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    SARS-CoV-2 Beta variant infection elicits potent lineage-specific and cross-reactive antibodies. Reincke S Momsen,Yuan Meng,Kornau Hans-Christian,Corman Victor M,van Hoof Scott,Sánchez-Sendin Elisa,Ramberger Melanie,Yu Wenli,Hua Yuanzi,Tien Henry,Schmidt Marie Luisa,Schwarz Tatjana,Jeworowski Lara Maria,Brandl Sarah E,Rasmussen Helle Foverskov,Homeyer Marie A,Stöffler Laura,Barner Martin,Kunkel Désirée,Huo Shufan,Horler Johannes,von Wardenburg Niels,Kroidl Inge,Eser Tabea M,Wieser Andreas,Geldmacher Christof,Hoelscher Michael,Gänzer Hannes,Weiss Günter,Schmitz Dietmar,Drosten Christian,Prüss Harald,Wilson Ian A,Kreye Jakob Science (New York, N.Y.) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Beta variant of concern (VOC) resists neutralization by major classes of antibodies from COVID-19 patients and vaccinated individuals. In this study, serum of Beta-infected patients revealed reduced cross-neutralization of wild-type virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of VOC-specific clonotypes and accommodation of mutations present in Beta and Omicron into a major antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with wild type-elicited antibodies, including a public VH1-58 clonotype that targets the RBD ridge. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift, with implications for design of next-generation vaccines and therapeutics. 10.1126/science.abm5835
    A lymphocyte-microglia-astrocyte axis in chronic active multiple sclerosis. Nature Multiple sclerosis (MS) lesions that do not resolve in the months after they form harbour ongoing demyelination and axon degeneration, and are identifiable in vivo by their paramagnetic rims on MRI scans. Here, to define mechanisms underlying this disabling, progressive neurodegenerative state and foster development of new therapeutic agents, we used MRI-informed single-nucleus RNA sequencing to profile the edge of demyelinated white matter lesions at various stages of inflammation. We uncovered notable glial and immune cell diversity, especially at the chronically inflamed lesion edge. We define 'microglia inflamed in MS' (MIMS) and 'astrocytes inflamed in MS', glial phenotypes that demonstrate neurodegenerative programming. The MIMS transcriptional profile overlaps with that of microglia in other neurodegenerative diseases, suggesting that primary and secondary neurodegeneration share common mechanisms and could benefit from similar therapeutic approaches. We identify complement component 1q (C1q) as a critical mediator of MIMS activation, validated immunohistochemically in MS tissue, genetically by microglia-specific C1q ablation in mice with experimental autoimmune encephalomyelitis, and therapeutically by treating chronic experimental autoimmune encephalomyelitis with C1q blockade. C1q inhibition is a potential therapeutic avenue to address chronic white matter inflammation, which could be monitored by longitudinal assessment of its dynamic biomarker, paramagnetic rim lesions, using advanced MRI methods. 10.1038/s41586-021-03892-7
    The emergent landscape of the mouse gut endoderm at single-cell resolution. Nature Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points-before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates and as they spatially converged within the nascent gut endoderm, which revealed these cells to be globally similar but retain aspects of their lineage history. We observed the regionalized identity of cells along the anterior-posterior axis of the emergent gut tube, which reflects their embryonic or extra-embryonic origin, and the coordinated patterning of these cells into organ-specific territories. 10.1038/s41586-019-1127-1
    Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Liu Lihong,Wang Pengfei,Nair Manoj S,Yu Jian,Rapp Micah,Wang Qian,Luo Yang,Chan Jasper F-W,Sahi Vincent,Figueroa Amir,Guo Xinzheng V,Cerutti Gabriele,Bimela Jude,Gorman Jason,Zhou Tongqing,Chen Zhiwei,Yuen Kwok-Yung,Kwong Peter D,Sodroski Joseph G,Yin Michael T,Sheng Zizhang,Huang Yaoxing,Shapiro Lawrence,Ho David D Nature The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2. 10.1038/s41586-020-2571-7
    Immune-evasive human islet-like organoids ameliorate diabetes. Yoshihara Eiji,O'Connor Carolyn,Gasser Emanuel,Wei Zong,Oh Tae Gyu,Tseng Tiffany W,Wang Dan,Cayabyab Fritz,Dai Yang,Yu Ruth T,Liddle Christopher,Atkins Annette R,Downes Michael,Evans Ronald M Nature Islets derived from stem cells hold promise as a therapy for insulin-dependent diabetes, but there remain challenges towards achieving this goal. Here we generate human islet-like organoids (HILOs) from induced pluripotent stem cells and show that non-canonical WNT4 signalling drives the metabolic maturation necessary for robust ex vivo glucose-stimulated insulin secretion. These functionally mature HILOs contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic NOD/SCID mice. Overexpression of the immune checkpoint protein programmed death-ligand 1 (PD-L1) protected HILO xenografts such that they were able to restore glucose homeostasis in immune-competent diabetic mice for 50 days. Furthermore, ex vivo stimulation with interferon-γ induced endogenous PD-L1 expression and restricted T cell activation and graft rejection. The generation of glucose-responsive islet-like organoids that are able to avoid immune detection provides a promising alternative to cadaveric and device-dependent therapies in the treatment of diabetes. 10.1038/s41586-020-2631-z
    Publisher Correction: Immune-evasive human islet-like organoids ameliorate diabetes. Yoshihara Eiji,O'Connor Carolyn,Gasser Emanuel,Wei Zong,Oh Tae Gyu,Tseng Tiffany W,Wang Dan,Cayabyab Fritz,Dai Yang,Yu Ruth T,Liddle Christopher,Atkins Annette R,Downes Michael,Evans Ronald M Nature 10.1038/s41586-020-03109-3
    DNA methylation atlas of the mouse brain at single-cell resolution. Liu Hanqing,Zhou Jingtian,Tian Wei,Luo Chongyuan,Bartlett Anna,Aldridge Andrew,Lucero Jacinta,Osteen Julia K,Nery Joseph R,Chen Huaming,Rivkin Angeline,Castanon Rosa G,Clock Ben,Li Yang Eric,Hou Xiaomeng,Poirion Olivier B,Preissl Sebastian,Pinto-Duarte Antonio,O'Connor Carolyn,Boggeman Lara,Fitzpatrick Conor,Nunn Michael,Mukamel Eran A,Zhang Zhuzhu,Callaway Edward M,Ren Bing,Dixon Jesse R,Behrens M Margarita,Ecker Joseph R Nature Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum. 10.1038/s41586-020-03182-8
    Epigenomic diversity of cortical projection neurons in the mouse brain. Nature Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties. 10.1038/s41586-021-03223-w
    CD8+ T cells target cerebrovasculature in children with cerebral malaria. Riggle Brittany A,Manglani Monica,Maric Dragan,Johnson Kory R,Lee Myoung-Hwa,Neto Osorio Lopes Abath,Taylor Terrie E,Seydel Karl B,Nath Avindra,Miller Louis H,McGavern Dorian B,Pierce Susan K The Journal of clinical investigation BACKGROUNDCerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.METHODSUsing multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or CM-), iRBC sequestration (Seqhi, Seqlo, Seq0) and HIV status (HIV+ or HIV-).RESULTSWe identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV- children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ > CM-, P = 0.004) and sequestration level (Seqhi > Seqlo, P = 0.010). HIV coinfection significantly increased T cell numbers (P = 0.017) and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.CONCLUSIONWithin the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.FUNDINGThis research was supported by the Intramural Research Program of the National Institutes of Health. 10.1172/JCI133474
    WDR5 is a conserved regulator of protein synthesis gene expression. Bryan Audra F,Wang Jing,Howard Gregory C,Guarnaccia Alissa D,Woodley Chase M,Aho Erin R,Rellinger Eric J,Matlock Brittany K,Flaherty David K,Lorey Shelly L,Chung Dai H,Fesik Stephen W,Liu Qi,Weissmiller April M,Tansey William P Nucleic acids research WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks-if any-that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis. 10.1093/nar/gkaa051
    InteractomeSeq: a web server for the identification and profiling of domains and epitopes from phage display and next generation sequencing data. Puccio Simone,Grillo Giorgio,Consiglio Arianna,Soluri Maria Felicia,Sblattero Daniele,Cotella Diego,Santoro Claudio,Liuni Sabino,Bellis Gianluca De,Lugli Enrico,Peano Clelia,Licciulli Flavio Nucleic acids research High-Throughput Sequencing technologies are transforming many research fields, including the analysis of phage display libraries. The phage display technology coupled with deep sequencing was introduced more than a decade ago and holds the potential to circumvent the traditional laborious picking and testing of individual phage rescued clones. However, from a bioinformatics point of view, the analysis of this kind of data was always performed by adapting tools designed for other purposes, thus not considering the noise background typical of the 'interactome sequencing' approach and the heterogeneity of the data. InteractomeSeq is a web server allowing data analysis of protein domains ('domainome') or epitopes ('epitome') from either Eukaryotic or Prokaryotic genomic phage libraries generated and selected by following an Interactome sequencing approach. InteractomeSeq allows users to upload raw sequencing data and to obtain an accurate characterization of domainome/epitome profiles after setting the parameters required to tune the analysis. The release of this tool is relevant for the scientific and clinical community, because InteractomeSeq will fill an existing gap in the field of large-scale biomarkers profiling, reverse vaccinology, and structural/functional studies, thus contributing essential information for gene annotation or antigen identification. InteractomeSeq is freely available at https://InteractomeSeq.ba.itb.cnr.it/. 10.1093/nar/gkaa363
    FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links. Awate Sanket,Sommers Joshua A,Datta Arindam,Nayak Sumeet,Bellani Marina A,Yang Olivia,Dunn Christopher A,Nicolae Claudia M,Moldovan George-Lucian,Seidman Michael M,Cantor Sharon B,Brosh Robert M Nucleic acids research FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage. 10.1093/nar/gkaa660
    Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction. Ruiz-Villalba Adrián,Romero Juan P,Hernández Silvia C,Vilas-Zornoza Amaia,Fortelny Nikolaus,Castro-Labrador Laura,San Martin-Uriz Patxi,Lorenzo-Vivas Erika,García-Olloqui Paula,Palacio Marcel,Gavira Juan José,Bastarrika Gorka,Janssens Stefan,Wu Ming,Iglesias Elena,Abizanda Gloria,de Morentin Xabier Martinez,Lasaga Miren,Planell Nuria,Bock Christoph,Alignani Diego,Medal Gema,Prudovsky Igor,Jin Yong-Ri,Ryzhov Sergey,Yin Haifeng,Pelacho Beatriz,Gomez-Cabrero David,Lindner Volkhard,Lara-Astiaso David,Prósper Felipe Circulation BACKGROUND:Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. METHODS:Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. RESULTS:We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. CONCLUSIONS:We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies as a novel regulator of the healing scar process and a target for future translational studies. 10.1161/CIRCULATIONAHA.119.044557
    IRF4 instructs effector Treg differentiation and immune suppression in human cancer. Alvisi Giorgia,Brummelman Jolanda,Puccio Simone,Mazza Emilia Mc,Tomada Elisa Paoluzzi,Losurdo Agnese,Zanon Veronica,Peano Clelia,Colombo Federico S,Scarpa Alice,Alloisio Marco,Vasanthakumar Ajithkumar,Roychoudhuri Rahul,Kallikourdis Marinos,Pagani Massimiliano,Lopci Egesta,Novellis Pierluigi,Blume Jonas,Kallies Axel,Veronesi Giulia,Lugli Enrico The Journal of clinical investigation The molecular mechanisms responsible for the high immunosuppressive capacity of CD4+ Tregs in tumors are not well known. High-dimensional single-cell profiling of T cells from chemotherapy-naive individuals with non-small-cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4+ effector Tregs with superior suppressive activity. In contrast to the IRF4- counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespective of the tumor type. 10.1172/JCI130426
    Microenvironmental Th9 and Th17 lymphocytes induce metastatic spreading in lung cancer. Salazar Ylia,Zheng Xiang,Brunn David,Raifer Hartmann,Picard Felix,Zhang Yajuan,Winter Hauke,Guenther Stefan,Weigert Andreas,Weigmann Benno,Dumoutier Laure,Renauld Jean-Christophe,Waisman Ari,Schmall Anja,Tufman Amanda,Fink Ludger,Brüne Bernhard,Bopp Tobias,Grimminger Friedrich,Seeger Werner,Pullamsetti Soni Savai,Huber Magdalena,Savai Rajkumar The Journal of clinical investigation Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the effect of tumor-infiltrating lymphocyte subpopulations on lung cancer biology by studying in vitro cocultures, in vivo mouse models, and human lung cancer tissue. Lymphocyte conditioned media (CM) induced epithelial-mesenchymal transition (EMT) and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue and correlated with poor survival. Coculturing lung cancer cells with Th9/Th17 cells or exposing them to the respective CM induced EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL-9 and IL-17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Coinjection of Th9/Th17 cells with tumor cells in WT, Rag1-/-, Il9r-/-, and Il17ra-/- mice altered tumor growth and metastasis. Accordingly, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration and metastatic spreading and offering potentially novel therapeutic strategies. 10.1172/JCI124037
    Protein phosphatase 2A B55β limits CD8+ T cell lifespan following cytokine withdrawal. Rodríguez-Rodríguez Noé,Madera-Salcedo Iris K,Cisneros-Segura J Alejandro,García-González H Benjamín,Apostolidis Sokratis A,Saint-Martin Abril,Esquivel-Velázquez Marcela,Nguyen Tran,Romero-Rodríguez Dámaris P,Tsokos George C,Alcocer-Varela Jorge,Rosetti Florencia,Crispín José C The Journal of clinical investigation How T cells integrate environmental cues into signals that limit the magnitude and length of immune responses is poorly understood. Here, we provide data that demonstrate that B55β, a regulatory subunit of protein phosphatase 2A, represents a molecular link between cytokine concentration and apoptosis in activated CD8+ T cells. Through the modulation of AKT, B55β induced the expression of the proapoptotic molecule Hrk in response to cytokine withdrawal. Accordingly, B55β and Hrk were both required for in vivo and in vitro contraction of activated CD8+ lymphocytes. We show that this process plays a role during clonal contraction, establishment of immune memory, and preservation of peripheral tolerance. This regulatory pathway may represent an unexplored opportunity to end unwanted immune responses or to promote immune memory. 10.1172/JCI129479
    Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D. DelGiorno Kathleen E,Chung Chi-Yeh,Vavinskaya Vera,Maurer H Carlo,Novak Sammy Weiser,Lytle Nikki K,Ma Zhibo,Giraddi Rajshekhar R,Wang Dezhen,Fang Linjing,Naeem Razia F,Andrade Leonardo R,Ali Wahida H,Tseng Hubert,Tsui Crystal,Gubbala Vikas B,Ridinger-Saison Maya,Ohmoto Makoto,Erikson Galina A,O'Connor Carolyn,Shokhirev Maxim Nikolaievich,Hah Nasun,Urade Yoshihiro,Matsumoto Ichiro,Kaech Susan M,Singh Pankaj K,Manor Uri,Olive Kenneth P,Wahl Geoffrey M Gastroenterology BACKGROUND & AIMS:Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. METHODS:We performed studies with LSL-Kras;Ptf1a mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. RESULTS:Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. CONCLUSIONS:In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type. 10.1053/j.gastro.2020.07.037
    First-dose mRNA vaccination is sufficient to reactivate immunological memory to SARS-CoV-2 in subjects who have recovered from COVID-19. Mazzoni Alessio,Di Lauria Nicoletta,Maggi Laura,Salvati Lorenzo,Vanni Anna,Capone Manuela,Lamacchia Giulia,Mantengoli Elisabetta,Spinicci Michele,Zammarchi Lorenzo,Kiros Seble Tekle,Rocca Arianna,Lagi Filippo,Colao Maria Grazia,Parronchi Paola,Scaletti Cristina,Turco Lucia,Liotta Francesco,Rossolini Gian Maria,Cosmi Lorenzo,Bartoloni Alessandro,Annunziato Francesco, The Journal of clinical investigation The characterization of the adaptive immune response to COVID-19 vaccination in individuals who recovered from SARS-CoV-2 infection may define current and future clinical practice. To determine the effect of the 2-dose BNT162b2 mRNA COVID-19 vaccination schedule in individuals who recovered from COVID-19 (COVID-19-recovered subjects) compared with naive subjects, we evaluated SARS-CoV-2 Spike-specific T and B cell responses, as well as specific IgA, IgG, IgM, and neutralizing antibodies titers in 22 individuals who received the BNT162b2 mRNA COVID-19 vaccine, 11 of whom had a previous history of SARS-CoV-2 infection. Evaluations were performed before vaccination and then weekly until 7 days after second injection. Data obtained clearly showed that one vaccine dose is sufficient to increase both cellular and humoral immune response in COVID-19-recovered subjects without any additional improvement after the second dose. On the contrary, the second dose proved mandatory in naive subjects to further enhance the immune response. These findings were further confirmed at the serological level in a larger cohort of naive (n = 68) and COVID-19-recovered (n = 29) subjects, tested up to 50 days after vaccination. These results question whether a second vaccine injection in COVID-19-recovered subjects is required, and indicate that millions of vaccine doses may be redirected to naive individuals, thus shortening the time to reach herd immunity. 10.1172/JCI149150
    Lung-resident memory B cells protect against bacterial pneumonia. The Journal of clinical investigation Lung-resident memory B cells (BRM cells) are elicited after influenza infections of mice, but connections to other pathogens and hosts - as well as their functional significance - have yet to be determined. We postulate that BRM cells are core components of lung immunity. To test this, we examined whether lung BRM cells are elicited by the respiratory pathogen pneumococcus, are present in humans, and are important in pneumonia defense. Lungs of mice that had recovered from pneumococcal infections did not contain organized tertiary lymphoid organs, but did have plasma cells and noncirculating memory B cells. The latter expressed distinctive surface markers (including CD69, PD-L2, CD80, and CD73) and were poised to secrete antibodies upon stimulation. Human lungs also contained B cells with a resident memory phenotype. In mice recovered from pneumococcal pneumonia, depletion of PD-L2+ B cells, including lung BRM cells, diminished bacterial clearance and the level of pneumococcus-reactive antibodies in the lung. These data define lung BRM cells as a common feature of pathogen-experienced lungs and provide direct evidence of a role for these cells in pulmonary antibacterial immunity. 10.1172/JCI141810
    Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity. Noël Grégory,Fontsa Mireille Langouo,Garaud Soizic,De Silva Pushpamali,de Wind Alexandre,Van den Eynden Gert G,Salgado Roberto,Boisson Anaïs,Locy Hanne,Thomas Noémie,Solinas Cinzia,Migliori Edoardo,Naveaux Céline,Duvillier Hugues,Lucas Sophie,Craciun Ligia,Thielemans Kris,Larsimont Denis,Willard-Gallo Karen The Journal of clinical investigation We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade. 10.1172/JCI139905
    Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis. Donadon Matteo,Torzilli Guido,Cortese Nina,Soldani Cristiana,Di Tommaso Luca,Franceschini Barbara,Carriero Roberta,Barbagallo Marialuisa,Rigamonti Alessandra,Anselmo Achille,Colombo Federico Simone,Maggi Giulia,Lleo Ana,Cibella Javier,Peano Clelia,Kunderfranco Paolo,Roncalli Massimo,Mantovani Alberto,Marchesi Federica The Journal of experimental medicine It has long been known that in vitro polarized macrophages differ in morphology. Stemming from a conventional immunohistology observation, we set out to test the hypothesis that morphology of tumor-associated macrophages (TAMs) in colorectal liver metastasis (CLM) represents a correlate of functional diversity with prognostic significance. Density and morphological metrics of TAMs were measured and correlated with clinicopathological variables. While density of TAMs did not correlate with survival of CLM patients, the cell area identified small (S-TAM) and large (L-TAM) macrophages that were associated with 5-yr disease-free survival rates of 27.8% and 0.2%, respectively (P < 0.0001). RNA sequencing of morphologically distinct macrophages identified LXR/RXR as the most enriched pathway in large macrophages, with up-regulation of genes involved in cholesterol metabolism, scavenger receptors, MERTK, and complement. In single-cell analysis of mononuclear phagocytes from CLM tissues, S-TAM and L-TAM signatures were differentially enriched in individual clusters. These results suggest that morphometric characterization can serve as a simple readout of TAM diversity with strong prognostic significance. 10.1084/jem.20191847
    Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids. Koundouros Nikos,Karali Evdoxia,Tripp Aurelien,Valle Adamo,Inglese Paolo,Perry Nicholas J S,Magee David J,Anjomani Virmouni Sara,Elder George A,Tyson Adam L,Dória Maria Luisa,van Weverwijk Antoinette,Soares Renata F,Isacke Clare M,Nicholson Jeremy K,Glen Robert C,Takats Zoltan,Poulogiannis George Cell Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT. 10.1016/j.cell.2020.05.053
    SARS-CoV-2 infection and vaccination trigger long-lived B and CD4+ T lymphocytes with implications for booster strategies. Mazzoni Alessio,Vanni Anna,Spinicci Michele,Lamacchia Giulia,Kiros Seble Tekle,Rocca Arianna,Capone Manuela,Di Lauria Nicoletta,Salvati Lorenzo,Carnasciali Alberto,Mantengoli Elisabetta,Farahvachi Parham,Zammarchi Lorenzo,Lagi Filippo,Colao Maria Grazia,Liotta Francesco,Cosmi Lorenzo,Maggi Laura,Bartoloni Alessandro,Rossolini Gian Maria,Annunziato Francesco The Journal of clinical investigation BACKGROUNDImmunization against SARS-CoV-2, the causative agent of COVID-19, occurs via natural infection or vaccination. However, it is currently unknown how long infection- or vaccination-induced immunological memory will last.METHODSWe performed a longitudinal evaluation of immunological memory to SARS-CoV-2 up to 1 year after infection and following mRNA vaccination in naive individuals and individuals recovered from COVID-19 infection.RESULTSWe found that memory cells are still detectable 8 months after vaccination, while antibody levels decline significantly, especially in naive individuals. We also found that a booster injection is efficacious in reactivating immunological memory to spike protein in naive individuals, whereas it was ineffective in previously SARS-CoV-2-infected individuals. Finally, we observed a similar kinetics of decay of humoral and cellular immunity to SARS-CoV-2 up to 1 year following natural infection in a cohort of unvaccinated individuals.CONCLUSIONShort-term persistence of humoral immunity, together with the reduced neutralization capacity versus the currently prevailing SARS-CoV-2 variants, may account for reinfections and breakthrough infections. Long-lived memory B and CD4+ T cells may protect from severe disease development. In naive individuals, a booster dose restored optimal anti-spike immunity, whereas the needs for vaccinated individuals who have recovered from COVID-19 have yet to be defined.FUNDINGThis study was supported by funds to the Department of Experimental and Clinical Medicine, University of Florence (Project Excellence Departments 2018-2022), the University of Florence (project RICTD2122), the Italian Ministry of Health (COVID-2020-12371849), and the region of Tuscany (TagSARS CoV 2). 10.1172/JCI157990
    Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer. Katzenelenbogen Yonatan,Sheban Fadi,Yalin Adam,Yofe Ido,Svetlichnyy Dmitry,Jaitin Diego Adhemar,Bornstein Chamutal,Moshe Adi,Keren-Shaul Hadas,Cohen Merav,Wang Shuang-Yin,Li Baoguo,David Eyal,Salame Tomer-Meir,Weiner Assaf,Amit Ido Cell Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1 Trem2 regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8 T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors. 10.1016/j.cell.2020.06.032
    Interleukin-10 contributes to reservoir establishment and persistence in SIV-infected macaques treated with antiretroviral therapy. The Journal of clinical investigation Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV. 10.1172/JCI155251
    αβγδ T cells play a vital role in fetal human skin development and immunity. Reitermaier René,Krausgruber Thomas,Fortelny Nikolaus,Ayub Tanya,Vieyra-Garcia Pablo Augusto,Kienzl Philip,Wolf Peter,Scharrer Anke,Fiala Christian,Kölz Marita,Hiess Manuela,Vierhapper Martin,Schuster Christopher,Spittler Andreas,Worda Christof,Weninger Wolfgang,Bock Christoph,Eppel Wolfgang,Elbe-Bürger Adelheid The Journal of experimental medicine T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αβ and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αβγδ T cells displayed little overlap of CDR3 sequences with single-positive αβ T cells. Gene signatures, cytokine profiles and in silico receptor-ligand interaction studies indicate their contribution to early skin development. DP αβγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin. 10.1084/jem.20201189
    Lymph node fibroblastic reticular cells preserve a tolerogenic niche in allograft transplantation through laminin α4. The Journal of clinical investigation Lymph node (LN) fibroblastic reticular cells (FRCs) define LN niches and regulate lymphocyte homeostasis through producing diverse extracellular matrix (ECM) components. We examined the role of ECM laminin α4 (Lama4) using FRC-Lama4 conditional KO Pdgfrb-Cre-/- × Lama4fl/fl mice. Single-cell RNA-sequencing (scRNA-Seq) data showed the promoter gene Pdgfrb was exclusively expressed in FRCs. Depleting FRC-Lama4 reduced Tregs and dendritic cells, decreased high endothelial venules, impaired the conduit system, and downregulated T cell survival factors in LNs. FRC-Lama4 depletion impaired the homing of lymphocytes to LNs in homeostasis and after allografting. Alloantigen-specific T cells proliferated, were activated to greater degrees in LNs lacking FRC-Lama4, and were more prone to differentiate into effector phenotypes relative to the Treg phenotype. In murine cardiac transplantation, tolerogenic immunosuppression was not effective in FRC-Lama4 recipients, which produced more alloantibodies than WT. After lung transplantation, FRC-Lama4-KO mice had more severe graft rejection with fewer Tregs in their LNs. Overall, FRC-Lama4 critically contributes to a tolerogenic LN niche by supporting T cell migration, constraining T cell activation and proliferation, and promoting Treg differentiation. Hence, it serves as a therapeutic target for immunoengineering. 10.1172/JCI156994
    Mechanistic dissection of dominant AIRE mutations in mouse models reveals AIRE autoregulation. The Journal of experimental medicine The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression. 10.1084/jem.20201076
    Lipid-loaded tumor-associated macrophages sustain tumor growth and invasiveness in prostate cancer. The Journal of experimental medicine Tumor-associated macrophages (TAMs) are correlated with the progression of prostatic adenocarcinoma (PCa). The mechanistic basis of this correlation and therapeutic strategies to target TAMs in PCa remain poorly defined. Here, single-cell RNA sequencing was used to profile the transcriptional landscape of TAMs in human PCa, leading to identification of a subset of macrophages characterized by dysregulation in transcriptional pathways associated with lipid metabolism. This subset of TAMs correlates positively with PCa progression and shorter disease-free survival and is characterized by an accumulation of lipids that is dependent on Marco. Mechanistically, cancer cell-derived IL-1β enhances Marco expression on macrophages, and reciprocally, cancer cell migration is promoted by CCL6 released by lipid-loaded TAMs. Moreover, administration of a high-fat diet to tumor-bearing mice raises the abundance of lipid-loaded TAMs. Finally, targeting lipid accumulation by Marco blockade hinders tumor growth and invasiveness and improves the efficacy of chemotherapy in models of PCa, pointing to combinatorial strategies that may influence patient outcomes. 10.1084/jem.20210564
    PRMT1 promotes the tumor suppressor function of p14 and is indicative for pancreatic cancer prognosis. Repenning Antje,Happel Daniela,Bouchard Caroline,Meixner Marion,Verel-Yilmaz Yesim,Raifer Hartmann,Holembowski Lena,Krause Eberhard,Kremmer Elisabeth,Feederle Regina,Keber Corinna U,Lohoff Michael,Slater Emily P,Bartsch Detlef K,Bauer Uta-Maria The EMBO journal The p14 protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14 is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14 undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14 as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14 . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14 . Genotoxic stress causes augmented interaction between PRMT1 and p14 , accompanied by arginine methylation of p14 . PRMT1-dependent NLS/NoLS methylation promotes the release of p14 from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14 cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14 's stress-induced tumor-suppressive function. 10.15252/embj.2020106777
    Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain. Lee Dongjin R,Rhodes Christopher,Mitra Apratim,Zhang Yajun,Maric Dragan,Dale Ryan K,Petros Timothy J eLife The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain. 10.7554/eLife.71864
    LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma. Cell Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies. 10.1016/j.cell.2022.03.011
    Single-cell transcriptome reveals the novel role of T-bet in suppressing the immature NK gene signature. Yang Chao,Siebert Jason R,Burns Robert,Zheng Yongwei,Mei Ao,Bonacci Benedetta,Wang Demin,Urrutia Raul A,Riese Matthew J,Rao Sridhar,Carlson Karen-Sue,Thakar Monica S,Malarkannan Subramaniam eLife The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through Akt-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-Akt-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells. 10.7554/eLife.51339
    Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity. Cell Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4 and CD8 T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4 and CD8 T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4 and CD8 T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4 T cell, CD8 T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2. 10.1016/j.cell.2020.09.038
    Early detection of cerebrovascular pathology and protective antiviral immunity by MRI. eLife Central nervous system (CNS) infections are a major cause of human morbidity and mortality worldwide. Even patients that survive, CNS infections can have lasting neurological dysfunction resulting from immune and pathogen induced pathology. Developing approaches to noninvasively track pathology and immunity in the infected CNS is crucial for patient management and development of new therapeutics. Here, we develop novel MRI-based approaches to monitor virus-specific CD8+ T cells and their relationship to cerebrovascular pathology in the living brain. We studied a relevant murine model in which a neurotropic virus (vesicular stomatitis virus) was introduced intranasally and then entered the brain via olfactory sensory neurons - a route exploited by many pathogens in humans. Using T2*-weighted high-resolution MRI, we identified small cerebral microbleeds as an early form of pathology associated with viral entry into the brain. Mechanistically, these microbleeds occurred in the absence of peripheral immune cells and were associated with infection of vascular endothelial cells. We monitored the adaptive response to this infection by developing methods to iron label and track individual virus specific CD8+ T cells by MRI. Transferred antiviral T cells were detected in the brain within a day of infection and were able to reduce cerebral microbleeds. These data demonstrate the utility of MRI in detecting the earliest pathological events in the virally infected CNS as well as the therapeutic potential of antiviral T cells in mitigating this pathology. 10.7554/eLife.74462
    Immune Checkpoint-Bioengineered Beta Cell Vaccine Reverses Early-Onset Type 1 Diabetes. Advanced materials (Deerfield Beach, Fla.) Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that results from autoreactive T cells destroying insulin-producing pancreatic beta (β) cells. The development of T1DM is associated with the deficiency of co-inhibitory immune checkpoint ligands (e.g., PD-L1, CD86, and Gal-9) in β cells. Here, a new translational approach based on metabolic glycoengineering and bioorthogonal click chemistry, which bioengineers β cells with co-inhibitory immune checkpoint molecules that induce antigen-specific immunotolerance and reverse early-onset hyperglycemia is reported. To achieve this goal, a subcutaneous injectable acellular pancreatic extracellular matrix platform for localizing the bioengineered β cells while creating a pancreas-like immunogenic microenvironment, in which the autoreactive T cells can interface with the β cells, is devised. 10.1002/adma.202101253
    REST Inhibits Direct Reprogramming of Pancreatic Exocrine to Endocrine Cells by Preventing PDX1-Mediated Activation of Endocrine Genes. Elhanani Ofer,Salame Tomer Meir,Sobel Jonathan,Leshkowitz Dena,Povodovski Lital,Vaknin Itay,Kolodkin-Gal Dror,Walker Michael D Cell reports The emerging appreciation of plasticity among pancreatic lineages has created interest in harnessing cellular reprogramming for β cell replacement therapy of diabetes. Current reprogramming methodologies are inefficient, largely because of a limited understanding of the underlying mechanisms. Using an in vitro reprogramming system, we reveal the transcriptional repressor RE-1 silencing transcription factor (REST) as a barrier for β cell gene expression in the reprogramming of pancreatic exocrine cells. We observe that REST-bound loci lie adjacent to the binding sites of multiple key β cell transcription factors, including PDX1. Accordingly, a loss of REST function combined with PDX1 expression results in the synergistic activation of endocrine genes. This is accompanied by increased histone acetylation and PDX1 binding at endocrine gene loci. Collectively, our data identify a mechanism for REST activity involving the prevention of PDX1-mediated activation of endocrine genes and uncover REST downregulation and the resulting chromatin alterations as key events in β cell reprogramming. 10.1016/j.celrep.2020.107591
    CD28 Regulates Metabolic Fitness for Long-Lived Plasma Cell Survival. Utley Adam,Chavel Colin,Lightman Shivana,Holling G Aaron,Cooper James,Peng Peng,Liu Wensheng,Barwick Benjamin G,Gavile Catherine M,Maguire Orla,Murray-Dupuis Megan,Rozanski Cheryl,Jordan Martha S,Kambayashi Taku,Olejniczak Scott H,Boise Lawrence H,Lee Kelvin P Cell reports Durable humoral immunity against epidemic infectious disease requires the survival of long-lived plasma cells (LLPCs). LLPC longevity is dependent on metabolic programs distinct from short-lived plasma cells (SLPCs); however, the mechanistic basis for this difference is unclear. We have previously shown that CD28, the prototypic T cell costimulatory receptor, is expressed on both LLPCs and SLPCs but is essential only for LLPC survival. Here we show that CD28 transduces pro-survival signaling specifically in LLPCs through differential SLP76 expression. CD28 signaling in LLPCs increased glucose uptake, mitochondrial mass/respiration, and reactive oxygen species (ROS) production. Unexpectedly, CD28-mediated regulation of mitochondrial respiration, NF-κB activation, and survival was ROS dependent. IRF4, a target of NF-κB, was upregulated by CD28 activation in LLPCs and decreased IRF4 levels correlated with decreased glucose uptake, mitochondrial mass, ROS, and CD28-mediated survival. Altogether, these data demonstrate that CD28 signaling induces a ROS-dependent metabolic program required for LLPC survival. 10.1016/j.celrep.2020.107815
    Excessive Astrocytic GABA Causes Cortical Hypometabolism and Impedes Functional Recovery after Subcortical Stroke. Nam Min-Ho,Cho Jongwook,Kwon Dae-Hyuk,Park Ji-Young,Woo Junsung,Lee Jung Moo,Lee Sangwon,Ko Hae Young,Won Woojin,Kim Ra Gyung,Song Hanlim,Oh Soo-Jin,Choi Ji Won,Park Ki Duk,Park Eun Kyung,Jung Haejin,Kim Hyung-Seok,Lee Min-Cheol,Yun Mijin,Lee C Justin,Kim Hyoung-Ihl Cell reports Glucose hypometabolism in cortical structures after functional disconnection is frequently reported in patients with white matter diseases such as subcortical stroke. However, the molecular and cellular mechanisms have been poorly elucidated. Here we show, in an animal model of internal capsular infarct, that GABA-synthesizing reactive astrocytes in distant cortical areas cause glucose hypometabolism via tonic inhibition of neighboring neurons. We find that reversal of aberrant astrocytic GABA synthesis, by pharmacological inhibition and astrocyte-specific gene silencing of MAO-B, reverses the reduction in cortical glucose metabolism. Moreover, induction of aberrant astrocytic GABA synthesis by cortical injection of putrescine or adenovirus recapitulates cortical hypometabolism. Furthermore, MAO-B inhibition causes a remarkable recovery from post-stroke motor deficits when combined with a rehabilitation regimen. Collectively, our data indicate that cortical glucose hypometabolism in subcortical stroke is caused by aberrant astrocytic GABA and MAO-B inhibition and that attenuating cortical hypometabolism can be a therapeutic approach in subcortical stroke. 10.1016/j.celrep.2020.107861
    Immune Checkpoint Ligand Bioengineered Schwann Cells as Antigen-Specific Therapy for Experimental Autoimmune Encephalomyelitis. Au Kin Man,Tisch Roland,Wang Andrew Z Advanced materials (Deerfield Beach, Fla.) Failure to establish immune tolerance leads to the development of autoimmune disease. The ability to regulate autoreactive T cells without inducing systemic immunosuppression represents a major challenge in the development of new strategies to treat autoimmune disease. Here, a translational method for bioengineering programmed death-ligand 1 (PD-L1)- and cluster of differentiation 86 (CD86)-functionalized mouse Schwann cells (SCs) to prevent and ameliorate multiple sclerosis (MS) in established mouse models of chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE) is described. It is shown that the intravenous (i.v.) administration of immune checkpoint ligand functionalized mouse SCs modifies the course of disease and ameliorates EAE. Further, it is found that such bioengineered mouse SCs inhibit the differentiation of myelin-specific helper T cells into pathogenic T helper type-1 (T 1) and type-17 (T 17) cells, promote the development of tolerogenic myelin-specific regulatory T (T ) cells, and resolve inflammatory central nervous system microenvironments without inducing systemic immunosuppression. 10.1002/adma.202107392
    Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nature medicine An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8 T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas. 10.1038/s41591-020-1073-3
    Dynamically linking influenza virus infection kinetics, lung injury, inflammation, and disease severity. Myers Margaret A,Smith Amanda P,Lane Lindey C,Moquin David J,Aogo Rosemary,Woolard Stacie,Thomas Paul,Vogel Peter,Smith Amber M eLife Influenza viruses cause a significant amount of morbidity and mortality. Understanding host immune control efficacy and how different factors influence lung injury and disease severity are critical. We established and validated dynamical connections between viral loads, infected cells, CD8 T cells, lung injury, inflammation, and disease severity using an integrative mathematical model-experiment exchange. Our results showed that the dynamics of inflammation and virus-inflicted lung injury are distinct and nonlinearly related to disease severity, and that these two pathologic measurements can be independently predicted using the model-derived infected cell dynamics. Our findings further indicated that the relative CD8 T cell dynamics paralleled the percent of the lung that had resolved with the rate of CD8 T cell-mediated clearance rapidly accelerating by over 48,000 times in 2 days. This complimented our analyses showing a negative correlation between the efficacy of innate and adaptive immune-mediated infected cell clearance, and that infection duration was driven by CD8 T cell magnitude rather than efficacy and could be significantly prolonged if the ratio of CD8 T cells to infected cells was sufficiently low. These links between important pathogen kinetics and host pathology enhance our ability to forecast disease progression, potential complications, and therapeutic efficacy. 10.7554/eLife.68864
    The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins. Nature genetics Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery. 10.1038/s41588-019-0559-8
    Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes. Han Patrick,Hanlon Douglas,Arshad Najla,Lee Jung Seok,Tatsuno Kazuki,Robinson Eve,Filler Renata,Sobolev Olga,Cote Christine,Rivera-Molina Felix,Toomre Derek,Fahmy Tarek,Edelson Richard Science advances Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies. 10.1126/sciadv.aaz1580
    ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation. Cell reports Endometriosis affects 1 in 10 women and is characterized by the presence of abnormal endometrium at ectopic sites. ARID1A mutations are observed in deeply invasive forms of the disease, often correlating with malignancy. To identify epigenetic dependencies driving invasion, we use an unbiased approach to map chromatin state transitions accompanying ARID1A loss in the endometrium. We show that super-enhancers marked by high H3K27 acetylation are strongly associated with ARID1A binding. ARID1A loss leads to H3K27 hyperacetylation and increased chromatin accessibility and enhancer RNA transcription at super-enhancers, but not typical enhancers, indicating that ARID1A normally prevents super-enhancer hyperactivation. ARID1A co-localizes with P300 at super-enhancers, and genetic or pharmacological inhibition of P300 in ARID1A mutant endometrial epithelia suppresses invasion and induces anoikis through the rescue of super-enhancer hyperacetylation. Among hyperactivated super-enhancers, SERPINE1 (PAI-1) is identified as an essential target gene driving ARID1A mutant endometrial invasion. Broadly, our findings provide rationale for therapeutic strategies targeting super-enhancers in ARID1A mutant endometrium. 10.1016/j.celrep.2020.108366
    Opposing immune and genetic mechanisms shape oncogenic programs in synovial sarcoma. Nature medicine Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies. 10.1038/s41591-020-01212-6
    Heterogeneous Escape from X Chromosome Inactivation Results in Sex Differences in Type I IFN Responses at the Single Human pDC Level. Hagen Sven Hendrik,Henseling Florian,Hennesen Jana,Savel Hélène,Delahaye Solenne,Richert Laura,Ziegler Susanne Maria,Altfeld Marcus Cell reports Immune responses differ between women and men, and type I interferon (IFN) responses following Toll-like receptor 7 (TLR7) stimulation are higher in women. The precise mechanisms driving these sex differences in immunity are unknown. To investigate possible genetic factors, we quantify escape from X chromosome inactivation (XCI) for TLR7 and four other genes (RPS6KA3, CYBB, BTK, and IL13RA1) at the single plasmacytoid dendritic cell (pDC) level. We observe escape from XCI for all investigated genes, leading to biallelic expression patterns. pDCs with biallelic gene expression have significantly higher mRNA levels of the respective genes. Unstimulated pDCs with biallelic TLR7 expression exhibit significantly higher IFNα/β mRNA levels, and IFNα exposure results in significantly increased IFNα/β protein production by pDCs. These results identify unanticipated heterogeneity in escape from XCI of several genes in pDCs and highlight the important contribution of X chromosome factors to sex differences in type I IFN responses, which might explain observed sex differences in human diseases. 10.1016/j.celrep.2020.108485
    Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets. Psaila Bethan,Wang Guanlin,Rodriguez-Meira Alba,Li Rong,Heuston Elisabeth F,Murphy Lauren,Yee Daniel,Hitchcock Ian S,Sousos Nikolaos,O'Sullivan Jennifer,Anderson Stacie,Senis Yotis A,Weinberg Olga K,Calicchio Monica L, ,Iskander Deena,Royston Daniel,Milojkovic Dragana,Roberts Irene,Bodine David M,Thongjuea Supat,Mead Adam J Molecular cell Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34 lineage hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis. 10.1016/j.molcel.2020.04.008
    Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice. Kay Jennifer E,Corrigan Joshua J,Armijo Amanda L,Nazari Ilana S,Kohale Ishwar N,Torous Dorothea K,Avlasevich Svetlana L,Croy Robert G,Wadduwage Dushan N,Carrasco Sebastian E,Dertinger Stephen D,White Forest M,Essigmann John M,Samson Leona D,Engelward Bevin P Cell reports N-Nitrosodimethylamine (NDMA) is a DNA-methylating agent that has been discovered to contaminate water, food, and drugs. The alkyladenine DNA glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we study Aag-knockout (Aag) and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine [3MeA]) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switches from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrate a suite of molecular, cellular, and physiological analyses. We find that unrepaired 3MeA is somewhat toxic, but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tip the balance between blocks and breaks and thus dictate the disease consequences of DNA damage. 10.1016/j.celrep.2021.108864
    Reprogramming competence of OCT factors is determined by transactivation domains. Science advances OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components. 10.1126/sciadv.aaz7364
    Induction of osteogenesis by bone-targeted Notch activation. Xu Cong,Dinh Van Vuong,Kruse Kai,Jeong Hyun-Woo,Watson Emma C,Adams Susanne,Berkenfeld Frank,Stehling Martin,Rasouli Seyed Javad,Fan Rui,Chen Rui,Bedzhov Ivan,Chen Qi,Kato Katsuhiro,Pitulescu Mara E,Adams Ralf H eLife Declining bone mass is associated with aging and osteoporosis, a disease characterized by progressive weakening of the skeleton and increased fracture incidence. Growth and lifelong homeostasis of bone rely on interactions between different cell types including vascular cells and mesenchymal stromal cells (MSCs). As these interactions involve Notch signaling, we have explored whether treatment with secreted Notch ligand proteins can enhance osteogenesis in adult mice. We show that a bone-targeting, high affinity version of the ligand Delta-like 4, termed Dll4, induces bone formation in male mice without causing adverse effects in other organs, which are known to rely on intact Notch signaling. Due to lower bone surface and thereby reduced retention of Dll4, the same approach failed to promote osteogenesis in female and ovariectomized mice but strongly enhanced trabecular bone formation in combination with parathyroid hormone. Single cell analysis of stromal cells indicates that Dll4 primarily acts on MSCs and has comparably minor effects on osteoblasts, endothelial cells, or chondrocytes. We propose that activation of Notch signaling by bone-targeted fusion proteins might be therapeutically useful and can avoid detrimental effects in Notch-dependent processes in other organs. 10.7554/eLife.60183
    Safety and effectiveness of up to 3 years' bulevirtide monotherapy in patients with HDV-related cirrhosis. Journal of hepatology The entry inhibitor bulevirtide (BLV) received conditional approval from the EMA in July 2020 for the treatment of adult patients with compensated chronic hepatitis delta. However, the effectiveness and safety of BLV administered as monotherapy beyond 48 weeks in difficult-to-treat patients with HDV-related cirrhosis is presently unknown. Herein, we describe the first patients with HDV-related compensated cirrhosis who were treated with BLV (10 mg/day as a starting dose) for up to 3 years on a compassionate use program. Patients were also monitored for HBcrAg and HBV RNA levels, and HDV- and HBV-specific T-cell markers. In the patient who stopped BLV at week 48, after achieving a virological and biochemical response, the initial virological and biochemical rebound was followed by alanine aminotransferase normalization coupled with low HDV RNA and HBsAg levels. In the 2 patients treated continuously for 3 years, virological and biochemical responses were maintained throughout the treatment period even after dose reduction. In a patient with advanced compensated cirrhosis, liver function tests significantly improved, esophageal varices disappeared, and histological/laboratory features of autoimmune hepatitis resolved. Overall, no safety issues were recorded, as bile salt increase was asymptomatic. While serum HBV RNA levels remained undetectable in all patients, HBV core-related antigen levels showed a progressive, yet modest decline during long-term BLV treatment. No HDV-specific interferon-γ-producing T cells were detected, neither after HDV reactivation (after BLV withdrawn in Patient 1) nor during 3 years of BLV treatment. In conclusion, this report shows that continuous administration of BLV monotherapy for 3 years leads to excellent virological and clinical responses in patients with HDV-related cirrhosis who had contraindications to interferon-based therapies. 10.1016/j.jhep.2021.10.012
    NKG2A expression identifies a subset of human Vδ2 T cells exerting the highest antitumor effector functions. Cazzetta Valentina,Bruni Elena,Terzoli Sara,Carenza Claudia,Franzese Sara,Piazza Rocco,Marzano Paolo,Donadon Matteo,Torzilli Guido,Cimino Matteo,Simonelli Matteo,Bello Lorenzo,Villa Anna,Tan Likai,Ravens Sarina,Prinz Immo,Supino Domenico,Colombo Federico S,Lugli Enrico,Marcenaro Emanuela,Vivier Eric,Della Bella Silvia,Mikulak Joanna,Mavilio Domenico Cell reports Human Vδ2 cells are innate-like γδ T effectors performing potent immune surveillance against tumors. The constitutive expression of NKG2A identifies a subset of Vδ2 T cells licensed with an intrinsic hyper-responsiveness against cancer. Indeed, the transcriptomic profiles of NKG2A and NKG2A cells characterize two distinct "intralineages" of Vδ2 T lymphocytes that appear early during development, keep their phenotypes, and show self-renewal capabilities in adult life. The hyper-responsiveness of NKG2A Vδ2 T cells is counterbalanced by the inhibitory signaling delivered by human leukocyte antigen E (HLA-E) expressed on malignant cells as a tumor-escape mechanism. However, either masking or knocking out NKG2A restores the capacity of Vδ2 T cells to exert the highest effector functions even against HLA-E tumors. This is highly relevant in the clinic, as the different degrees of engagement of the NKG2A-HLA-E checkpoint in hepatocellular carcinoma, glioblastoma, and non-small cell lung cancer directly impact patients' overall survival. These findings open avenues for developing combined cellular and immunologic anticancer therapies. 10.1016/j.celrep.2021.109871
    Deciphering epiblast lumenogenesis reveals proamniotic cavity control of embryo growth and patterning. Science advances During the peri-implantation stages, the mouse embryo radically changes its appearance, transforming from a hollow-shaped blastocyst to an egg cylinder. At the same time, the epiblast gets reorganized from a simple ball of cells to a cup-shaped epithelial monolayer enclosing the proamniotic cavity. However, the cavity's function and mechanism of formation have so far been obscure. Through investigating the cavity formation, we found that in the epiblast, the process of lumenogenesis is driven by reorganization of intercellular adhesion, vectoral fluid transport, and mitotic paracellular water influx from the blastocoel into the emerging proamniotic cavity. By experimentally blocking lumenogenesis, we found that the proamniotic cavity functions as a hub for communication between the early lineages, enabling proper growth and patterning of the postimplantation embryo. 10.1126/sciadv.abe1640
    Epithelial-myeloid exchange of MHC class II constrains immunity and microbiota composition. Cell reports Intestinal epithelial cells (IECs) have long been understood to express high levels of major histocompatibility complex class II (MHC class II) molecules but are not considered canonical antigen-presenting cells, and the impact of IEC-MHC class II signaling on gut homeostasis remains enigmatic. As IECs serve as the primary barrier between underlying host immune cells, we reasoned that IEC-intrinsic antigen presentation may play a role in responses toward the microbiota. Mice with an IEC-intrinsic deletion of MHC class II (IEC) are healthy but have fewer microbial-bound IgA, regulatory T cells (Tregs), and immune repertoire selection. This was associated with increased interindividual microbiota variation and altered proportions of two taxa in the ileum where MHC class II on IECs is highest. Intestinal mononuclear phagocytes (MNPs) have similar MHC class II transcription but less surface MHC class II and are capable of acquiring MHC class II from IECs. Thus, epithelial-myeloid interactions mediate development of adaptive responses to microbial antigens within the gastrointestinal tract. 10.1016/j.celrep.2021.109916
    SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity. Rosa Annachiara,Pye Valerie E,Graham Carl,Muir Luke,Seow Jeffrey,Ng Kevin W,Cook Nicola J,Rees-Spear Chloe,Parker Eleanor,Dos Santos Mariana Silva,Rosadas Carolina,Susana Alberto,Rhys Hefin,Nans Andrea,Masino Laura,Roustan Chloe,Christodoulou Evangelos,Ulferts Rachel,Wrobel Antoni G,Short Charlotte-Eve,Fertleman Michael,Sanders Rogier W,Heaney Judith,Spyer Moira,Kjær Svend,Riddell Andy,Malim Michael H,Beale Rupert,MacRae James I,Taylor Graham P,Nastouli Eleni,van Gils Marit J,Rosenthal Peter B,Pizzato Massimo,McClure Myra O,Tedder Richard S,Kassiotis George,McCoy Laura E,Doores Katie J,Cherepanov Peter Science advances The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite. 10.1126/sciadv.abg7607
    Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target. Journal of hepatology BACKGROUND AND AIMS:The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). METHODS:We used high-dimensional single cell technologies to characterise the T cell and myeloid compartments of iCCA comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology specifically in iCCA. RESULTS:We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of CD4+ Tregs characterized by a hyperactivated state. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, abundance of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of iCCA patients. CONCLUSIONS:Interfering with hyperactivated Tregs should be explored to enhance anti-tumor immunity in iCCA. LAY SUMMARY:By high-dimensional profiling, we identify increased frequency of a suppressive population of lymphocytes called Tregs that are highly active within intrahepatic cholangiocarcinoma. MEOX1, a transcription factor novel to Treg biology, promotes their activity. The genetic program controlled by MEOX1 strongly correlates with worse disease progression, suggesting this specific protein is a potential target of immunotherapy. 10.1016/j.jhep.2022.05.043
    In utero origin of myelofibrosis presenting in adult monozygotic twins. Nature medicine The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs. 10.1038/s41591-022-01793-4
    A T cell resilience model associated with response to immunotherapy in multiple tumor types. Nature medicine Despite breakthroughs in cancer immunotherapy, most tumor-reactive T cells cannot persist in solid tumors due to an immunosuppressive environment. We developed Tres (tumor-resilient T cell), a computational model utilizing single-cell transcriptomic data to identify signatures of T cells that are resilient to immunosuppressive signals, such as transforming growth factor-β1, tumor necrosis factor-related apoptosis-inducing ligand and prostaglandin E2. Tres reliably predicts clinical responses to immunotherapy in melanoma, lung cancer, triple-negative breast cancer and B cell malignancies using bulk T cell transcriptomic data from pre-treatment tumors from patients who received immune-checkpoint inhibitors (n = 38), infusion products for chimeric antigen receptor T cell therapies (n = 34) and pre-manufacture samples for chimeric antigen receptor T cell or tumor-infiltrating lymphocyte therapies (n = 84). Further, Tres identified FIBP, whose functions are largely unknown, as the top negative marker of tumor-resilient T cells across many solid tumor types. FIBP knockouts in murine and human donor CD8 T cells significantly enhanced T cell-mediated cancer killing in in vitro co-cultures. Further, Fibp knockout in murine T cells potentiated the in vivo efficacy of adoptive cell transfer in the B16 tumor model. Fibp knockout T cells exhibit reduced cholesterol metabolism, which inhibits effector T cell function. These results demonstrate the utility of Tres in identifying biomarkers of T cell effectiveness and potential therapeutic targets for immunotherapies in solid tumors. 10.1038/s41591-022-01799-y
    Interleukin-11 signaling promotes cellular reprogramming and limits fibrotic scarring during tissue regeneration. Allanki Srinivas,Strilic Boris,Scheinberger Lilly,Onderwater Yeszamin L,Marks Alora,Günther Stefan,Preussner Jens,Kikhi Khrievono,Looso Mario,Stainier Didier Y R,Reischauer Sven Science advances Damage-induced fibrotic scarring limits tissue regeneration in mammals and is a leading cause of morbidity. In contrast, species like zebrafish can regenerate damaged tissues without excessive fibrosis. However, whether specific signaling pathways can both limit fibrosis and promote regeneration is unclear. Here, we show that interleukin-11 (Il-11)/Stat3 signaling has such a dual function. Zebrafish lacking Il-11 receptor function display severely compromised heart, fin, and scale regeneration. Deep phenotyping and transcriptional analysis of adult hearts and fins show that Il-11 signaling drives cellular reprogramming to orchestrate global and tissue-specific regenerative programs and broadly antagonizes hallmarks of adult mammalian scarring. Mechanistically, our data indicate that IL-11 signaling in endothelial cells antagonizes profibrotic transforming growth factor–β signaling and endothelial-to-mesenchymal transition, limiting scarring and promoting cardiomyocyte repopulation, after injury. Overall, our findings position damage-induced Il-11/Stat3 signaling in a key role limiting fibrosis and promoting regeneration, revealing novel targets for regenerative therapies. 10.1126/sciadv.abg6497
    Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing. Karunakaran Mohindar M,Willcox Carrie R,Salim Mahboob,Paletta Daniel,Fichtner Alina S,Noll Angela,Starick Lisa,Nöhren Anna,Begley Charlotte R,Berwick Katie A,Chaleil Raphaël A G,Pitard Vincent,Déchanet-Merville Julie,Bates Paul A,Kimmel Brigitte,Knowles Timothy J,Kunzmann Volker,Walter Lutz,Jeeves Mark,Mohammed Fiyaz,Willcox Benjamin E,Herrmann Thomas Immunity Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner. 10.1016/j.immuni.2020.02.014
    Intestinal inflammation alters the antigen-specific immune response to a skin commensal. Cell reports Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4 regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4 circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4Il1r1 mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases. 10.1016/j.celrep.2022.110891
    Specific mesoderm subset derived from human pluripotent stem cells ameliorates microvascular pathology in type 2 diabetic mice. Science advances Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDRCD56APLNR (KNA) expression. KNA cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice ( mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of mice injected with either control or diabetic donor-derived KNA cells showed correction of aberrant signaling in retinas toward normal healthy retina. These data provide "proof of principle" that KNA cells restore perfusion and correct vascular dysfunction in mice. 10.1126/sciadv.abm5559
    Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19. Peng Yanchun,Mentzer Alexander J,Liu Guihai,Yao Xuan,Yin Zixi,Dong Danning,Dejnirattisai Wanwisa,Rostron Timothy,Supasa Piyada,Liu Chang,López-Camacho César,Slon-Campos Jose,Zhao Yuguang,Stuart David I,Paesen Guido C,Grimes Jonathan M,Antson Alfred A,Bayfield Oliver W,Hawkins Dorothy E D P,Ker De-Sheng,Wang Beibei,Turtle Lance,Subramaniam Krishanthi,Thomson Paul,Zhang Ping,Dold Christina,Ratcliff Jeremy,Simmonds Peter,de Silva Thushan,Sopp Paul,Wellington Dannielle,Rajapaksa Ushani,Chen Yi-Ling,Salio Mariolina,Napolitani Giorgio,Paes Wayne,Borrow Persephone,Kessler Benedikt M,Fry Jeremy W,Schwabe Nikolai F,Semple Malcolm G,Baillie J Kenneth,Moore Shona C,Openshaw Peter J M,Ansari M Azim,Dunachie Susanna,Barnes Eleanor,Frater John,Kerr Georgina,Goulder Philip,Lockett Teresa,Levin Robert,Zhang Yonghong,Jing Ronghua,Ho Ling-Pei, , ,Cornall Richard J,Conlon Christopher P,Klenerman Paul,Screaton Gavin R,Mongkolsapaya Juthathip,McMichael Andrew,Knight Julian C,Ogg Graham,Dong Tao Nature immunology The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4 and/or CD8 epitopes, including six immunodominant regions. Six optimized CD8 epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8 T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design. 10.1038/s41590-020-0782-6
    Two subsets of stem-like CD8 memory T cell progenitors with distinct fate commitments in humans. Galletti Giovanni,De Simone Gabriele,Mazza Emilia M C,Puccio Simone,Mezzanotte Claudia,Bi Timothy M,Davydov Alexey N,Metsger Maria,Scamardella Eloise,Alvisi Giorgia,De Paoli Federica,Zanon Veronica,Scarpa Alice,Camisa Barbara,Colombo Federico S,Anselmo Achille,Peano Clelia,Polletti Sara,Mavilio Domenico,Gattinoni Luca,Boi Shannon K,Youngblood Benjamin A,Jones Rhiannon E,Baird Duncan M,Gostick Emma,Llewellyn-Lacey Sian,Ladell Kristin,Price David A,Chudakov Dmitriy M,Newell Evan W,Casucci Monica,Lugli Enrico Nature immunology T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8 memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8 memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8 memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines. 10.1038/s41590-020-0791-5
    Different human resting memory CD4 T cell subsets show similar low inducibility of latent HIV-1 proviruses. Kwon Kyungyoon J,Timmons Andrew E,Sengupta Srona,Simonetti Francesco R,Zhang Hao,Hoh Rebecca,Deeks Steven G,Siliciano Janet D,Siliciano Robert F Science translational medicine The latent reservoir of HIV-1 in resting CD4 T cells is a major barrier to cure. It is unclear whether the latent reservoir resides principally in particular subsets of CD4 T cells, a finding that would have implications for understanding its stability and developing curative therapies. Recent work has shown that proliferation of HIV-1-infected CD4 T cells is a major factor in the generation and persistence of the latent reservoir and that latently infected T cells that have clonally expanded in vivo can proliferate in vitro without producing virions. In certain CD4 memory T cell subsets, the provirus may be in a deeper state of latency, allowing the cell to proliferate without producing viral proteins, thus permitting escape from immune clearance. To evaluate this possibility, we used a multiple stimulation viral outgrowth assay to culture resting naïve, central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4 T cells from 10 HIV-1-infected individuals on antiretroviral therapy. On average, only 1.7% of intact proviruses across all T cell subsets were induced to transcribe viral genes and release replication-competent virus after stimulation of the cells. We found no consistent enrichment of intact or inducible proviruses in any T cell subset. Furthermore, we observed notable plasticity among the canonical memory T cell subsets after activation in vitro and saw substantial person-to-person variability in the inducibility of infectious virus release. This finding complicates the vision for a targeted approach for HIV-1 cure based on T cell memory subsets. 10.1126/scitranslmed.aax6795
    Dysbiosis exacerbates colitis by promoting ubiquitination and accumulation of the innate immune adaptor STING in myeloid cells. Immunity Alterations in the cGAS-STING DNA-sensing pathway affect intestinal homeostasis. We sought to delineate the functional role of STING in intestinal inflammation. Increased STING expression was a feature of intestinal inflammation in mice with colitis and in humans afflicted with inflammatory bowel disease. Mice bearing an allele rendering STING constitutively active exhibited spontaneous colitis and dysbiosis, as well as progressive chronic intestinal inflammation and fibrosis. Bone marrow chimera experiments revealed STING accumulation in intestinal macrophages and monocytes as the initial driver of inflammation. Depletion of Gram-negative bacteria prevented STING accumulation in these cells and alleviated intestinal inflammation. STING accumulation occurred at the protein rather than transcript level, suggesting post-translational stabilization. We found that STING was ubiquitinated in myeloid cells, and this K63-linked ubiquitination could be elicited by bacterial products, including cyclic di-GMP. Our findings suggest a positive feedback loop wherein dysbiosis foments the accumulation of STING in intestinal myeloid cells, driving intestinal inflammation. 10.1016/j.immuni.2021.05.008
    Publisher Correction: The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens. Canton Johnathan,Blees Hanna,Henry Conor M,Buck Michael D,Schulz Oliver,Rogers Neil C,Childs Eleanor,Zelenay Santiago,Rhys Hefin,Domart Marie-Charlotte,Collinson Lucy,Alloatti Andres,Ellison Cara J,Amigorena Sebastian,Papayannopoulos Venizelos,Thomas David C,Randow Felix,Reis e Sousa Caetano Nature immunology 10.1038/s41590-021-00885-6
    The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens. Nature immunology Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8 T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity. 10.1038/s41590-020-00824-x
    Microglia use TAM receptors to detect and engulf amyloid β plaques. Huang Youtong,Happonen Kaisa E,Burrola Patrick G,O'Connor Carolyn,Hah Nasun,Huang Ling,Nimmerjahn Axel,Lemke Greg Nature immunology Two microglial TAM receptor tyrosine kinases, Axl and Mer, have been linked to Alzheimer's disease, but their roles in disease have not been tested experimentally. We find that in Alzheimer's disease and its mouse models, induced expression of Axl and Mer in amyloid plaque-associated microglia was coupled to induced plaque decoration by the TAM ligand Gas6 and its co-ligand phosphatidylserine. In the APP/PS1 mouse model of Alzheimer's disease, genetic ablation of Axl and Mer resulted in microglia that were unable to normally detect, respond to, organize or phagocytose amyloid-β plaques. These major deficits notwithstanding, TAM-deficient APP/PS1 mice developed fewer dense-core plaques than APP/PS1 mice with normal microglia. Our findings reveal that the TAM system is an essential mediator of microglial recognition and engulfment of amyloid plaques and that TAM-driven microglial phagocytosis does not inhibit, but rather promotes, dense-core plaque development. 10.1038/s41590-021-00913-5
    Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide. Montiel Virginie,Bella Ramona,Michel Lauriane Y M,Esfahani Hrag,De Mulder Delphine,Robinson Emma L,Deglasse Jean-Philippe,Tiburcy Malte,Chow Pak Hin,Jonas Jean-Christophe,Gilon Patrick,Steinhorn Benjamin,Michel Thomas,Beauloye Christophe,Bertrand Luc,Farah Charlotte,Dei Zotti Flavia,Debaix Huguette,Bouzin Caroline,Brusa Davide,Horman Sandrine,Vanoverschelde Jean-Louis,Bergmann Olaf,Gilis Dimitri,Rooman Marianne,Ghigo Alessandra,Geninatti-Crich Simonetta,Yool Andrea,Zimmermann Wolfram H,Roderick H Llewelyn,Devuyst Olivier,Balligand Jean-Luc Science translational medicine Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal is a major isoform. expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (HO), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of HO through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by HO Deletion of or selective blockade of the AQP1 intrasubunit pore inhibited HO transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of HO by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies. 10.1126/scitranslmed.aay2176
    DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity. Immunity Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging. 10.1016/j.immuni.2021.10.001
    TGF-β-mediated silencing of genomic organizer SATB1 promotes Tfh cell differentiation and formation of intra-tumoral tertiary lymphoid structures. Immunity The immune checkpoint receptor PD-1 on T follicular helper (Tfh) cells promotes Tfh:B cell interactions and appropriate positioning within tissues. Here, we examined the impact of regulation of PD-1 expression by the genomic organizer SATB1 on Tfh cell differentiation. Vaccination of CD4Satb1 mice enriched for antigen-specific Tfh cells, and TGF-β-mediated repression of SATB1 enhanced Tfh differentiation of human T cells. Mechanistically, high Icos expression in Satb1 CD4 T cells promoted Tfh cell differentiation by preventing T follicular regulatory cell skewing and resulted in increased isotype-switched B cell responses in vivo. Ovarian tumors in CD4Satb1 mice accumulated tumor antigen-specific, LIGHTCXCL13IL-21 Tfh cells and tertiary lymphoid structures (TLS). TLS formation decreased tumor growth in a CD4 T cell and CXCL13-dependent manner. The transfer of Tfh cells, but not naive CD4 T cells, induced TLS at tumor beds and decreased tumor growth. Thus, TGF-β-mediated silencing of Satb1 licenses Tfh cell differentiation, providing insight into the genesis of TLS within tumors. 10.1016/j.immuni.2021.12.007
    Impaired immune cell cytotoxicity in severe COVID-19 is IL-6 dependent. Mazzoni Alessio,Salvati Lorenzo,Maggi Laura,Capone Manuela,Vanni Anna,Spinicci Michele,Mencarini Jessica,Caporale Roberto,Peruzzi Benedetta,Antonelli Alberto,Trotta Michele,Zammarchi Lorenzo,Ciani Luca,Gori Leonardo,Lazzeri Chiara,Matucci Andrea,Vultaggio Alessandra,Rossi Oliviero,Almerigogna Fabio,Parronchi Paola,Fontanari Paolo,Lavorini Federico,Peris Adriano,Rossolini Gian Maria,Bartoloni Alessandro,Romagnani Sergio,Liotta Francesco,Annunziato Francesco,Cosmi Lorenzo The Journal of clinical investigation BACKGROUNDCoronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODSWe performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTSPatients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSIONThe association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDINGThis study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the "Excellence Departments 2018-2022 Project") derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy). 10.1172/JCI138554
    An immunodominant NP-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease. Peng Yanchun,Felce Suet Ling,Dong Danning,Penkava Frank,Mentzer Alexander J,Yao Xuan,Liu Guihai,Yin Zixi,Chen Ji-Li,Lu Yongxu,Wellington Dannielle,Wing Peter A C,Dominey-Foy Delaney C C,Jin Chen,Wang Wenbo,Hamid Megat Abd,Fernandes Ricardo A,Wang Beibei,Fries Anastasia,Zhuang Xiaodong,Ashley Neil,Rostron Timothy,Waugh Craig,Sopp Paul,Hublitz Philip,Beveridge Ryan,Tan Tiong Kit,Dold Christina,Kwok Andrew J,Rich-Griffin Charlotte,Dejnirattisa Wanwisa,Liu Chang,Kurupati Prathiba,Nassiri Isar,Watson Robert A,Tong Orion,Taylor Chelsea A,Kumar Sharma Piyush,Sun Bo,Curion Fabiola,Revale Santiago,Garner Lucy C,Jansen Kathrin,Ferreira Ricardo C,Attar Moustafa,Fry Jeremy W,Russell Rebecca A, ,Stauss Hans J,James William,Townsend Alain,Ho Ling-Pei,Klenerman Paul,Mongkolsapaya Juthathip,Screaton Gavin R,Dendrou Calliope,Sansom Stephen N,Bashford-Rogers Rachael,Chain Benny,Smith Geoffrey L,McKeating Jane A,Fairfax Benjamin P,Bowness Paul,McMichael Andrew J,Ogg Graham,Knight Julian C,Dong Tao Nature immunology NP-B*07:02-specific CD8 T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design. 10.1038/s41590-021-01084-z
    Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation. Cell metabolism Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4 T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes. 10.1016/j.cmet.2020.04.015
    Nov/CCN3 Enhances Cord Blood Engraftment by Rapidly Recruiting Latent Human Stem Cell Activity. Cell stem cell Umbilical cord blood (UCB) has had considerable impact in pediatric stem cell transplantation, but its wider use is limited in part by unit size. Long-term ex vivo culture offers one approach to increase engraftment capacity by seeking to expand stem and progenitor cells. Here, we show brief incubation (8 h) of UCB CD34+ cells with the matricellular regulator Nov (CCN3) increases the frequency of serially transplantable hematopoietic stem cells (HSCs) 6-fold. This rapid response suggests recruitment rather than expansion of stem cells; accordingly, in single-cell assays, Nov increases the clonogenicity of phenotypic HSCs without increasing their number through cell division. Recruitment is associated with both metabolic and transcriptional changes, and tracing of cell divisions demonstrates that the increased clonogenic activity resides within the undivided fraction of cells. Harnessing latent stem cell potential through recruitment-based approaches will inform understanding of stem cell state transitions with implications for translation to the clinic. 10.1016/j.stem.2020.02.012
    Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer. Fumagalli Arianna,Oost Koen C,Kester Lennart,Morgner Jessica,Bornes Laura,Bruens Lotte,Spaargaren Lisa,Azkanaz Maria,Schelfhorst Tim,Beerling Evelyne,Heinz Maria C,Postrach Daniel,Seinstra Danielle,Sieuwerts Anieta M,Martens John W M,van der Elst Stefan,van Baalen Martijn,Bhowmick Debajit,Vrisekoop Nienke,Ellenbroek Saskia I J,Suijkerbuijk Saskia J E,Snippert Hugo J,van Rheenen Jacco Cell stem cell Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5 and formed distant metastases in which Lgr5 CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5 cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors. 10.1016/j.stem.2020.02.008
    Bacterial-Driven Inflammation and Mutant Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy. DeStefano Shields Christina E,White James R,Chung Liam,Wenzel Alyssa,Hicks Jessica L,Tam Ada J,Chan June L,Dejea Christine M,Fan Hongni,Michel John,Maiuri Ashley R,Sriramkumar Shruthi,Podicheti Ram,Rusch Douglas B,Wang Hao,De Marzo Angelo M,Besharati Sepideh,Anders Robert A,Baylin Stephen B,O'Hagan Heather M,Housseau Franck,Sears Cynthia L Cancer discovery Colorectal cancer is multifaceted, with subtypes defined by genetic, histologic, and immunologic features that are potentially influenced by inflammation, mutagens, and/or microbiota. Colorectal cancers with activating mutations in are associated with distinct clinical characteristics, although the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (Min) enterotoxigenic (ETBF) murine model is characterized by IL17-dependent, distal colon adenomas. Herein, we report that the addition of the mutation to this model results in the emergence of a distinct locus of midcolon tumors. In ETBF-colonized Min (BLM) mice, tumors have similarities to human tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8 T cells, express IFNγ signatures, and are sensitive to anti-PD-L1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in colorectal cancer pathogenesis and sensitivity to immunotherapy. SIGNIFICANCE: Colorectal cancers with mutations have distinct characteristics. We present evidence of specific colorectal cancer gene-microbial interactions in which colonization with toxigenic bacteria drives tumorigenesis in Min mice, wherein tumors phenocopy aspects of human -mutated tumors and have a distinct IFNγ-dominant immune microenvironment uniquely responsive to immune checkpoint blockade.. 10.1158/2159-8290.CD-20-0770
    Dnmt1 has de novo activity targeted to transposable elements. Haggerty Chuck,Kretzmer Helene,Riemenschneider Christina,Kumar Abhishek Sampath,Mattei Alexandra L,Bailly Nina,Gottfreund Judith,Giesselmann Pay,Weigert Raha,Brändl Björn,Giehr Pascal,Buschow René,Galonska Christina,von Meyenn Ferdinand,Pappalardi Melissa B,McCabe Michael T,Wittler Lars,Giesecke-Thiel Claudia,Mielke Thorsten,Meierhofer David,Timmermann Bernd,Müller Franz-Josef,Walter Jörn,Meissner Alexander Nature structural & molecular biology DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development. 10.1038/s41594-021-00603-8
    Anti-Inflammatory Drugs Remodel the Tumor Immune Environment to Enhance Immune Checkpoint Blockade Efficacy. Pelly Victoria S,Moeini Agrin,Roelofsen Lisanne M,Bonavita Eduardo,Bell Charlotte R,Hutton Colin,Blanco-Gomez Adrian,Banyard Antonia,Bromley Christian P,Flanagan Eimear,Chiang Shih-Chieh,Jørgensen Claus,Schumacher Ton N,Thommen Daniela S,Zelenay Santiago Cancer discovery Identifying strategies to improve the efficacy of immune checkpoint blockade (ICB) remains a major clinical need. Here, we show that therapeutically targeting the COX2/PGE/EP2-4 pathway with widely used nonsteroidal and steroidal anti-inflammatory drugs synergized with ICB in mouse cancer models. We exploited a bilateral surgery model to distinguish responders from nonresponders shortly after treatment and identified acute IFNγ-driven transcriptional remodeling in responder mice, which was also associated with patient benefit to ICB. Monotherapy with COX2 inhibitors or EP2-4 PGE receptor antagonists rapidly induced this response program and, in combination with ICB, increased the intratumoral accumulation of effector T cells. Treatment of patient-derived tumor fragments from multiple cancer types revealed a similar shift in the tumor inflammatory environment to favor T-cell activation. Our findings establish the COX2/PGE/EP2-4 axis as an independent immune checkpoint and a readily translatable strategy to rapidly switch the tumor inflammatory profile from cold to hot. SIGNIFICANCE: Through performing in-depth profiling of mice and human tumors, this study identifies mechanisms by which anti-inflammatory drugs rapidly alter the tumor immune landscape to enhance tumor immunogenicity and responses to immune checkpoint inhibitors... 10.1158/2159-8290.CD-20-1815
    The global response to the COVID-19 pandemic: how have immunology societies contributed? Nature reviews. Immunology The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 10.1038/s41577-020-00428-4
    Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern. Science translational medicine The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain–RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern. 10.1126/scitranslmed.abj5413
    Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells. Cancer discovery Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II-mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance and . YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1-CDK9 complex elicited RNA mA modification-dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1-CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. SIGNIFICANCE: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti-PD-1 response through regulating the mA modification program, linking epigenetic regulation to immunomodulatory function in GBM.. 10.1158/2159-8290.CD-20-1848
    The COVID-19 puzzle: deciphering pathophysiology and phenotypes of a new disease entity. Osuchowski Marcin F,Winkler Martin S,Skirecki Tomasz,Cajander Sara,Shankar-Hari Manu,Lachmann Gunnar,Monneret Guillaume,Venet Fabienne,Bauer Michael,Brunkhorst Frank M,Weis Sebastian,Garcia-Salido Alberto,Kox Matthijs,Cavaillon Jean-Marc,Uhle Florian,Weigand Markus A,Flohé Stefanie B,Wiersinga W Joost,Almansa Raquel,de la Fuente Amanda,Martin-Loeches Ignacio,Meisel Christian,Spinetti Thibaud,Schefold Joerg C,Cilloniz Catia,Torres Antoni,Giamarellos-Bourboulis Evangelos J,Ferrer Ricard,Girardis Massimo,Cossarizza Andrea,Netea Mihai G,van der Poll Tom,Bermejo-Martín Jesús F,Rubio Ignacio The Lancet. Respiratory medicine The zoonotic SARS-CoV-2 virus that causes COVID-19 continues to spread worldwide, with devastating consequences. While the medical community has gained insight into the epidemiology of COVID-19, important questions remain about the clinical complexities and underlying mechanisms of disease phenotypes. Severe COVID-19 most commonly involves respiratory manifestations, although other systems are also affected, and acute disease is often followed by protracted complications. Such complex manifestations suggest that SARS-CoV-2 dysregulates the host response, triggering wide-ranging immuno-inflammatory, thrombotic, and parenchymal derangements. We review the intricacies of COVID-19 pathophysiology, its various phenotypes, and the anti-SARS-CoV-2 host response at the humoral and cellular levels. Some similarities exist between COVID-19 and respiratory failure of other origins, but evidence for many distinctive mechanistic features indicates that COVID-19 constitutes a new disease entity, with emerging data suggesting involvement of an endotheliopathy-centred pathophysiology. Further research, combining basic and clinical studies, is needed to advance understanding of pathophysiological mechanisms and to characterise immuno-inflammatory derangements across the range of phenotypes to enable optimum care for patients with COVID-19. 10.1016/S2213-2600(21)00218-6
    An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples. Maas Roeltje R,Soukup Klara,Klemm Florian,Kornete Mara,Bowman Robert L,Bedel Romain,Marie Damien N,Álvarez-Prado Ángel F,Labes Danny,Wilson Anne,Brouland Jean-Philippe,Daniel Roy T,Hegi Monika E,Joyce Johanna A Nature protocols Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment. 10.1038/s41596-021-00594-2
    Aberrant gut-microbiota-immune-brain axis development in premature neonates with brain damage. Seki David,Mayer Margareta,Hausmann Bela,Pjevac Petra,Giordano Vito,Goeral Katharina,Unterasinger Lukas,Klebermaß-Schrehof Katrin,De Paepe Kim,Van de Wiele Tom,Spittler Andreas,Kasprian Gregor,Warth Benedikt,Berger Angelika,Berry David,Wisgrill Lukas Cell host & microbe Premature infants are at substantial risk for suffering from perinatal white matter injury. Though the gut microbiota has been implicated in early-life development, a detailed understanding of the gut-microbiota-immune-brain axis in premature neonates is lacking. Here, we profiled the gut microbiota, immunological, and neurophysiological development of 60 extremely premature infants, which received standard hospital care including antibiotics and probiotics. We found that maturation of electrocortical activity is suppressed in infants with severe brain damage. This is accompanied by elevated γδ T cell levels and increased T cell secretion of vascular endothelial growth factor and reduced secretion of neuroprotectants. Notably, Klebsiella overgrowth in the gut is highly predictive for brain damage and is associated with a pro-inflammatory immunological tone. These results suggest that aberrant development of the gut-microbiota-immune-brain axis may drive or exacerbate brain injury in extremely premature neonates and represents a promising target for novel intervention strategies. 10.1016/j.chom.2021.08.004
    Persistent B cell memory after SARS-CoV-2 vaccination is functional during breakthrough infections. Terreri Sara,Piano Mortari Eva,Vinci Maria Rosaria,Russo Cristina,Alteri Claudia,Albano Christian,Colavita Francesca,Gramigna Giulia,Agrati Chiara,Linardos Giulia,Coltella Luana,Colagrossi Luna,Deriu Gloria,Ciofi Degli Atti Marta,Rizzo Caterina,Scarsella Marco,Brugaletta Rita,Camisa Vincenzo,Santoro Annapaola,Roscilli Giuseppe,Pavoni Emiliano,Muzi Alessia,Magnavita Nicola,Scutari Rossana,Villani Alberto,Raponi Massimiliano,Locatelli Franco,Perno Carlo Federico,Zaffina Salvatore,Carsetti Rita Cell host & microbe Breakthrough SARS-CoV-2 infections in fully vaccinated individuals are considered a consequence of waning immunity. Serum antibodies represent the most measurable outcome of vaccine-induced B cell memory. When antibodies decline, memory B cells are expected to persist and perform their function, preventing clinical disease. We investigated whether BNT162b2 mRNA vaccine induces durable and functional B cell memory in vivo against SARS-CoV-2 3, 6, and 9 months after the second dose in a cohort of health care workers (HCWs). While we observed physiological decline of SARS-CoV-2-specific antibodies, memory B cells persist and increase until 9 months after immunization. HCWs with breakthrough infections had no signs of waning immunity. In 3-4 days, memory B cells responded to SARS-CoV-2 infection by producing high levels of specific antibodies in the serum and anti-Spike IgA in the saliva. Antibodies to the viral nucleoprotein were produced with the slow kinetics typical of the response to a novel antigen. 10.1016/j.chom.2022.01.003
    Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment. Schulte-Schrepping Jonas,Reusch Nico,Paclik Daniela,Baßler Kevin,Schlickeiser Stephan,Zhang Bowen,Krämer Benjamin,Krammer Tobias,Brumhard Sophia,Bonaguro Lorenzo,De Domenico Elena,Wendisch Daniel,Grasshoff Martin,Kapellos Theodore S,Beckstette Michael,Pecht Tal,Saglam Adem,Dietrich Oliver,Mei Henrik E,Schulz Axel R,Conrad Claudia,Kunkel Désirée,Vafadarnejad Ehsan,Xu Cheng-Jian,Horne Arik,Herbert Miriam,Drews Anna,Thibeault Charlotte,Pfeiffer Moritz,Hippenstiel Stefan,Hocke Andreas,Müller-Redetzky Holger,Heim Katrin-Moira,Machleidt Felix,Uhrig Alexander,Bosquillon de Jarcy Laure,Jürgens Linda,Stegemann Miriam,Glösenkamp Christoph R,Volk Hans-Dieter,Goffinet Christine,Landthaler Markus,Wyler Emanuel,Georg Philipp,Schneider Maria,Dang-Heine Chantip,Neuwinger Nick,Kappert Kai,Tauber Rudolf,Corman Victor,Raabe Jan,Kaiser Kim Melanie,Vinh Michael To,Rieke Gereon,Meisel Christian,Ulas Thomas,Becker Matthias,Geffers Robert,Witzenrath Martin,Drosten Christian,Suttorp Norbert,von Kalle Christof,Kurth Florian,Händler Kristian,Schultze Joachim L,Aschenbrenner Anna C,Li Yang,Nattermann Jacob,Sawitzki Birgit,Saliba Antoine-Emmanuel,Sander Leif Erik, Cell Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19. 10.1016/j.cell.2020.08.001
    Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection. Triana Sergio,Stanifer Megan L,Metz-Zumaran Camila,Shahraz Mohammed,Mukenhirn Markus,Kee Carmon,Serger Clara,Koschny Ronald,Ordoñez-Rueda Diana,Paulsen Malte,Benes Vladimir,Boulant Steeve,Alexandrov Theodore Molecular systems biology Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single-cell RNA-Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis. 10.15252/msb.20209833
    Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos. Molecular systems biology The correct wiring of neuronal circuits is one of the most complex processes in development, since axons form highly specific connections out of a vast number of possibilities. Circuit structure is genetically determined in vertebrates and invertebrates, but the mechanisms guiding each axon to precisely innervate a unique pre-specified target cell are poorly understood. We investigated Drosophila embryonic motoneurons using single-cell genomics, imaging, and genetics. We show that a cell-specific combination of homeodomain transcription factors and downstream immunoglobulin domain proteins is expressed in individual cells and plays an important role in determining cell-specific connections between differentiated motoneurons and target muscles. We provide genetic evidence for a functional role of five homeodomain transcription factors and four immunoglobulins in the neuromuscular wiring. Knockdown and ectopic expression of these homeodomain transcription factors induces cell-specific synaptic wiring defects that are partly phenocopied by genetic modulations of their immunoglobulin targets. Taken together, our data suggest that homeodomain transcription factor and immunoglobulin molecule expression could be directly linked and function as a crucial determinant of neuronal circuit structure. 10.15252/msb.202110255
    Single-cell analysis of human B cell maturation predicts how antibody class switching shapes selection dynamics. King Hamish W,Orban Nara,Riches John C,Clear Andrew J,Warnes Gary,Teichmann Sarah A,James Louisa K Science immunology Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre-germinal center state primed to undergo class switch recombination and dissect how antibody class-dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell-mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection. 10.1126/sciimmunol.abe6291
    Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2. Harly Christelle,Joyce Stephen Paul,Domblides Charlotte,Bachelet Thomas,Pitard Vincent,Mannat Charlotte,Pappalardo Angela,Couzi Lionel,Netzer Sonia,Massara Layal,Obre Emilie,Hawchar Omar,Lartigue Lydia,Claverol Stéphane,Cano Carla,Moreau Jean-François,Mahouche Isabelle,Soubeyran Isabelle,Rossignol Rodrigue,Viollet Benoit,Willcox Carrie R,Mohammed Fiyaz,Willcox Benjamin E,Faustin Benjamin,Déchanet-Merville Julie Science immunology Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer. 10.1126/sciimmunol.aba9010
    Author Correction: BCAT1 restricts αKG levels in AML stem cells leading to IDH-like DNA hypermethylation. Raffel Simon,Falcone Mattia,Kneisel Niclas,Hansson Jenny,Wang Wei,Lutz Christoph,Bullinger Lars,Poschet Gernot,Nonnenmacher Yannic,Barnert Andrea,Bahr Carsten,Zeisberger Petra,Przybylla Adriana,Sohn Markus,Tönjes Martje,Erez Ayelet,Adler Lital,Jensen Patrizia,Scholl Claudia,Fröhling Stefan,Cocciardi Sibylle,Wuchter Patrick,Thiede Christian,Flörcken Anne,Westermann Jörg,Ehninger Gerhard,Lichter Peter,Hiller Karsten,Hell Rüdiger,Herrmann Carl,Ho Anthony D,Krijgsveld Jeroen,Radlwimmer Bernhard,Trumpp Andreas Nature In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online. 10.1038/s41586-018-0403-9
    Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus. Bashant Kathleen R,Aponte Angel M,Randazzo Davide,Rezvan Sangsari Paniz,Wood Alexander Jt,Bibby Jack A,West Erin E,Vassallo Arlette,Manna Zerai G,Playford Martin P,Jordan Natasha,Hasni Sarfaraz,Gucek Marjan,Kemper Claudia,Conway Morris Andrew,Morgan Nicole Y,Toepfner Nicole,Guck Jochen,Mehta Nehal N,Chilvers Edwin R,Summers Charlotte,Kaplan Mariana J Annals of the rheumatic diseases OBJECTIVES:Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. METHODS:Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. RESULTS:Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. CONCLUSIONS:Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy. 10.1136/annrheumdis-2020-218338
    Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. Maude Shannon L,Laetsch Theodore W,Buechner Jochen,Rives Susana,Boyer Michael,Bittencourt Henrique,Bader Peter,Verneris Michael R,Stefanski Heather E,Myers Gary D,Qayed Muna,De Moerloose Barbara,Hiramatsu Hidefumi,Schlis Krysta,Davis Kara L,Martin Paul L,Nemecek Eneida R,Yanik Gregory A,Peters Christina,Baruchel Andre,Boissel Nicolas,Mechinaud Francoise,Balduzzi Adriana,Krueger Joerg,June Carl H,Levine Bruce L,Wood Patricia,Taran Tetiana,Leung Mimi,Mueller Karen T,Zhang Yiyun,Sen Kapildeb,Lebwohl David,Pulsipher Michael A,Grupp Stephan A The New England journal of medicine BACKGROUND:In a single-center phase 1-2a study, the anti-CD19 chimeric antigen receptor (CAR) T-cell therapy tisagenlecleucel produced high rates of complete remission and was associated with serious but mainly reversible toxic effects in children and young adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). METHODS:We conducted a phase 2, single-cohort, 25-center, global study of tisagenlecleucel in pediatric and young adult patients with CD19+ relapsed or refractory B-cell ALL. The primary end point was the overall remission rate (the rate of complete remission or complete remission with incomplete hematologic recovery) within 3 months. RESULTS:For this planned analysis, 75 patients received an infusion of tisagenlecleucel and could be evaluated for efficacy. The overall remission rate within 3 months was 81%, with all patients who had a response to treatment found to be negative for minimal residual disease, as assessed by means of flow cytometry. The rates of event-free survival and overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 months. The median duration of remission was not reached. Persistence of tisagenlecleucel in the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were suspected to be related to tisagenlecleucel occurred in 73% of patients. The cytokine release syndrome occurred in 77% of patients, 48% of whom received tocilizumab. Neurologic events occurred in 40% of patients and were managed with supportive care, and no cerebral edema was reported. CONCLUSIONS:In this global study of CAR T-cell therapy, a single infusion of tisagenlecleucel provided durable remission with long-term persistence in pediatric and young adult patients with relapsed or refractory B-cell ALL, with transient high-grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849 .). 10.1056/NEJMoa1709866
    Chemotherapy-induced infiltration of neutrophils promotes pancreatic cancer metastasis via Gas6/AXL signalling axis. Bellomo Gaia,Rainer Carolyn,Quaranta Valeria,Astuti Yuliana,Raymant Meirion,Boyd Elzbieta,Stafferton Ruth,Campbell Fiona,Ghaneh Paula,Halloran Christopher M,Hammond Dean E,Morton Jennifer P,Palmer Daniel,Vimalachandran Dale,Jones Robert,Mielgo Ainhoa,Schmid Michael C Gut OBJECTIVE:Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease and cytotoxic chemotherapy is the standard of care treatment for patients with advanced disease. Here, we investigate how the microenvironment in PDAC liver metastases reacts to chemotherapy and its role in metastatic disease progression post-treatment, an area which is poorly understood. DESIGN:The impact of chemotherapy on metastatic disease progression and immune cell infiltrates was characterised using flow and mass cytometry combined with transcriptional and histopathological analysis in experimental PDAC liver metastases mouse models. Findings were validated in patient derived liver metastases and in an autochthonous PDAC mouse model. Human and murine primary cell cocultures and ex vivo patient-derived liver explants were deployed to gain mechanistical insights on whether and how chemotherapy affects the metastatic tumour microenvironment. RESULTS:We show that in vivo, chemotherapy induces an initial infiltration of proinflammatory macrophages into the liver and activates cytotoxic T cells, leading only to a temporary restraining of metastatic disease progression. However, after stopping treatment, neutrophils are recruited to the metastatic liver via CXCL1 and 2 secretion by metastatic tumour cells. These neutrophils express growth arrest specific 6 (Gas6) which leads to AXL receptor activation on tumour cells enabling their regrowth. Disruption of neutrophil infiltration or inhibition of the Gas6/AXL signalling axis in combination with chemotherapy inhibits metastatic growth. Chemotherapy increases Gas6 expression in circulating neutrophils from patients with metastatic pancreatic cancer and recombinant Gas6 is sufficient to promote tumour cell proliferation ex vivo, in patient-derived metastatic liver explants. CONCLUSION:Combining chemotherapy with Gas6/AXL or neutrophil targeted therapy could provide a therapeutic benefit for patients with metastatic pancreatic cancer. 10.1136/gutjnl-2021-325272
    Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas. Molecular cell Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems. 10.1016/j.molcel.2022.05.023