Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate pre-metastatic niche and bone tropism is largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a pre-metastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interacting with their receptor LGR4, promoted osteoclastic pre-metastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating WNT inhibitor DKK1 through Gαq and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppressing its receptor low-density lipoprotein-related receptors 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibiting canonical WNT signaling. In clinical samples, RSPO2, LGR4 and DKK1 expression showed positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of pre-metastatic niche for BCa bone metastasis, indicate RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.
Zhiying Yue, Xin Niu, Zengjin Yuan, Qin Qin, Wenhao Jiang, Liang He, Jingduo Gao, Yi Ding, Yanxi Liu, Ziwei Xu, Zhenxi Li, Zhengfeng Yang, Rong Li, Xiwen Xue, Yankun Gao, Fei Yue, Xiang H.-F. Zhang, Guohong Hu, Yi Wang, Yi Li, Geng Chen, Stefan Siwko, Alison Gartland, Ning Wang, Jianru Xiao, Mingyao Liu, Jian Luo
Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-β1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.
Muhammad Nasir Hayat Malik, Syed F. Hassnain Waqas, Jana Zeitvogel, Jingyuan Cheng, Robert Geffers, Zeinab Abu-Elbaha Gouda, Ahmed Mahrous Elsaman, Ahmed R. Radwan, Matthias Schefzyk, Peter Braubach, Bernd Auber, Ruth Olmer, Mathias Müsken, Lennart M. Roesner, Gisa Gerold, Sven Schuchardt, Sylvia Merkert, Ulrich Martin, Felix Meissner, Thomas Werfel, Frank Pessler
Memory B cells (MBC) can provide a recall response able to supplement waning antibodies with an affinity-matured response better able to neutralise variant viruses. We studied a cohort of elderly care home residents and younger staff (median age 87yrs and 56yrs respectively) who had survived COVID-19 outbreaks with only mild/asymptomatic infection. The cohort was selected to enrich for a high proportion who had lost neutralising antibodies (nAb), to specifically investigate the reserve immunity from SARS-CoV-2-specific MBC in this setting. Class-switched spike and RBD-tetramer-binding MBC persisted five months post-mild/asymptomatic SARS-CoV-2 infection, irrespective of age. The majority of spike/RBD-specific MBC had a classical phenotype but activated memory B cells, that may indicate ongoing antigenic stimulation or inflammation, were expanded in the elderly. Spike/RBD-specific MBC remained detectable in the majority who had lost nAb, although at lower frequencies and with a reduced IgG/IgA isotype ratio. Functional spike/S1/RBD-specific recall was also detectable by ELISpot in some who had lost nAb, but was significantly impaired in the elderly. Our findings demonstrate a reserve of SARS-CoV-2-specific MBC persists beyond loss of nAb, but highlight the need for careful monitoring of functional defects in spike/RBD-specific B cell immunity in the elderly.
Anna Jeffery-Smith, Alice R. Burton, Sabela Lens, Chloe Rees-Spear, Jessica Davies, Monika Patel, Robin Gopal, Luke Muir, Felicity Aiano, Katie J. Doores, J. Yimmy Chow, Shamez N. Ladhani, Maria Zambon, Laura E. McCoy, Mala K. Maini.
Vast numbers of differentially expressed genes and perturbed networks have been identified in Alzheimer’s disease (AD), however neither disease- nor brain region-specificity of these transcriptome alterations have been explored. Using RNA sequencing data from 231 temporal cortex and 224 cerebellum samples of patients with AD and progressive supranuclear palsy (PSP), a tauopathy, we identify a striking correlation in the directionality and magnitude of gene expression changes between these two neurodegenerative proteinopathies. Further, the transcriptome changes in AD and PSP are highly conserved between the temporal and cerebellar cortices, indicating highly similar transcriptional changes occur in pathologically affected and grossly less affected, albeit functionally connected, areas of the brain. Shared up- or down-regulated genes in AD and PSP are enriched in biological pathways. Many of these genes also have concordant protein changes and evidence of epigenetic control. These conserved transcriptomic alterations of two distinct proteinopathies in brain regions with and without significant gross neuropathology have broad implications. AD and other neurodegenerative diseases are likely characterized by common disease or compensatory pathways with widespread perturbations in the whole brain. These findings can be leveraged to develop multifaceted therapies and biomarkers that address these common, complex and ubiquitous molecular alterations in neurodegenerative diseases.
Xue Wang, Mariet Allen, Özkan İş, Joseph S. Reddy, Frederick Q. Tutor-New, Monica Castanedes Casey, Minerva M. Carrasquillo, Stephanie R. Oatman, Yuhao Min, Yan W. Asmann, Cory Funk, Thuy Nguyen, Charlotte C.G. Ho, Kimberly G. Malphrus, Nicholas T. Seyfried, Allan I. Levey, Steven G. Younkin, Melissa E. Murray, Dennis W. Dickson, Nathan D. Price, Todd E. Golde, Nilufer Ertekin-Taner
While negative selection of developing B cells in the periphery is well described, yet poorly understood, evidence of naïve B cell positive selection remains elusive. Using two humanized mouse models, we demonstrate that there is strong skewing of expressed immunoglobulin repertoire upon transit into the peripheral naïve B cell pool. This positive selection of expanded naïve B cells in humanized mice resembled that in healthy donors and was independent of autologous thymic tissue. In contrast, negative selection of autoreactive B cells required thymic-derived regulatory T cells (Tregs) and MHC class II-restricted self-antigen presentation by B cells. Indeed, both defective MHC class II expression on their B cells in rare bare lymphocyte syndrome patients or prevention of self-antigen presentation via HLA-DM inhibition in humanized mice result in the production of autoreactive naïve B cells. These latter observations suggest that Tregs repress autoreactive naïve B cells continuously produced by the bone marrow. Thus, a model emerges in which both positive and negative selection shape the human naïve B cell repertoire and that each process is mediated by fundamentally different molecular and cellular mechanisms.
Jeff W. Chen, Jean-Nicolas Schickel, Nikolaos Tsakiris, Joel Sng, Florent Arbogast, Delphine Bouis, Daniele Parisi, Ruchi Gera, Joshua M. Boeckers, Fabien R. Delmotte, Margaret Veselits, Catharina Schuetz, Eva-Maria Jacobsen, Carsten Posovszky, Ansgar S. Schulz, Klaus Schwarz, Marcus R. Clark, Laurence Menard, Eric Meffre
In chronic lymphocytic leukemia (CLL), the B-cell receptor (BCR) plays a critical role in disease development and progression as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlining BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each co-expressed by > 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (IG) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization linked with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B-cell fate decisions, possibly opening new avenues for CLL therapy.
Andrea N. Mazzarello, Eva Gentner-Göbel, Marcus Dühren-von Minden, Tatyana N. Tarasenko, Antonella Nicolò, Gerardo Ferrer, Stefano Vergani, Yun Liu, Davide Bagnara, Kanti R. Rai, Jan A. Burger, Peter J. McGuire, Palash C. Maity, Hassan Jumaa, Nicholas Chiorazzi
Various population of cells are recruited to the heart after cardiac injury but little is known about whether the cardiomyocyte directly regulates heart repair. In a murine model of ischemic cardiac injury, we demonstrate that the cardiomyocyte plays a pivotal role in heart repair by regulating nucleotide metabolism and fates of non-myocytes. Cardiac injury induced the expression of the ectonucleotidase ENPP1 that hydrolyzes extracellular ATP to form AMP. In response to AMP, the cardiomyocyte released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at OMP synthesis step, induced genotoxic stress and a p53 mediated cell death of cycling non-myocytes. As non-myocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors on small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in non-myocyte cells, augmented cardiac repair and post infarct heart function. These observations demonstrate that the cardiac muscle cell by releasing adenine and specific nucleosides after heart injury regulates pyrimidine metabolism in non-muscle cells and provide insight into how inter-cellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.
Shen Li, Tomohiro Yokota, Ping Wang, Johanna ten Hoeve, Feiyang Ma, Thuc M. Le, Evan R. Abt, Yonggang Zhou, Rimao Wu, Maxine Nanthavongdouangsy, Abraham Rodriguez, Yijie Wang, Yen-Ju Lin, Hayato Muranaka, Mark Sharpley, Demetrios T. Braddock, Vicky E. MacRae, Utpal Banerjee, Pei-Yu Chiou, Marcus Seldin, Dian Huang, Michael Teitell, Ilya Gertsman, Michael Jung, Steven J. Bensinger, Robert Damoiseaux, Kym Faull, Matteo Pellegrini, Aldons Lusis, Thomas G. Graeber, Caius G. Radu, Arjun Deb
BACKGROUND. Neoantigen-driven recognition and T cell-mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with Tumor-Infiltrating Lymphocytes (TILs). Yet, how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined. METHODS. Using barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic mela-noma patients who received ACT. RESULTS. We identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with in-creased survival, and that detection of engrafted CD8+ T cells in post-treatment (i.e. originating from the TIL infusion product) were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product. CONCLUSIONS. These data support previous case studies of neoepitope-specific CD8+ T cells in melanoma, and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells. FUNDING. NEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.
Nikolaj Pagh Kristensen, Christina Heeke, Siri A. Tvingsholm, Annie Borch, Arianna Draghi, Michael D. Crowther, Ibel Carri, Kamilla K. Munk, Jeppe Sejerø Holm, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Andrea M. Marquard, Zoltan Szallasi, Nicholas McGranahan, Rikke Andersen, Morten Nielsen, Göran B. Jönsson, Marco Donia, Inge Marie Svane, Sine Reker Hadrup
Food allergy affects an estimated 8% of children in the US. Oral immunotherapy (OIT) is a recently approved treatment, with outcomes ranging from sustained tolerance to food allergen to no apparent benefit. The immunological underpinnings that influence clinical outcomes of OIT still remain largely unresolved. Using single-cell RNA sequencing and paired TCRα/β sequencing, we assessed the transcriptomes of CD154+ and CD137+ peanut-reactive T helper cells from 12 peanut-allergic patients longitudinally throughout OIT. We observed expanded populations of cells expressing Th1, Th2, and Th17 signatures that further separated into six clonally distinct subsets. Four of these subsets demonstrated convergence of TCR sequences, suggesting antigen-driven T cell fate. Over the course of OIT, we observed suppression of Th2 and Th1 gene signatures in effector clonotypes but not Tfh-like clonotypes. Positive outcomes were associated with stronger suppression of Th2 signatures in Th2A-like cells, while treatment failure was associated with the expression of baseline inflammatory gene signatures that were present in Th1 and Th17 populations and unmodulated by OIT. These results demonstrate that differential clinical responses to OIT are associated both with pre-existing characteristics of peanut-reactive CD4+ T cells and with suppression of a subset of Th2 cells.
Brinda Monian, Ang A. Tu, Bert Ruiter, Duncan M. Morgan, Patrick M. Petrossian, Neal P. Smith, Todd M. Gierahn, Julia H. Ginder, Wayne G. Shreffler, J. Christopher Love
Tertiary lymphoid tissues (TLTs) facilitate local T- and B-cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here we identify TNF superfamily CD153-CD30 signaling between two unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL21 and IFNγ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153-CD30 signaling in TLT formation and propose targeting CD153-CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.
Yuki Sato, Akiko Oguchi, Yuji Fukushima, Kyoko Masuda, Naoya Toriu, Keisuke Taniguchi, Takahisa Yoshikawa, Xiaotong Cui, Makiko Kondo, Takeshi Hosoi, Shota Komidori, Yoko Shimizu, Harumi Fujita, Li Jiang, Yingyi Kong, Takashi Yamanashi, Jun Seita, Takuya Yamamoto, Shinya Toyokuni, Yoko Hamazaki, Masakazu Hattori, Yasunobu Yoshikai, Peter Boor, Jürgen Floege, Hiroshi Kawamoto, Yasuhiro Murakawa, Nagahiro Minato, Motoko Yanagita
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