C Analysis of Genomic Organization (CHiCAGO) is a computational pipeline developed specifically for Capture Hi-C analysis. It implements a statistical model accounting for biological and technical background components, as well as bespoke normalization and multiple testing procedures for this data type. Here we provide a step-by-step guide to the CHiCAGO workflow that is aimed at users with basic experience of the command line and R. We also describe more advanced strategies for tuning the key parameters for custom experiments and provide guidance on data preprocessing and downstream analysis using companion tools. In a typical experiment, CHiCAGO takes ~2-3 h to run, although pre- and postprocessing steps may take much longer.

+view abstract Nature protocols , PMID: 34373652

Köfeler HC, Eichmann TO, Ahrends R, Bowden JA, Danne-Rasche N, Dennis EA, Fedorova M, Griffiths WJ, Han X, Hartler J, Holčapek M, Jirásko R, Koelmel JP, Ejsing CS, Liebisch G, Ni Z, O'Donnell VB, Quehenberger O, Schwudke D, Shevchenko A, Wakelam MJO, Wenk MR, Wolrab D, Ekroos K Signalling , Lipidomics

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+view abstract Nature communications , PMID: 34362906

Lê Cao KA, Abadi AJ, Davis-Marcisak EF, Hsu L, Arora A, Coullomb A, Deshpande A, Feng Y, Jeganathan P, Loth M, Meng C, Mu W, Pancaldi V, Sankaran K, Singh A, Sodicoff JS, Stein-O'Brien GL, Subramanian A, Welch JD, You Y, Argelaguet R, Carey VJ, Dries R, Greene CS, Holmes S, Love MI, Ritchie ME, Yuan GC, Culhane AC, Fertig E Epigenetics

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+view abstract Genome biology , PMID: 34353350

Rayon T, Maizels RJ, Barrington C, Briscoe J Epigenetics

The spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly distinct neuronal subtypes generated in a characteristic spatiotemporal arrangement from progenitors in the embryonic neural tube. To gain insight into the diversity and complexity of cells in the developing human neural tube, we used single-cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie stages (CS) CS12, CS14, CS17 and CS19 from gestational weeks 4-7. Analysis of progenitor and neuronal populations from the neural tube and dorsal root ganglia identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with mouse revealed overall similarity of mammalian neural tube development while highlighting some human-specific features. These data provide a catalogue of gene expression and cell type identity in the human neural tube that will support future studies of sensory and motor control systems. The data can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

+view abstract Development , PMID: 34351410

Li Z, Ktistakis NT Signalling

Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements: the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.

+view abstract Methods in cell biology , PMID: 34311864

Hopkins EL, Gu W, Kobe B, Coleman MP

Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

+view abstract Frontiers in molecular biosciences , PMID: 34307460

Hopkins EL, Gu W, Kobe B, Coleman MP

Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

+view abstract Frontiers in molecular biosciences , PMID: 34307460

Calderón L, Schindler K, Malin SG, Schebesta A, Sun Q, Schwickert T, Alberti C, Fischer M, Jaritz M, Tagoh H, Ebert A, Minnich M, Liston A, Cochella L, Busslinger M Immunology

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving -targeting microRNAs. Additional PTEN loss in double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

+view abstract Science immunology , PMID: 34301800

Junius S, Mavrogiannis AV, Lemaitre P, Gerbaux M, Staels F, Malviya V, Burton O, Gergelits V, Singh K, Tito Tadeo RY, Raes J, Humblet-Baron S, Liston A, Schlenner SM Immunology

Regulatory T cells (T) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. T can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (T) characteristics, a process referred to as T plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that T stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of T after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the T population, with the nonconverting majority of T being resistant to plasticity upon secondary stability challenge. The unstable T fraction is a complex mixture of phenotypically distinct T, enriched for naïve and neuropilin-1-negative T, and includes peripherally induced T and recent thymic emigrant T These results suggest that a "purging" process can be used to purify stable T that are capable of robust fate retention, with potential implications for improving cell transfer therapy.

+view abstract Science immunology , PMID: 34301799

Prescott JA, Mitchell JP, Cook SJ Signalling

Cells must adapt to changes in their environment to maintain cell, tissue and organismal integrity in the face of mechanical, chemical or microbiological stress. Nuclear factor-κB (NF-κB) is one of the most important transcription factors that controls inducible gene expression as cells attempt to restore homeostasis. It plays critical roles in the immune system, from acute inflammation to the development of secondary lymphoid organs, and also has roles in cell survival, proliferation and differentiation. Given its role in such critical processes, NF-κB signalling must be subject to strict spatiotemporal control to ensure measured and context-specific cellular responses. Indeed, deregulation of NF-κB signalling can result in debilitating and even lethal inflammation and also underpins some forms of cancer. In this review, we describe the homeostatic feedback mechanisms that limit and 're-set' inducible activation of NF-κB. We first describe the key components of the signalling pathways leading to activation of NF-κB, including the prominent role of protein phosphorylation and protein ubiquitylation, before briefly introducing the key features of feedback control mechanisms. We then describe the array of negative feedback loops targeting different components of the NF-κB signalling cascade including controls at the receptor level, post-receptor signalosome complexes, direct regulation of the critical 'inhibitor of κB kinases' (IKKs) and inhibitory feedforward regulation of NF-κB-dependent transcriptional responses. We also review post-transcriptional feedback controls affecting RNA stability and translation. Finally, we describe the deregulation of these feedback controls in human disease and consider how feedback may be a challenge to the efficacy of inhibitors.

+view abstract The Biochemical journal , PMID: 34269817

Capatina N, Hemberger M, Burton GJ, Watson ED, Yung HW

Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1 ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number, developmental delay, and is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1 heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecologic conditions. ABSTRACT: Women with pre-gestational health conditions (e.g., obesity, diabetes) or gynaecological problems (e.g., endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, preeclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPR ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1 mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPR pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPR pathway inhibitors rescued this effect. While the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1 blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1 homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1 homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1 heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population, and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecologic conditions. This article is protected by copyright. All rights reserved.

+view abstract The Journal of physiology , PMID: 34269420

Kumari R, Roy U, Desai S, Nilavar NM, Van Nieuwenhuijze A, Paranjape A, Radha G, Bawa P, Srivastava M, Nambiar M, Balaji KN, Liston A, Choudhary B, Raghavan SC Immunology

Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes. We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer.

+view abstract Cell reports , PMID: 34260911

Baizan-Edge A, Stubbs BA, Stubbington MJT, Bolland DJ, Tabbada K, Andrews S, Corcoran AE Immunology , Bioinformatics

Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences V gene selection during V-to-DJ recombination. We find skewing toward 3' V genes during de novo V-to-DJ recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in D-to-J recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of D and V antisense transcription in IL-7Rα B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.

+view abstract Cell reports , PMID: 34260907

Durgan J, Florey O Signalling

Atg8-family protein lipidation is the most commonly used marker for monitoring autophagy. During macroautophagy, Atg8-family proteins are specifically conjugated to phosphatidylethanolamine (PE) in forming, double-membrane autophagosomes. A distinct, non-canonical autophagy pathway also operates, characterized by the Conjugation of ATG8s to endolysosomal Single Membranes (CASM). In our new study, we show that CASM is associated with the alternative conjugation of Atg8-family proteins to phosphatidylserine (PS), and PE, in response to various cellular stimuli. We also discover differences in the regulation of conjugation to PE and PS by ATG4s, and altered dynamics between the two species. The identification of alternative Atg8-family protein PS lipidation opens up exciting new questions on the roles, regulation and biology of Atg8-family proteins during non-canonical autophagy.

+view abstract Autophagy , PMID: 34251968

Eisenbarth SC, Baumjohann D, Craft J, Fazilleau N, Ma CS, Tangye SG, Vinuesa CG, Linterman MA Immunology

T follicular helper (Tfh) cells cognately guide differentiation of antigen-primed B cells in secondary lymphoid tissues. 'Tfh-like' populations not expressing the canonical Tfh cell transcription factor BCL6 have also been described, which can aid particular aspects of B cell differentiation. Tfh and Tfh-like cells are essential for protective and pathological humoral immunity. These CD4 T cells that help B cells are polarized to produce diverse combinations of cytokines and chemokine receptors and can be grouped into distinct subsets that promote antibodies of different isotype, affinity, and duration, according to the nature of immune challenge. However, unified nomenclature to describe the distinct functional Tfh and Tfh-like cells does not exist. While explicitly acknowledging cellular plasticity, we propose categorizing these cell states into three groups based on phenotype and function, paired with their anatomical site of action.

+view abstract Trends in immunology , PMID: 34244056

Vanderbeke L, Van Mol P, Van Herck Y, De Smet F, Humblet-Baron S, Martinod K, Antoranz A, Arijs I, Boeckx B, Bosisio FM, Casaer M, Dauwe D, De Wever W, Dooms C, Dreesen E, Emmaneel A, Filtjens J, Gouwy M, Gunst J, Hermans G, Jansen S, Lagrou K, Liston A, Lorent N, Meersseman P, Mercier T, Neyts J, Odent J, Panovska D, Penttila PA, Pollet E, Proost P, Qian J, Quintelier K, Raes J, Rex S, Saeys Y, Sprooten J, Tejpar S, Testelmans D, Thevissen K, Van Buyten T, Vandenhaute J, Van Gassen S, Velásquez Pereira LC, Vos R, Weynand B, Wilmer A, Yserbyt J, Garg AD, Matthys P, Wouters C, Lambrechts D, Wauters E, Wauters J Immunology

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

+view abstract Nature communications , PMID: 34226537

Monzón-Casanova E, Bates KJ, Smith CWJ, Turner M

The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self-reactivity. At the molecular level this is regulated by signalling from membrane immunoglobulin and the BAFF-receptor which sustain a pro-survival programme of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here we show that the polypyrimidine tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high-density of binding to 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the maturation of quiescent B cells by regulating gene expression at the post-transcriptional level. This article is protected by copyright. All rights reserved.

+view abstract European journal of immunology , PMID: 34214192

Canovas S, Ivanova E, Hamdi M, Perez-Sanz F, Rizos D, Kelsey G, Coy P Epigenetics

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25-25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.

+view abstract International journal of molecular sciences , PMID: 34204008

Fra-Bido S, Walker SA, Innocentin S, Linterman MA Immunology , Imaging

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).

+view abstract STAR protocols , PMID: 34195671

Collier DA, Ferreira IATM, Kotagiri P, Datir R, Lim E, Touizer E, Meng B, Abdullahi A, , Elmer A, Kingston N, Graves B, Le Gresley E, Caputo D, Bergamaschi L, Smith KGC, Bradley JR, Ceron-Gutierrez L, Cortes-Acevedo P, Barcenas-Morales G, Linterman MA, McCoy L, Davis C, Thomson E, Lyons PA, McKinney E, Doffinger R, Wills M, Gupta RK Immunology

Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, data are scarce on vaccine efficacy against variants of concern (VOC) in individuals above 80 years of age. Here we analysed immune responses following vaccination with mRNA vaccine BNT162b2 in elderly participants and younger health care workers. Serum neutralisation and binding IgG/IgA after the first vaccine dose diminished with increasing age, with a marked drop in participants over 80 years old. Sera from participants above 80 showed significantly lower neutralisation potency against B.1.1.7, B.1.351 and P.1. variants of concern as compared to wild type and were more likely to lack any neutralisation against VOC following the first dose. However, following the second dose, neutralisation against VOC was detectable regardless of age. Frequency of SARS-CoV-2 Spike specific B-memory cells was higher in elderly responders versus non-responders after first dose. Elderly participants demonstrated clear reduction in somatic hypermutation of class switched cells. SARS-CoV-2 Spike specific T- cell IFNγ and IL-2 responses decreased with increasing age, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high risk population that warrant specific measures to boost vaccine responses, particularly where variants of concern are circulating.

+view abstract Nature , PMID: 34192737