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- Toll2011 at Lago di Garda: studying danger sensors by the guard towers at the lake
- Nat Immunol 12(9):805-808 (2011)
Nature Immunology | Meeting Report Toll2011 at Lago di Garda: studying danger sensors by the guard towers at the lake * Søren R Paludan1 * Ofer Levy2 * Andrew G Bowie3Journal name:Nature ImmunologyVolume: 12,Pages:805–808Year published:(2011)DOI:doi:10.1038/ni.0911-805Published online18 August 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg At the Toll2011 Meeting, recent advances in the field of innate immunity were presented and discussed. These insights have set new directions for both basic and translational research in this important field. View full text Author information * Abstract * Author information Affiliations * Søren R. Paludan is in the Department of Biomedicine, Aarhus University, Aarhus, Denmark. * Ofer Levy is in Division of Infectious Diseases, Children's Hospital Boston & Harvard Medical School, Boston, Massachusetts, USA. * Andrew G. Bowie is in the School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Søren R Paludan Author Details * Søren R Paludan Contact Søren R Paludan Search for this author in: * NPG journals * PubMed * Google Scholar * Ofer Levy Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew G Bowie Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - IDO: more than an enzyme
- Nat Immunol 12(9):809-811 (2011)
Article preview View full access options Nature Immunology | News and Views IDO: more than an enzyme * WanJun Chen1Journal name:Nature ImmunologyVolume: 12,Pages:809–811Year published:(2011)DOI:doi:10.1038/ni.2088Published online18 August 2011 In addition to its recognized function as an enzyme that catalyzes tryptophan, indoleamine 2,3-dioxygenase (IDO) acts as an intracellular signal transducer, in response to transforming growth factor-β (TGF-β), to induce a stably regulatory phenotype in plasmacytoid dendritic cells. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * WanJun Chen is in the Mucosal Immunology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * WanJun Chen Author Details * WanJun Chen Contact WanJun Chen Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - A 'Tsc, Tsc' keeps the kids quie(scen)t and holds down ROS
- Nat Immunol 12(9):811-812 (2011)
Article preview View full access options Nature Immunology | News and Views A 'Tsc, Tsc' keeps the kids quie(scen)t and holds down ROS * Mark Boothby1 * Keunwook Lee1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:811–812Year published:(2011)DOI:doi:10.1038/ni.2092Published online18 August 2011 Tuberins, the tumor-suppressor proteins that regulate signaling via the kinase mTOR, now emerge as essential enforcers of T cell quiescence that promote survival. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Mark Boothby and Keunwook Lee are in the Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mark Boothby Author Details * Mark Boothby Contact Mark Boothby Search for this author in: * NPG journals * PubMed * Google Scholar * Keunwook Lee Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - IL-17R signaling: new players get in on the Act1
- Nat Immunol 12(9):813-815 (2011)
Article preview View full access options Nature Immunology | News and Views IL-17R signaling: new players get in on the Act1 * Michael J May1Journal name:Nature ImmunologyVolume: 12,Pages:813–815Year published:(2011)DOI:doi:10.1038/ni.2093Published online18 August 2011 Proinflammatory signaling by the interleukin 17 receptor requires the adaptor Act1. Two studies identify an additional function for Act1 and identify new participants that regulate the inflammatory effects of IL-17. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Michael J. May is in the Department of Animal Biology, The School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Michael J May Author Details * Michael J May Contact Michael J May Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - IKKα takes control of canonical NF-κB activation
- Nat Immunol 12(9):815-816 (2011)
Article preview View full access options Nature Immunology | News and Views IKKα takes control of canonical NF-κB activation * Christiane Pelzer1 * Margot Thome1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:815–816Year published:(2011)DOI:doi:10.1038/ni.2082Published online18 August 2011 The kinase IKKα has a well-described activating role in the noncanonical transcription factor NF-κB pathway. Evidence now suggests that IKKα also inhibits the canonical NF-κB pathway by phosphorylating the scaffold protein TAX1BP1 to promote the assembly of the A20 ubiquitin-editing complex. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Christiane Pelzer and Margot Thome are in the Department of Biochemistry, University of Lausanne, Epalinges, Switzerland. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Margot Thome Author Details * Christiane Pelzer Search for this author in: * NPG journals * PubMed * Google Scholar * Margot Thome Contact Margot Thome Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - NLR functions in plant and animal immune systems: so far and yet so close
- Nat Immunol 12(9):817-826 (2011)
Nature Immunology | Review NLR functions in plant and animal immune systems: so far and yet so close * Takaki Maekawa1 * Thomas A Kufer2 * Paul Schulze-Lefert1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:817–826Year published:(2011)DOI:doi:10.1038/ni.2083Published online18 August 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In plants and animals, the NLR family of receptors perceives non-self and modified-self molecules inside host cells and mediates innate immune responses to microbial pathogens. Despite their similar biological functions and protein architecture, animal NLRs are normally activated by conserved microbe- or damage-associated molecular patterns, whereas plant NLRs typically detect strain-specific pathogen effectors. Plant NLRs recognize either the effector structure or effector-mediated modifications of host proteins. The latter indirect mechanism for the perception of non-self, as well as the within-species diversification of plant NLRs, maximize the capacity to recognize non-self through the use of a finite number of innate immunoreceptors. We discuss recent insights into NLR activation, signal initiation through the homotypic association of N-terminal domains and subcellular receptor dynamics in plants and compare those with NLR functions in animals. View full text Figures at a glance * Figure 1: Mode of action of NLRs in plant and animal innate immune systems. () Simplified tripartite modular structures of plant NLRs () and animal NLRs (). PYD, pyrin domain; BIRs, baculovirus inhibitor-of-apoptosis repeats. () Detection of strain-specific pathogen effectors ('Avr') by plant NLRs. Bacterial pathogens usually secrete effectors via the bacterial type III secretion system (T3SS), whereas filamentous pathogens often export effectors from specialized feeding structures called 'haustoria'. The pathogen effectors included here are thought to suppress PRR-triggered resistance responses. A subset of NLRs shuttle between the nucleus and cytoplasm, whereas others are invariably tethered to the plasma membrane. The lipase-like protein EDS1 shuttles between the cytoplasm and nucleus and serves as convergence point in the signaling of resistance responses initiated by TIR-type NLRs. NDR1 and RIN4 are membrane-associated proteins that interacting with RPM1. Some NLRs associate with transcriptional repressors (TR), which is thought to amplify 'def! ense' gene expression activated by transcription factors (TF). () Functions of various well-characterized vertebrate NLRs in a human cell. Exposure to bacterial pathogens, which in some cases can access the host cytoplasm, activates NLR proteins. This results in the expression of proinflammatory cytokines and antimocrobial peptides. Activation of pyrin domain–containing NLRs such as NLRP1 and NLRP3 can also result in activation of the protease caspase-1, which in turn cleaves the zymogens of IL-1β and IL-18 for subsequent secretion. Activation of these NLRs by particular bacteria can also induce the caspase-1-dependent cell-death program of pyroptosis. NLRP3 can also be activated by DAMPs, especially by membrane damage. Nuclear shuttling has been reported for some animal NLRs, such as CIITA and NLRC5. This results in the activation of genes encoding major histocompatibility complex (MHC) class I and II molecules involved in the presentation of antigens to T cells of the ! adaptive immune system. PAMPs, pathogen-associated molecular p! attern; MAPK, mitogen-activated protein kinase. * Figure 2: Polymorphic surface patches of N-terminal TIR and CC domains of plant NLRs are critical for receptor function. () Mapping of individual residues of flax L6 (ref. 78), tobacco N77, 95 or Arabidopsis RPS439 onto the monomeric structure of the plant TIR domain from Arabidopsis thaliana96 (AtTIR; Protein Data Bank accession code 3JRN); TIR domain surface residues needed for disease resistance mediated by the NLRs are in red. The L6 TIR domain functions as homodimer78. () Mapping of CC domain surface residues needed for disease resistance provided by MLA10 (ref. 79) and RPM1 (ref. 100) onto the MLA10 dimer structure79 (red; Protein Data Bank accession code 3QFL). The RPM1 CC domain is assumed to adopt a dimer fold similar to the MLA10 CC dimer79. The EDVID motif (blue) is present in many CC-type NLRs. Protomers (structural units of the oligomeric proteins) are green and brown. * Figure 3: NLR signal initiation mediated by the N-terminal module. In healthy plants, the N-terminal NLR module is kept in an inactive state (orange oval) through intramolecular interactions with other NLR domains and/or intermolecular interaction(s) with other host proteins (guardee or decoy). After pathogen effector–mediated modification of the guardee or decoy, a conformational change in the receptor exposes the active N-terminal receptor domain (orange hexagon), which enables associations with signal transducers (gray hexagon) for signal initiation (Post-activation complex). This might result in the relocation of the receptor inside the cell. Author information * Abstract * Author information Affiliations * Max-Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, Köln, Germany. * Takaki Maekawa & * Paul Schulze-Lefert * Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Köln, Germany. * Thomas A Kufer Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paul Schulze-Lefert Author Details * Takaki Maekawa Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas A Kufer Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Schulze-Lefert Contact Paul Schulze-Lefert Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Recognition of β-linked self glycolipids mediated by natural killer T cell antigen receptors
- Nat Immunol 12(9):827-833 (2011)
Nature Immunology | Article Recognition of β-linked self glycolipids mediated by natural killer T cell antigen receptors * Daniel G Pellicci1, 6 * Andrew J Clarke2, 6 * Onisha Patel2 * Thierry Mallevaey3 * Travis Beddoe2 * Jérôme Le Nours2 * Adam P Uldrich1 * James McCluskey1 * Gurdyal S Besra4 * Steven A Porcelli5 * Laurent Gapin3 * Dale I Godfrey1 * Jamie Rossjohn2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:827–833Year published:(2011)DOI:doi:10.1038/ni.2076Received20 April 2011Accepted22 June 2011Published online31 July 2011Corrected online04 August 2011 Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of N! KT cell TCRs to β-linked antigens. View full text Figures at a glance * Figure 1: Hierarchical recognition of multiple CD1d-antigen complexes by NKT cell TCRs. Flow cytometry of the TCRαβ− 5KC mouse hybridoma transduced with the Vα14Jα18 invariant chain and various engineered TCRβ chains and stained with five different CD1d tetramers: α-GalCer, β-GalCer, β-LacCer, iGb3 or Gb3. Above, CDR2β and CDR3β sequences of the TCRβ chains; 'Self' (top row), CD1d tetramers made of CD1d monomers affinity-purified from the supernatants of human 293 cells transduced with a lentivirus encoding mouse CD1d with no external antigen added before the formation of tetramers; right, structures of antigens used in CD1d tetramers. β-GalCer was purified from bovine galactocerebrosides; β-LacCer was purified from porcine red blood cells; both contain heterogeneous fatty acid length and saturation (R). Numbers in top right quadrants indicate percent CD1d tetramer–positive cells. Data are representative of two independent experiments. * Figure 2: Overview of the structures of NKT cell TCR–CD1d–β-linked antigens. () Auto-Vα24 NKT cell TCR–CD1d–β-GalCer. Cα, α-chain constant region; Cβ, β-chain constant region; β2m, β2-microglobulin; hCD1d, human CD1d. () Auto-Vα14 NKT cell TCR–CD1d–β-GalCer. mCD1d, mouse CD1d. () Mouse NKT cell TCR–CD1d–α-GalCer12. () Auto-Vα14 NKT cell TCR–CD1d–iGb3. () Auto-Vα14 NKT cell TCR–CD1d–Gb3. () Auto-Vα14 NKT cell TCR–CD1d–β-LacCer. Below (–), associated footprints on the CD1d-antigen complexes: pink, CDR1α loops; purple, CDR3α loops; red, CDR2β loops; orange, CDR3β loops; black, framework (FW) contributions. (,) CDR3β loops in the binding of auto-Vα14 and auto-Vα24 NKT cell TCRs to mouse () or human () CD1d: gold, human NKT cell TCRα chain; green, human NKT cell TCRβ chain; blue, mouse NKT cell TCRα chain; brown, mouse NKT cell TCRβ chain; yellow, β-linked ligands and α-GalCer. * Figure 3: Recognition of β-GalCer by human and mouse NKT cell TCRs. () Auto-Vα14 NKT cell TCR–CD1d–β-GalCer. () Auto-Vα24 NKT cell TCR–CD1d–β-GalCer. () Vα14-Vβ8.2 NKT cell TCR–CD1d–α-GalCer12. () Vα24-Vβ11 NKT cell TCR–CD1d–α-GalCer12. () Conformational adjustments of the β-GalCer head group (relative to sulfatide; Protein Data Bank accession code 2AKR26) after ligation of the NKT cell TCR. * Figure 4: Recognition of bulky β-linked ligands by NKT cell TCRs. () Auto-Vα14 NKT cell TCR–CD1d–iGb3. () Auto-Vα14 NKT cell TCR–CD1d–Gb3. () Auto-Vα14 NKT cell TCR–CD1d–β-LacCer. () Conformational adjustments of the iGb3 head group after ligation of the NKT cell TCR (gray, not ligand bound; yellow, ligand bound). The position of the third sugar head group of iGb3 in the binary complex (green) was not resolved in the crystal structure of the CD1d-iGb3 complex27. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Protein Data Bank * 3HUJ * 3SCM * 3SDA * 3SDC * 3SDD * 3SDX * 2AKR * 3HUJ * 3SCM * 3SDA * 3SDC * 3SDD * 3SDX * 2AKR Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationErratum 04 August 2011In the version of this article initially published online, in the diagrams at right in Figure 1, α-GalCer incorrectly included a second NH group and the acyl chain length of iGb3 was incorrect. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Daniel G Pellicci & * Andrew J Clarke Affiliations * Department of Microbiology & Immunology, University of Melbourne, Parkville, Victoria, Australia. * Daniel G Pellicci, * Adam P Uldrich, * James McCluskey & * Dale I Godfrey * The Protein Crystallography Unit, Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University,Clayton, Victoria, Australia. * Andrew J Clarke, * Onisha Patel, * Travis Beddoe, * Jérôme Le Nours & * Jamie Rossjohn * Department of Immunology, University of Colorado Denver and National Jewish Health, Denver, Colorado, USA. * Thierry Mallevaey & * Laurent Gapin * School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK. * Gurdyal S Besra * Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA. * Steven A Porcelli Contributions D.G.P. and A.J.C., isolation and characterization of NKT cell TCR–CD1d–β-antigen complexes; T.B. and A.P.U., surface plasmon resonance studies; J.L.N., crystallographic analyses; T.M., functional studies; O.P., crystallization and solution of the structure of the human NKT cell TCR complex; G.S.B., S.A.P. and J.M., intellectual input; and L.G., D.I.G. and J.R., investigation leadership and project conception. L.G., D.I.G. and J.R. contributed equally to this work. Competing financial interests S.A.P. has received payments as a consultant for Vaccinex for work related to the development of therapeutics based on CD1d-presented glycolipids. Corresponding authors Correspondence to: * Dale I Godfrey or * Jamie Rossjohn Author Details * Daniel G Pellicci Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew J Clarke Search for this author in: * NPG journals * PubMed * Google Scholar * Onisha Patel Search for this author in: * NPG journals * PubMed * Google Scholar * Thierry Mallevaey Search for this author in: * NPG journals * PubMed * Google Scholar * Travis Beddoe Search for this author in: * NPG journals * PubMed * Google Scholar * Jérôme Le Nours Search for this author in: * NPG journals * PubMed * Google Scholar * Adam P Uldrich Search for this author in: * NPG journals * PubMed * Google Scholar * James McCluskey Search for this author in: * NPG journals * PubMed * Google Scholar * Gurdyal S Besra Search for this author in: * NPG journals * PubMed * Google Scholar * Steven A Porcelli Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Gapin Search for this author in: * NPG journals * PubMed * Google Scholar * Dale I Godfrey Contact Dale I Godfrey Search for this author in: * NPG journals * PubMed * Google Scholar * Jamie Rossjohn Contact Jamie Rossjohn Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–3 and Tables 1–7 Additional data - The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1
- Nat Immunol 12(9):834-843 (2011)
Nature Immunology | Article The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1 * Noula Shembade1 * Rajeshree Pujari1 * Nicole S Harhaj1 * Derek W Abbott2 * Edward W Harhaj1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:834–843Year published:(2011)DOI:doi:10.1038/ni.2066Received02 February 2011Published online17 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKβ, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation. View full text Figures at a glance * Figure 1: Phosphorylation of TAX1BP1 in response to proinflammatory stimuli. () Immunoblot analysis (IB) of lysates of MEFs transfected with expression vector for Flag-tagged TAX1BP1 (Flag-TAX1BP1), then left untreated (NT) or treated for 30 min with TNF or LPS (10 ng/ml). α-, anti-; p-, phosphorylated. () Immunoblot analysis of lysates of MEFs treated with calf intestinal alkaline phosphatase (CIP) and left unstimulated (−) or stimulated for 30 min (+) with TNF (10 ng/ml). () In vivo kinase assay of MEFs labeled with 32P-orthophosphate and left untreated (−) or treated for 15 min (+) with TNF (10 ng/ml), followed by immunoprecipitation of proteins from lysates with anti-TAX1BP1 and autoradiography for visualization of phosphorylated TAX1BP1. kDa, kilodaltons. () Immunoblot analysis of lysates of 293T cells transfected with expression vector for Flag-TAX1BP1 and left untransfected (−) or transfected with expression vector for hemagglutinin (HA)-tagged IKKα, IKKβ or IKKγ (above lanes). () Immunoblot analysis of lysates of wild-type (WT), Ikk! a−/−, Ikbkb−/− or Ikbkg−/− MEFs left untransfected (−) or transfected (+) with expression vector for Flag-TAX1BP1 and left untreated (−) or treated (+) for 15 min with TNF. Anti-β-actin serves as a loading control throughout. () In vitro kinase assay of recombinant active IKKα or IKKβ, with a fusion of GST and TAX1BP1 amino acids 205–599 (GST-TAX1BP1(205–599)) or IκBα amino acids 1–54 (GST-IκBα(1–54)) as the substrate. Far left (−), GST fusion proteins alone; bottom blots in each, immunoblot analysis of GST fusion proteins with anti-GST. () Immunoassay of TAX1BP1 interactions in Ikka−/− MEFs transfected with expression vectors for HA-IKKα or HA-IKKα(K44M) and left untreated or treated for 15 min with TNF or IL-1, detected in lysates after immunoprecipitation (IP) with anti-TAX1BP1 (top two blots) by immunoblot analysis with anti-HA or anti-TAX1BP1; below, immunoblot analysis of total cell lysates with anti-HA, anti-IκBα or anti-β-ac! tin. () Immunoblot analysis of lysates of Ikka−/− MEFs tra! nsfected with empty vector (EV) or expression vectors for Flag-TAX1BP1, plus HA-IKKα or HA-IKKα(K44M), then left untreated (−) or treated (+) for 15 min with TNF (left) or IL-1 (right); blots were probed with anti-Flag, anti-β-actin, anti-IκBα or anti-IKKα. Data are representative of at least three experiments. * Figure 2: Phosphorylation of TAX1BP1 on Ser593 and Ser624 in response to stimulation with TNF or IL-1. () Consensus IKK-phosphorylation sites in IκBα (Ser32 and Ser36, ) and putative IKK-phosphorylation sites in TAX1BP1 (Ser212, Ser254 and Ser593, ). Top, consensus phosphorylation sites, with phosphorylated serine (pS) at position 0 and acidic amino acids aspartic acid (D) upstream at position −5 and glutamic acid (E) downstream at position +3. pT, phosphorylated threonine; L/I, leucine or isoleucine; pT/D, phosphorylated threonine or aspartic acid; Y, tyrosine; X, any amino acid. () TAX1BP1 deletion mutants (left): SKICH, SKIP (skeletal muscle and kidney enriched inositol phosphatase) carboxyl homology domain; CC, coiled coil domain; ZnF, zinc-finger domain; far left, amino acids. Right, phosphorylation results. () Immunoblot analysis of lysates of 293T cells transfected with expression vector for Flag-tagged TAX1BP1 deletion mutants in , with or without HA-tagged IKKα (above lanes). () Silver-stained gel of 293T cells transfected with expression vector for Flag-TAX1BP1! and left untreated or treated for 30 min with TNF (top), and identification of phosphorylated peptides in those cells by matrix-assisted laser desorption-ionization quadrupole ion–trap time-of-flight (bottom): far left, observed mass; right, amino acid sequence (phosphorylated residues underlined). () Immunoblot analysis of lysates of 293T cells transfected with empty vector or expression vector for wild-type TAX1BP1 or TAX1BP1 (S593A), with or without IKKα (above lanes). () Immunoblot analysis of lysates of Tax1bp1−/− MEFs transfected with expression vector for wild-type or mutant TAX1BP1 (below blots) and left untreated or treated with TNF or IL-1 (above lanes); blots were probed with anti-Flag, anti-β-actin or anti-IκBα. () Sequence alignment of the phosphorylation site of TAX1BP1 (; human Ser624) in various species. () In vivo kinase assay of Tax1bp1−/− MEFs reconstituted with empty vector or expression vector for Flag-tagged wild-type TAX1BP1 or TAX1BP1(! S593A,S624A) (AA), labeled with 32P-orthophosphate and left un! treated or treated for 15 min with TNF (10 ng/ml), followed by immunoprecipitation and autoradiography (as in Fig. 1c); blots were probed with anti-TAX1BP1 (after immunoprecipitation with anti-TAX1BP1) or anti-β-actin and anti-IκBα (total lysates). () Immunoblot analysis of lysates of 293T cells transfected with various expression vectors (above lanes), probed with antibody specific for TAX1BP1 phosphorylated at Ser593 (α-p-TAX1BP1), anti-Flag or anti-HA. () Immunoblot analysis of lysates of Tax1bp1+/− and Tax1bp1−/− MEFs treated for 0–120 min with TNF, probed with the phosphorylation-specific antibody in , anti-IκBα or anti-β-actin. () Immunoblot analysis of lysates of mouse BMDMs treated for 0–60 min with TNF or IL-1, probed with the phosphorylation-specific antibody in , anti-TAX1BP1, anti-IκBα or anti-β-actin. Data are representative of at least three experiments. * Figure 3: Phosphorylation of TAX1BP1 is essential for the termination of NF-κB signaling, Jnk phosphorylation and RIP1 ubiquitination. (,) Immunoassay of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged wild-type TAX1BP1 or Flag-TAX1BP1(S593A,S624A), then treated for 0–120 min with TNF () or IL-1 (), assessed after immunoprecipitation with anti-RIP1 by immunoblot analysis with antibody specific for K63-linked ubiquitin (K63-Ub) and anti-RIP1 () or after immunoprecipitation with anti-TRAF6 by immunoblot analysis with antibody specific for K63-linked ubiquitin and anti-TRAF6 (). Below (Lysates), immunoblot analysis of total cell lysates with antibodies along left margin. () Immunoblot analysis of lysates of mouse BMDMs transfected with siRNA specific for mouse Tax1bp1, together with empty vector or expression vector for Flag-tagged wild-type human TAX1BP1 or TAX1BP1(S593A,S624A), and treated for 0–60 min with TNF, probed with anti-TAX1BP1, anti-IκBα or anti-β-actin. Data are representative of at least three experiments. * Figure 4: Phosphorylation of TAX1BP1 regulates recruitment of the A20 ubiquitin-editing complex to TRAF2 and TRAF6. Immunoassay of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged TAX1BP1 or TAX1BP1(S593A,S624A), then treated for 0–120 min with TNF () or IL-1 (), assessed after immunoprecipitation with anti-TRAF2 () or anti-TRAF6 () by immunoblot analysis with antibodies along left margins (,). Below (Lysates), immunoblot analysis of total cell lysates with antibodies along left margins. Data are representative of at least three experiments. * Figure 5: Phosphorylation of TAX1BP1 is required for assembly of the A20 ubiquitin-editing complex. (,) NF-κB luciferase activity in lysates of Tax1bp1−/− MEFs transfected with an NF-κB firefly luciferase reporter and a renilla luciferase vector reporter, plus empty vector or expression vector for Flag-tagged wild-type TAX1BP1, TAX1BP1(S593A), TAX1BP1(S624A) or TAX1BP1(S593A,S624A), then treated for 8 h with TNF () or IL-1 (); results are presented relative to renilla luciferase activity. Below, immunoblot analysis of the cells above with anti-Flag. (,) Immunoassay of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged wild-type TAX1BP1, TAX1BP1(S593A), TAX1BP1(S624A) or TAX1BP1(S593A,S624A), then treated for 0–120 min with TNF () or IL-1 (), assessed after immunoprecipitation with anti-RNF11 by immunoblot analysis with antibodies along left margins. Below (Lysates), immunoblot analysis of total cell lysates with antibodies along left margins. Data are representative of at least three experiments (error bars (,), s.e.m.! of triplicates). * Figure 6: IKKα is essential for assembly of the A20 ubiquitin-editing complex and termination of NF-κB signaling. () Immunoassay of lysates of wild-type, Ikka−/−, Ikbkb−/− and Ikbkg−/− MEFs, left untreated or treated for 15 min with TNF, assessed after immunoprecipitation with anti-RIP1 or antibody to isotype-matched control immunoglobulin (α-Ig) by immunoblot analysis with anti-A20 or anti-RIP1. () Immunoassay of lysates of wild-type and Ikka−/− MEFs treated as in , assessed after immunoprecipitation with anti-TAX1BP1 or control antibody (as in ) by immunoblot analysis with anti-Itch or anti-TAX1BP1. Below (Lysates; ,), immunoblot analysis of total cell lysates with anti-IκBα and anti-β-actin. (,) Immunoassay of lysates of wild-type MEFs transfected with control siRNA (Ctrl) or IKKα-specific siRNA and treated with TNF () or IL-1 (), assessed after immunoprecipitation with anti-A20 or anti-TAX1BP1 by immunoblot analysis with antibodies along left margins. Anti-IκBα, anti-IKKα or anti-β-actin performed on total cell lysates (,). (,) Immunoassay of lysates of mous! e BMDMs treated for 0–120 min with TNF () or IL-1 (), assessed after immunoprecipitation with anti-RIP1 by immunoblot analysis with antibody specific for K63-linked ubiquitin and anti-RIP1 () or after immunoprecipitation with anti-TRAF6 by immunoblot analysis with antibody specific for K63-linked ubiquitin or anti-TRAF6 (). Below (Lysates), immunoblot analysis of total cell lysates with anti-IKKα, anti-IκBα or anti-β-actin. Ig HC (), immunoglobulin heavy chain. Data are representative of at least three experiments. * Figure 7: Phospho-mimetic TAX1BP1 bypasses the requirement for IKKα in terminating NF-κB signaling. Immunoblot analysis of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged wild-type TAX1BP1, TAX1BP1(S593A,S624A) or TAX1BP1(S593E,S624E), together with control siRNA or IKKα-specific siRNA, then treated for 0–30 min with TNF () or IL-1 (); blots were probed with anti-Flag, anti-IκBα, anti-IKKα or anti-β-actin. Data are representative of two experiments. * Figure 8: HTLV-I Tax blocks the phosphorylation of TAX1BP1. () Immunoblot analysis of lysates of wild-type MEFs transfected with expression vector for Flag-tagged TAX1BP1 plus plasmid encoding Tax, then treated with TNF or IL-1, probed with anti-Flag, anti-β-actin plus anti-IκBα or anti-Tax. () Immunoblot analysis of lysates of wild-type MEFs transfected with empty plasmid or plasmid encoding Tax, then treated for 0–120 min with IL-1 or TNF, probed with antibody specific for TAX1BP1 phosphorylated at Ser593, anti-TAX1BP1, anti-β-actin plus anti-IκBα or anti-Tax. () Immunoassay of lysates of wild-type MEFs transfected with empty plasmid or plasmid encoding Tax, then treated for 30 min with TNF or IL-1 (above lanes), assessed after immunoprecipitation with anti-TAX1BP1 by immunoblot analysis with anti-IKKα or anti-TAX1BP1. Below (Lysates), immunoblot analysis of total cell lysates with anti-IκBα, anti-Tax or anti-β-actin. () Immunoblot analysis of lysates of the adult T cell leukemia cell lines TL-OM1, MT-2 and C8166 and of! Jurkat Tax Tet-on cells left untreated (−) or treated (+) for 15 min with PMA and ionomycin (P-I) with (+) or without (−) treatment for 16 h with doxycycline (Dox; 1 μg/ml); blots were probed with antibody specific for TAX1BP1 phosphorylated at Ser593, anti-TAX1BP1, anti-Tax, antibody to phosphorylated IκBα or anti-β-actin. () NF-κB luciferase assay of lysates of Tax1bp1−/− MEFs transfected with an NF-κB firefly luciferase reporter and a renilla luciferase vector reporter, plus empty vector or expression vector for Flag-tagged wild-type TAX1BP1 or TAX1BP1(S593E,S624E) (EE), with (Tax) or without (−) plasmid encoding Tax; results are presented relative to renilla luciferase activity. Below, immunoblot analysis with anti-TAX1BP1 or anti-Tax. Data are representative of at least three experiments (error bars (), s.e.m. of triplicates). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Microbiology and Immunology, Sylvester Comprehensive Cancer Center, The University of Miami, Miller School of Medicine, Miami, Florida, USA. * Noula Shembade, * Rajeshree Pujari, * Nicole S Harhaj & * Edward W Harhaj * Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA. * Derek W Abbott Contributions N.S., R.P. and N.S.H. did experiments and analyzed data; D.W.A. provided reagents and did bioinformatic analyses; and E.W.H. designed and supervised the experiments, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Edward W Harhaj Author Details * Noula Shembade Search for this author in: * NPG journals * PubMed * Google Scholar * Rajeshree Pujari Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole S Harhaj Search for this author in: * NPG journals * PubMed * Google Scholar * Derek W Abbott Search for this author in: * NPG journals * PubMed * Google Scholar * Edward W Harhaj Contact Edward W Harhaj Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–8 and Methods Additional data - The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation
- Nat Immunol 12(9):844-852 (2011)
Nature Immunology | Article The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation * Katarzyna Bulek1, 6 * Caini Liu1, 6 * Shadi Swaidani1 * Liwen Wang2 * Richard C Page3 * Muhammet F Gulen1 * Tomasz Herjan1 * Amina Abbadi1 * Wen Qian1 * Dongxu Sun1 * Mark Lauer4 * Vincent Hascall4 * Saurav Misra3 * Mark Chance2 * Mark Aronica5 * Thomas Hamilton1 * Xiaoxia Li1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:844–852Year published:(2011)DOI:doi:10.1038/ni.2080Received16 December 2010Accepted28 June 2011Published online07 August 2011 Abstract * Abstract * Accession codes * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1–TRAF6–transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a k! inase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function. View full text Figures at a glance * Figure 1: IKKi forms a complex with Act1 after stimulation with IL-17. () Immunoassay of Act1-deficient MEFs infected with a retroviral construct encoding wild-type Act1 (Act1-KO + WT Act1 reconst), then left untreated (0) or treated for 15, 30 or 60 min with IL-17 (50 ng/ml); lysates immunoprecipitated (IP) with anti-Act1 or immunoglobulin G (IgG; above) and whole-cell lysates (WCL) were analyzed immunoblot (IB) with anti-IKKi, anti-Act1 and anti-GAPDH (antibody to glyceraldehyde phosphate dehydrogenase; loading control). () Immunoblot analysis of MEFs reconstituted as in and treated for 0, 15 or 30 min with IL-17 (50 ng/ml), then left untreated (UT) or treated for 1 h with phosphatase (CIP); blots were probed with anti-Act1 and anti-GAPDH. () Immunoassay of wild-type (Act1-WT) and Act1-deficient (Act1-KO) MEFs left untreated (0) or treated for 15 or 30 min with IL-17 (50 ng/ml); lysates immunoprecipitated with anti-IKKi (top and middle) and whole-cell lysates (bottom) were analyzed immunoblot analysis with anti-Act1 and anti-IKKi. Data are re! presentative of three independent experiments. * Figure 2: IKKi is required for the IL-17-mediated expression of genes encoding proinflammatory molecules. () Real-time PCR analysis of Cxcl1, Cxcl2, Tnf, Il6 and Csf3 in wild-type (IKKi-WT) and IKKi-deficient (IKKi-KO) airway epithelial cells left untreated (0) or stimulated for 4 or 8 h at the basal surface with TNF (10 ng/ml), IL-17A (50 ng/ml) or IL-17F (50 ng/ml) alone or with TNF plus IL-17A or IL-17F; results are presented relative to those at time 0. () Immunoblot analysis of phosphorylated (p-) and/or total Jnk, Jnk1, p38, Erk, Erk1, p65, IκB, IKKi and GAPDH in lysates of wild-type and IKKi-deficient airway epithelial cells left untreated (0) or treated for 15–60 min with IL-17A (50 ng/ml) or TNF (10 ng/ml). Data are representative of three independent experiments (error bars (), s.d.). * Figure 3: IKKi is required for IL-17-mediated pulmonary inflammation. () Total BAL and differential cell counts in samples from wild-type and IKKi-deficient mice (n = 6 per group) left unchallenged (Control) or challenged for 24 h by intranasal injection of IL-17 (1 μg). *P < 0.05 (two-tailed t-test). Below, cytospins prepared from the BAL fluid of IL-17-challenged wild-type and IKKi-deficient mice, stained with the nuclear stain Hema3. Arrows indicate neutrophils. Original magnification, ×400. () Lung sections of IL-17-challenged wild-type and IKKi-deficient mice (as in ), stained with hematoxylin and eosin. Original magnification, ×100. () Enzyme-linked immunosorbent assay of CXCL1 in BAL fluid from control or IL-17-challenged wild-type and IKKi-deficient mice. () Real-time PCR analysis of Cxcl1, Cxcl2, Tnf, Il6 and Csf3 in lung tissue from control or IL-17-treated wild-type and IKKi-deficient mice; results are presented in arbitrary units relative to the expression of mRNA encoding β-actin. *P < 0.05 (two-tailed t-test). Data are repres! entative of two independent experiments (mean and s.d. in ,,). * Figure 4: The kinase activity of IKKi is required for stabilization of chemokine mRNA. () RNA blot analysis of CXCL1 and GAPDH mRNA among total RNA from wild-type and IKKi-deficient MEFs left untreated (NT) or pretreated for 1 h with TNF (10 ng/ml), followed by treatment for 0–4 h with IL-17 and actinomycin D (ActD; 5 μg/ml). () RNA blot analysis of CXCL1 and GAPDH mRNA in HeLa Tet-Off cells transfected with 1 μg pTRE2 CXCL1Δ4 and 1 μg pcDNA3.1 plasmid encoding wild-type IKKi (IKKi(WT)) or kinase-inactive IKKi (IKKi(K38A)) or empty plasmid (Vector), then treated for 0–90 min with doxycycline (Dox; 1 μg/ml). () RNA blot analysis of CXCL1 and GAPDH mRNA in HeLa Tet-Off cells transfected with 1 μg pTRE2 CXCL1Δ4 and 1 μg empty plasmid (Vector) or plasmid encoding Act1 alone (Act1), or Act1 plus wild-type IKKi or IKKi(K38A), then treated for 0–90 min with doxycycline (1 μg/ml). Below left (–), abundance of CXCL1 mRNA over time; below right (,), immunoblot analysis of cell lysates to ensure similar protein expression. Data are representative of thre! e independent experiments. * Figure 5: The kinase activity of IKKi is required for IL-17-induced phosphorylation of Act1. () Immunoblot analysis of Act1, IKKi and GAPDH in lysates of wild-type and IKKi-deficient airway epithelial cells left untreated (0) or treated for 15 or 30 min with IL-17A (50 ng/ml) or IL-17F (50 ng/ml). () Immunoblot analysis of lysates of HEK293 cells transfected with empty pcDNA3 plasmid alone (Control) Act1 plus empty pcDNA3 (Act1), or Act1 plus pcDNA3 encoding wild-type IKKi or IKKi(K38A), then left untreated (UT) or treated for 1 h with phosphatase (CIP); blots were probed with anti-Act1, anti-IKKi and anti-GAPDH. () In vitro kinase assay of lysates of wild-type or Act1-deficient MEFs after immunoprecipitation of proteins from lysates, analyzed with (+) or without (−) recombinant IKKi–glutathione S-transferase. () In vitro kinase assay of lysates of wild-type MEFs (top) or wild-type or Act1-deficient kidney epithelial cells (below) left untreated (0) or treated for 15 or 30 min with IL-17 (50 ng/ml), followed by immunoprecipitation of proteins from lysates with a! nti-IKKi. () Immunoblot analysis (top) and real-time PCR analysis (below) of IKKi (encoded by Ikbke) and GAPDH in lysates of wild-type airway epithelial cells left untreated (0) or treated for 0.5–24 h (above lanes; top) or 4 or 8 h (below) with IL-17A (50 ng/ml). Data are representative of three independent experiments (error bars (), s.d.). * Figure 6: Identification of the Ser311-phosphorylation site of Act1 by mass spectrometry. () Tandem mass spectrometry (ms2) of precursor ions in the phosphorylated Act1 peptide (amino acids 305–322; sequence VILNDSSpPQDQEERPAQR where 'Sp' (red) indicates phosphorylated serine) at a m/z of 721.33 Da. Blue lines (top) indicate peptide cleavage. () Tandem mass spectrometry of precursor ions of an unmodified peptide of the same sequence as in (VILNDSSpQDQEERPAQR) at a m/z of 695.01 Da. () Immunoblot analysis of Act1-deficient MEFs infected with retroviral constructs for wild-type Act1 (WT) or Act1(S11A) (S311A), then left untreated (0) or treated for 10 or 30 min with IL-17 (50 ng/ml), or of 293 cells transfected with plasmid encoding IKKi (far right) plus wild-type Act1 or Act1(S311A) (above lanes); blots were probed with anti-Act1 or anti-actin. () Real-time PCR analysis of Cxcl1 and Il6 in Act1-deficient MEFs infected with retroviral constructs for wild-type Act1 (Act1 (WT)) or Act1(S311A), followed by treatment for 0, 1 or 3 h with IL-17A (50 ng/ml). *P < 0.05 ! (two-tailed t-test). () Immunoblot analysis of Act1-deficient MEFs infected as in and left untreated or treated for 0, 15, 30 or 60 min with IL-17 (50 ng/ml), probed with various antibodies (left margin). () RNA blot analysis of CXCL1 and GAPDH mRNA among total RNA from HeLa Tet-Off cells transfected with 1 μg pTRE2 CXCL1Δ4 and 1 μg plasmid encoding wild-type Act1 (Act1(WT)) or Act1(S311A), then treated for 0, 45 or 90 min with doxycycline (1 μg/ml). Right, abundance of CXCL1 mRNA over time. Data are representative of three independent experiments (error bars (), s.d.). * Figure 7: The effect of phosphorylation of Act1 Ser311 on the interaction of Act1 with TRAF proteins. () Immunoassay of wild-type and IKKi-deficient kidney epithelial cells left untreated (0) or treated for 15 or 30 min with IL-17 (50 ng/ml); lysates immunoprecipitated with anti-Act1 (above) and whole-cell lysates (below) were analyzed by immunoblot with anti-TRAF2, anti-TRAF5, anti-TRAF6, anti-IKKi and anti-Act1. () Immunoassay of Act1-deficient MEFs infected with retroviral construct for wild-type Act1 or Act1(S311A), left untreated (0) or treated for 15 min with IL-17A (50 ng/ml), followed by immunoprecipitate and immunoblot analysis as . () Computational modeling of the interaction of the Act1 TRAF-binding motif (human Act1 amino acids 327–334) with the structures of the TRAF domains of TRAF6 (yellow) or TRAF2 (pink) or a homology model of the TRAF domain of TRAF5 (orange). Dashed red circles outline the Act1 target serine or phosphorylated serine. (–) Models of the Act1 TRAF-binding motifs docked onto the TRAF domains of TRAF6 (yellow; ), TRAF5 (orange; ) or TRAF2 (! pink; ). Act1-interacting side chains of the TRAF domains are debold; residues of Act1 are in bold; the key Act1 serine or phosphorylated serine is in red bold; dashed lines indicate putative hydrogen bonds or salt bridges between Act1 and TRAF-domain side chains (hydrogen bonds between Act1 and TRAF-domain backbone atoms are also dashed lines but for clarity the TRAF-domain backbone atoms are not presented). Data are from one representative of three independent experiments (,). Accession codes * Abstract * Accession codes * Author information Referenced accessions Protein Data Bank * 1CA9 * 1D00 * 1D01 * 1D0A * 1CZY * 1CZZ * 1QSC * 1LB5 * 1LB6 * 1CA9 * 1D00 * 1D01 * 1D0A * 1CZY * 1CZZ * 1QSC * 1LB5 * 1LB6 Author information * Abstract * Accession codes * Author information Primary authors * These authors contributed equally to this work. * Katarzyna Bulek & * Caini Liu Affiliations * Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA. * Katarzyna Bulek, * Caini Liu, * Shadi Swaidani, * Muhammet F Gulen, * Tomasz Herjan, * Amina Abbadi, * Wen Qian, * Dongxu Sun, * Thomas Hamilton & * Xiaoxia Li * Center for Proteomics & Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. * Liwen Wang & * Mark Chance * Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA. * Richard C Page & * Saurav Misra * Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA. * Mark Lauer & * Vincent Hascall * Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA. * Mark Aronica Contributions K.B., C.L., S.S., L.W., M.F.G., T.H., W.Q. and D.S., experiments; K.B. and C.L., experimental design, performance and interpretation and writing of the manuscript; L.W. and M.C., mass spectrometry study; R.C.P. and S.M., structure modeling; A.A., M.L. and V.H., assistance with the primary mouse airway epithelial cell culture; M.A., assistance with in vivo experiments; X.L. and T.H., design and interpretation of experiments and writing of the manuscript; all authors, review of the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Xiaoxia Li Author Details * Katarzyna Bulek Search for this author in: * NPG journals * PubMed * Google Scholar * Caini Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Shadi Swaidani Search for this author in: * NPG journals * PubMed * Google Scholar * Liwen Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Richard C Page Search for this author in: * NPG journals * PubMed * Google Scholar * Muhammet F Gulen Search for this author in: * NPG journals * PubMed * Google Scholar * Tomasz Herjan Search for this author in: * NPG journals * PubMed * Google Scholar * Amina Abbadi Search for this author in: * NPG journals * PubMed * Google Scholar * Wen Qian Search for this author in: * NPG journals * PubMed * Google Scholar * Dongxu Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Lauer Search for this author in: * NPG journals * PubMed * Google Scholar * Vincent Hascall Search for this author in: * NPG journals * PubMed * Google Scholar * Saurav Misra Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Chance Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Aronica Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Hamilton Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoxia Li Contact Xiaoxia Li Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Treatment with IL-17 prolongs the half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicing-regulatory factor SF2 (ASF)
- Nat Immunol 12(9):853-860 (2011)
Nature Immunology | Article Treatment with IL-17 prolongs the half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicing-regulatory factor SF2 (ASF) * Dongxu Sun1 * Michael Novotny1 * Katarzyna Bulek1 * Caini Liu1 * Xiaoxia Li1 * Thomas Hamilton1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:853–860Year published:(2011)DOI:doi:10.1038/ni.2081Received16 December 2010Accepted30 June 2011Published online07 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin 17 (IL-17) promotes the expression of chemokines and cytokines via the induction of gene transcription and post-transcriptional stabilization of mRNA. We show here that IL-17 enhanced the stability of chemokine CXCL1 mRNA and other mRNAs through a pathway that involved the adaptor Act1, the adaptors TRAF2 or TRAF5 and the splicing factor SF2 (also known as alternative splicing factor (ASF)). TRAF2 and TRAF5 were necessary for IL-17 to signal the stabilization of CXCL1 mRNA. Furthermore, IL-17 promoted the formation of complexes of TRAF5-TRAF2, Act1 and SF2 (ASF). Overexpression of SF2 (ASF) shortened the half-life of CXCL1 mRNA, whereas depletion of SF2 (ASF) prolonged it. SF2 (ASF) bound chemokine mRNA in unstimulated cells, whereas the SF2 (ASF)-mRNA interaction was much lower after stimulation with IL-17. Our findings define an IL-17-induced signaling pathway that links to the stabilization of selected mRNA species through Act1, TRAF2-TRAF5 and the RNA-binding! protein SF2 (ASF). View full text Figures at a glance * Figure 1: Expression of TRAF2 or TRAF5 selectively prolongs the half-life of CXCL1 mRNA. () RNA blot analysis (top) of residual KCΔ4 and GAPDH mRNA in HeLa Tet-off cells transfected with KCΔ4 and treated for 0–135 min with doxycyline (Dox) alone (−) or in the presence of IL-17 (+; 25 ng/ml); below, abundance of CXCL1 mRNA (normalized to that of GAPDH mRNA), presented as decay over time (left) and half-life (right). () RNA blot analysis (top) of KCΔ4 mRNA in HeLa Tet-Off cells cotransfected with KCΔ4 plus empty vector (EV) or plasmid encoding TRAF2, TRAF5 or TRAF6, then treated for 0–135 min with doxycyline (1 μg/ml); below, mRNA abundance (as in ); inset (right), immunoblot analysis of TRAF2 (T2), TRAF5 (T5) and TRAF6 (T6) with anti-Flag (α-Flag). () RNA blot analysis (left) of KCΔ4 mRNA in HeLa Tet-Off cells cotransfected with KCΔ4 (top) or reporter plasmid encoding the 5′ UTR and coding region of mouse CXCL1 and the 3′ UTR of GM-CSF (KC-GM-CSF; below), along with empty vector or the TRAF5 expression plasmid, then treated for 0–135 min with ! doxycyline; right, abundance (as in ). Data are from three independent experiments (error bars, s.d.). * Figure 2: TRAF2 and TRAF5 are required for IL-17-induced stabilization of CXCL1 mRNA. () RNA blot analysis (left) of CXCL1 mRNA in wild-type MEFs (WT) and MEFs deficient in both TRAF2 and TRAF (T2T5-dKO) treated for 2 h with TNF (10 ng/ml) alone (TNF) or in the presence of IL-17 (TNF + IL-17), followed by the addition of actinomycin D (ActD; 5 μg/ml); right, mRNA half-life (as in Fig. 1a). () RNA blot analysis (left) of CXCL1 mRNA in MEFs deficient in both TRAF2 and TRAF5 (left) and doubly deficient MEFs reconstituted with both TRAF2 and TRAF5 (T2T5-dKO reconst; right), treated for 2 h with TNF plus IL-17 (10 ng/ml each), followed by the addition of actinomycin D (5 μg/ml); right, mRNA half-life (as in Fig. 1a). () RNA blot analysis (left) of KCΔ4 mRNA in HeLa Tet-Off cells cotransfected with control siRNA (Ctrl) or siRNA specific for TRAF2 (T2) or TRAF5 (T5) or both (T2T5), along with the KCΔ4 reporter, followed by treatment with doxycyline alone (−) or in the presence of IL-17 (+); top right, immunoblot analysis of residual TRAF expression; bottom rig! ht, mRNA half-life (as in Fig. 1a). Data are from four () or three () independent experiments (error bars, s.d.) or one of two independent experiments with similar results (; error bars, 1/2 range). * Figure 3: IL-17 promotes the interaction of Act1 with TRAF5. () Immunoassay of HeLa Tet-Off cells cotransfected with empty vector or plasmid encoding Flag-tagged TRAF2 (T2), TRAF5 (T5) or TRAF6 (T6), along with hemagglutinin-tagged Act1; lysates immunoprecipitated (IP) with anti-Flag (top) and whole-cell lysates (WCL (40 μg protein); below) were analyzed by immunoblot (IB) with anti-hemagglutinin and anti-Flag (right margin). () Immunoassay of HeLa Tet-Off cells treated for 0–30 min with IL-17; lysates immunoprecipitated with anti-TRAF5 (above) and whole-cell lysates (40 μg protein; below) were analyzed by immunoblot with anti-Act1 or anti-TRAF5. IgG (far left), immunoblot analysis of whole-cell lysates of cells treated for 5 min with IL-17, followed by immunoprecipitation with nonspecific IgG. Open arrowhead, unmodified TRAF5; filled arrowhead, immunoglobulin heavy chain (HC). () Immunoassay of wild-type MEFs treated for 0–60 min with IL-17, followed by immunoprecipitation with anti-Act1 and immunoblot analysis with anti-TRAF5 ! or anti-Act1. Results are representative of three (,) or two () independent experiments. * Figure 4: IL-17 induces a complex of TRAF2 or TRAF5 and SF2 (ASF). () Immunoprecipitation (with anti-Flag) of proteins from MEFs deficient in both TRAF2 and TRAF5 and reconstituted with Flag-TRAF2 and Flag-TRAF5, then left untreated or stimulated for 1 h with IL-17 (10 ng/ml); immunoprecipitates were separated by SDS-PAGE, followed by silver staining. Arrow (right margin), band analyzed further by mass spectrometry; Mr (left margin), relative molecular mass. () Immunoassay of HeLa Tet-Off cells left untreated (0) or treated for 5, 15 or 30 min with IL-17 (25 ng/ml); lysates immunoprecipitated with anti-TRAF5 (above) and whole-cell lysates (WCL (40 μg protein); below) were analyzed by immunoblot with anti-SF2. () Immunoassay of HeLa Tet-Off cells transfected to express Flag-tagged TRAF2 (T2), TRAF4 (T4), TRAF5 (T5) or TRAF6 (T6) and used for immunoprecipitation with anti-Flag; lysates immunoprecipitated with anti-Flag (above) and whole-cell lysates (40 μg protein; below) were analyzed by immunoblot with anti-SF2 or anti-Flag. () Immunoassa! y of HeLa Tet-Off cells cotransfected to express Flag-tagged TRAF5 (T5) or TRAF6 (T6) along with hemagglutinin-tagged TAK1; immunoprecipitates and whole-cell lysates as in were analyzed by immunoblot with anti-hemagglutinin (α-HA) and anti-Flag. () Immunoassay of HeLa Tet-Off cells treated for 0–60 min with IL-17 (50 ng/ml); lysates immunoprecipitated with anti-Act1 (above) and whole-cell lysates (below) were analyzed by immunoblot with anti-SF2, anti-TRAF5 and anti-Act1. Results are representative of three (,,) or two (,) independent experiments. * Figure 5: SF2 (ASF) promotes enhanced decay of CXCL1 mRNA. () RNA blot analysis (left) of residual KCΔ4 and KC-GM-CSF mRNA in Hela Tet-Off cells cotransfected with the KCΔ4 or KC-GM-CSF reporter plasmid, plus empty vector or vector encoding full-length SF2 (ASF), followed by treatment with doxycycline (1 μg/ml); middle, mRNA abundance, and right, half-life (as in Fig. 1a). () RNA blot analysis (bottom left) of KCΔ4 or KC-Clu-P2 mRNA in Hela Tet-Off cells cotransfected for 48 h with the KCΔ4 or KC-Clu-P2 reporter plasmid, along with control or SF2-specific siRNA, then treated with doxycycline (1 μg/ml); right, mRNA half-life (as in Fig. 1a). Top left, immunoblot analysis of SF2 (ASF) in whole-cell lysates (40 μg). () RNA blot analysis (bottom left) of CXCL1 in SF2 (ASF)-deficient MEFs reconstituted with hemagglutinin-tagged SF2 (ASF) under Tet-Off control, left untreated (No Dox) or treated for 2 d with doxycycline (+ Dox; 1 μg/ml), followed by stimulation for 2 h with TNF (10 ng/ml), then treatment for 0–90 min with actino! mycin D (5 μg/ml); right, mRNA half-life (as in Fig. 1a). Top left, immunoblot analysis of hemagglutinin-SF2 (ASF) in whole-cell lysates (40 μg) of untreated (0) and doxycycline-treated (1–3) cells. Data are from three () or two (,) independent experiments (error bars, s.d.). * Figure 6: SF2 (ASF) binds CXCL1 mRNA. () RNA blot analysis (left) of KCΔ4 mRNA in HeLa Tet-Off cells cotransfected with KCΔ4 and empty vector or expression plasmid encoding SF2 (ASF) with a C-terminal deletion (SF2 (ASF)-N) or N-terminal deletion (SF2 (ASF)-C), then treated with doxycycline (1 μg/ml); right, mRNA half-life (as in Fig. 1a). () RNA blot analysis (left) of CXCL1 in HeLa Tet-Off cells cotransfected with KCΔ4 and empty vector or the expression plasmid encoding SF2 (ASF) with C-terminal deletion (SF2 (ASF)-N), or with substitution of aspartic acid for two phenylalanine residues (SF2 (ASF)-N-FF-DD), followed by treatment with doxycycline (1 μg/ml); right, mRNA half-life (as in Fig. 1a). () RNA-immunoprecipitation and real-time RT-PCR analysis of KCΔ4 or KC-Clu-P2 mRNA in extracts of HeLa Tet-Off cells transfected with the KCΔ4 or KC-Clu-P2 reporter plasmid alone (left) or with KCΔ4 and hemagglutinin-tagged versions of the SF2 (ASF) expression plasmids in (right), or transfected with KCΔ4 and l! eft untreated or treated for 30 min with IL-17 (middle), followed by immunoprecipitation with anti-SF2 (ASF) or anti-hemagglutinin (for cells with transgenic expression of hemagglutinin-tagged versions of SF2 (ASF)); results are presented relative to results obtained by immunoprecipitation with nonspecific IgG (change in cycling threshold). Data are from three () or two (,) independent experiments (error bars, s.d. (,); mean and 1/2 range ()). * Figure 7: IL-17 promotes TRAF5 and SF2 (ASF) function in primary cells. () RNA blot analysis (left) of CXCL1, CXCL2 (MIP-2), CCL2 (MCP-1) and GAPDH mRNA in primary kidney epithelial cells treated for 2 h with TNF (10 ng/ml) alone (left) or in combination with IL-17 (25 ng/ml; right), followed by the addition of actinomycin D (5 μg/ml) for 0–90 min; below, RT-PCR analysis (27 cycles) of CSF3 and GAPDH mRNA in the same samples; right, mRNA abundance (as in Fig. 1a). () Immunoassay of primary kidney epithelial cells left untreated (0) or treated for 10 or 30 min with IL-17 (25 ng/ml); lysates immunoprecipitated with anti-TRAF5 (above) and whole-cell lysates (40 μg; below) were analyzed by immunoblot with anti-SF2 (ASF). () RNA-immunoprecipitation (with anti-SF2 (ASF)) and real-time RT-PCR analysis of CXCL1, CSF3, CXCL2 (MIP-2) and GAPDH mRNA in extracts of primary kidney epithelial cells treated for 2.5 h with TNF or TNF alone (TN) or for 2 h with TNF followed by IL-17 for 30 min (TNF + IL-17); results are presented relative to results obtained! by immunoprecipitation with nonspecific IgG (change in cycling threshold). Data are representative of two (,) or three () independent experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA. * Dongxu Sun, * Michael Novotny, * Katarzyna Bulek, * Caini Liu, * Xiaoxia Li & * Thomas Hamilton Contributions M.N., K.B. and C.L. did experiments; D.S. designed, did and interpreted experiments and participated in writing the manuscript; T.H. and X.L. designed and interpreted experiments and participated in writing the manuscript; and all authors reviewed the final version of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas Hamilton Author Details * Dongxu Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Novotny Search for this author in: * NPG journals * PubMed * Google Scholar * Katarzyna Bulek Search for this author in: * NPG journals * PubMed * Google Scholar * Caini Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoxia Li Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Hamilton Contact Thomas Hamilton Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (340K) Supplementary Figures 1–2 Additional data - The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-γ
- Nat Immunol 12(9):861-869 (2011)
Nature Immunology | Article The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-γ * Feng Ma1, 2, 5 * Sheng Xu1, 5 * Xingguang Liu1 * Qian Zhang1 * Xiongfei Xu1 * Mofang Liu3 * Minmin Hua3 * Nan Li1 * Hangping Yao2 * Xuetao Cao1, 2, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:861–869Year published:(2011)DOI:doi:10.1038/ni.2073Received06 December 2010Accepted20 June 2011Published online24 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interferon-γ (IFN-γ) has a critical role in immune responses to intracellular bacterial infection. MicroRNAs (miRNAs) are important in the regulation of innate and adaptive immunity. However, whether miRNAs can directly target IFN-γ and regulate IFN-γ production post-transcriptionally remains unknown. Here we show that infection of mice with Listeria monocytogenes or Mycobacterium bovis bacillus Calmette-Guérin (BCG) downregulated miR-29 expression in IFN-γ-producing natural killer cells, CD4+ T cells and CD8+ T cells. Moreover, miR-29 suppressed IFN-γ production by directly targeting IFN-γ mRNA. We developed mice with transgenic expression of a 'sponge' target to compete with endogenous miR-29 targets (GS29 mice). We found higher serum concentrations of IFN-γ and lower L. monocytogenes burdens in L. monocytogenes–infected GS29 mice than in their littermates. GS29 mice had enhanced T helper type 1 (TH1) responses and greater resistance to infection with BCG or Myc! obacterium tuberculosis. Therefore, miR-29 suppresses immune responses to intracellular pathogens by targeting IFN-γ. View full text Figures at a glance * Figure 1: Infection with an intracellular pathogen upregulates IFN-γ production but downregulates miR-29 expression in activated NK cells and T cells. () Quantitative RT-PCR assay of IFN-γ, miR-29a and miR-29b mRNA in splenocytes and NK cells from mice infected with L. monocytogenes (LM), presented relative to expression in uninfected splenocytes (0 h). () Quantitative RT-PCR assay of IFN-γ, miR-29a and miR-29b mRNA in splenocytes, splenic CD4+ T and CD8+ T cells from mice before (0 d) and 14 d after BCG infection, presented relative to expression in uninfected cells (0 d). *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.e.m.). * Figure 2: NF-κB mediates the transcriptional suppression of miR-29 in activated, IFN-γ-producing NK cells and T cells. () Quantitative RT-PCR assay of IFN-γ and miR-29a mRNA in NK cells left unstimulated (control (Ctrl)) or stimulated for 6 h with poly(I:C), IL-12 plus IL-18, or PMA plus ionomycin (iono), presented relative to expression in unstimulated NK cells. () Quantitative RT-PCR assay of IFN-γ and miR-29a mRNA in naive CD4+ T cells, various helper T cell subsets and natural regulatory T cells (Treg), presented relative to expression in naive CD4+ T cells. (,) Flow cytometry analysis of IFN-γ expression (left) and quantitative RT-PCR assay of miR-29a (right) in CD45.1− and CD45.1+ T cells from mice given transfer of CD45.1+ OT-II T cells () or OT-I T cells (), and then immunized with OVA plus complete Freund's adjuvant, assessed 5 d after immunization; mRNA results are presented relative to those of CD45.1− T cells. () Quantitative RT-PCR assay of IFN-γ and miR-29a mRNA in human naive CD4+ T and TH1 cells, presented relative to expression in naive CD4+ T cells. () Quantitative ! RT-PCR assay of miR-29a in mouse TH0 cells without pretreatment (−) or pretreated (+) for 30 min with PDTC (30 μM), then left unstimulated (−) or stimulated (+) for 12 h with mAb to CD3 (anti-CD3); results are presented relative to those of untreated TH0 cells. () Luciferase activity in lysates of RAW264.7 cells transfected with luciferase reporter plasmid for the Mir29b-1–Mir29a promoter, pretreated with PDTC or not, then left unstimulated or stimulated for 24 h with LPS or CpG; results are presented relative to renilla luciferase activity. () Chromatin-immunoprecipitation (ChIP) assay of the binding of p50 and c-Rel to the Mir29b-1–Mir29a promoter in TH1 cells stimulated for 2 h with mAb to CD3, assessed with two independent primers for the promoter. IgG, immunoglobulin G (immunoprecipitation control). () Luciferase activity in lysates of RAW264.7 cells transfected with Mir29b-1–Mir29a luciferase reporter plasmids mutation of the YY1- or NF-κB-binding site (ho! rizontal axis), then left unstimulated or stimulated for 24 h ! with LPS; results are presented relative to renilla luciferase activity. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (–,,; mean and s.e.m. (–) or mean and s.d. of four samples (,)) or one experiment representative of three independent experiments with similar results (). * Figure 3: Direct targeting of the 3′ UTR of IFN-γ mRNA by miR-29. () Conserved miR-29-binding sites (underlined) in the 3′ UTR of IFN-γ mRNA. () Luciferase activity in lysates of HEK293T cells transfected with construct encoding wild-type (WT) or mutated (Mut) IFN-γ 3′ UTR plus mimics (left) or inhibitors (right) of miR-29a, miR-29b or miR-29c (or the appropriate control). () Enzyme-linked immunosorbent assay (ELISA) of IFN-γ in supernatants of EL4 cells mock transfected or transfected with vector for T-bet and, 24 h later, stimulated for 24, 48 or 72 h with PMA plus ionomycin. () ELISA of IFN-γ in supernatants of EL4 cells transfected with T-bet vector and miR-29a mimic (left) or miR-29a inhibitor (right) or control and, 12 h later (mimic) or 24 h later (inhibitor), stimulated as in . () RT-PCR assay of NPM1 mRNA (positive control) and miR-29a mRNA (middle) and quantitative RT-PCR assay of IFN-γ mRNA (bottom) for RNA-binding proteins immunoprecipitated (RIP) with antibody to Ago2 or immunoglobulin G (IgG) from lysates of EL4 cell! s transfected with vector for T-bet and miR-29a mimic and, 24 h later, stimulated for 6 h with PMA plus ionomycin; results (bottom) are presented relative to those of EL4 cells transfected with the T-bet vector and control mimic, followed by immunoprecipitation with IgG. Top, immunoblot analysis of Ago2 protein in EL4 cell lysates (input control). *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.e.m.). * Figure 4: Downregulation of IFN-γ production in activated primary CD4+ and CD8+ T cells by miR-29. (,) Quantitative RT-PCR assay of miR-29a in OT-II or OT-I T cells transduced with miR-29a lentivirus (LV-29a; ), miR-29 sponge lentivirus (LV-29sponge; ) or the appropriate control lentivirus (LV-ctrl; ,); results are presented relative to those of untreated T cells () or LV-ctrl-transduced T cells (). (,) ELISA of IFN-γ in supernatants of OT-II () or OT-I () T cells transduced with LV-ctrl, LV-29a or LV-29sponge, then activated for 3 d with OVA peptide–pulsed dendritic cells. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.e.m.). * Figure 5: Generation of GS29 mice and verification of the involvement of miR-29 in regulating IFN-γ production in vivo. () Similarity between miR-29 family members and the miR-29 sponge sequence. Underlining indicates nucleotides that differ; superscripting represents bulge in sponge sequence. () Luciferase activity in lysates of HEK293T cells transfected with construct encoding the 3′ UTR of IFN-γ and increasing concentrations (wedge) of UBC-29sponge, presented relative to renilla luciferase activity. () ELISA of IFN-γ in supernatants of EL4 cells transfected with vector for T-bet plus increasing concentrations (wedge) of control vector and/or UBC-29sponge (key), then stimulated for 24 h with PMA plus ionomycin. () RT-PCR analysis (top) and quantitative RT-PCR assay (bottom) of GFP mRNA in peritoneal macrophages (MΦ) and CD4+ T cells from GS29 mice; results (bottom) are presented relative to expression in cells from littermates (control mice). () Luciferase activity in lysates of peritoneal macrophages from GS29 and control mice transfected with various reporter vectors (below); results! are presented relative to renilla luciferase activity. mut, mutant. () ELISA of IFN-γ in supernatants of NK cells obtained from GS29 and control mice and left unstimulated (Med) or stimulated for 6 h with PMA plus ionomycin, IL-12 plus IL-18 or poly(I:C). (,) ELISA of IFN-γ in supernatants of activated CD4+ T cells () and CD8+ T cells () from GS29 and control mice. THN, naive CD4+ T cells stimulated with mAb to CD3 and mAb to CD28. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.e.m.). * Figure 6: Greater resistances of GS29 mice to L. monocytogenes infection. () Survival of GS29 and control mice (n = 10 per genotype) injected intraperitoneally with 5 × 105 virulent L. monocytogenes. P < 0.01 (Wilcoxon test). (,) Bacterial burden in liver and spleen () and ELISA of serum IFN-γ () in GS29 and control mice 3 d after intraperitoneal injection of 5 × 104L. monocytogenes. CFU, colony-forming units. () Flow cytometry analysis of IFN-γ expression in NK cells from GS29 and control mice 24 h after infection with L. monocytogenes. Numbers adjacent to outlined areas indicate percent IFN-γ+NK1.1+ cells. (–) Quantitative RT-PCR assay of tumor necrosis factor (TNF) mRNA (), IL-6 mRNA () and inducible nitric oxide synthase (iNOS) mRNA () in peritoneal macrophages from GS29 and control mice 24 h and 48 h after infection with L. monocytogenes. *P < 0.01 (Student's t-test). Data are from three independent experiments (–,–; mean ± s.e.m.) or are representative of three independent experiments (). * Figure 7: More potent TH1 responses and delayed-type hypersensitivity in GS29 mice infected with BCG. () ELISA of IFN-γ and IL-4 in supernatants of splenocytes from GS29 and control mice infected with BCG and, 28 d later, restimulated for 3 d with various doses (horizontal axes) of PPD. () Flow cytometry analysis of IFN-γ-producing CD4+ and CD8+ T cells in splenocytes from GS29 and control mice infected with BCG and, 28 d later, restimulated with mAb to CD3. Numbers adjacent to outlined areas indicate percent IFN-γ+CD4+ cells (top) or IFN-γ+CD8+ cells (bottom). () Footpad swelling of BCG-infected GS29 and control mice 24 h after PPD challenge. Each symbol represents an individual mouse; small horizontal lines indicate the mean. () Histology of swollen footpads from the mice in . Original magnification, ×100. () BCG burden in lungs of GS29 and control mice 4 weeks after intravenous infection of BCG. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (,,; mean ± s.e.m.) or are representative of three independent experiments (,). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Feng Ma & * Sheng Xu Affiliations * National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China. * Feng Ma, * Sheng Xu, * Xingguang Liu, * Qian Zhang, * Xiongfei Xu, * Nan Li & * Xuetao Cao * Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China. * Feng Ma, * Hangping Yao & * Xuetao Cao * National Key Laboratory of Molecular Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. * Mofang Liu & * Minmin Hua * National Key Laboratory of Medical Molecular Biology, Chinese Academy of Medical Sciences, Beijing, China. * Xuetao Cao Contributions X.C. and F.M. designed the experiments; F.M., S.X., X.L., Q.Z., X.X., M.L., M.H., N.L. and H.Y. did the experiments; and X.C., F.M. and S.X. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Xuetao Cao Author Details * Feng Ma Search for this author in: * NPG journals * PubMed * Google Scholar * Sheng Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Xingguang Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Qian Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Xiongfei Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Mofang Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Minmin Hua Search for this author in: * NPG journals * PubMed * Google Scholar * Nan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Hangping Yao Search for this author in: * NPG journals * PubMed * Google Scholar * Xuetao Cao Contact Xuetao Cao Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–16 and Methods Additional data - Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells
- Nat Immunol 12(9):870-878 (2011)
Nature Immunology | Article Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells * Maria T Pallotta1 * Ciriana Orabona1 * Claudia Volpi1 * Carmine Vacca1 * Maria L Belladonna1 * Roberta Bianchi1 * Giuseppe Servillo2 * Cinzia Brunacci2 * Mario Calvitti1 * Silvio Bicciato3 * Emilia M C Mazza3 * Louis Boon4 * Fabio Grassi5 * Maria C Fioretti1 * Francesca Fallarino1, 6 * Paolo Puccetti1, 6 * Ursula Grohmann1, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:870–878Year published:(2011)DOI:doi:10.1038/ni.2077Received09 May 2011Accepted22 June 2011Published online31 July 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-β (TGF-β) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-β-driven tolerance in noninflammatory contexts. View full text Figures at a glance * Figure 1: IDO catalytic activity is not required for the IDO-dependent, immunoregulatory effects of pDCs conditioned with TGF-β in vitro. () Proliferation of CD4+ T cells in a CD4+CD25− T cell population cultured for 4 d together with pDCs transfected with scrambled sequence (Control) or siRNA targeting Ido1 (two separate siRNAs: Ido1-1 and Ido1-2) or Ido2, or treated with 1-MT; cultures were left untreated (None) or treated with IFN-γ or TGF-β and were assessed by cytofluorometry as the frequency of cells positive for the thymidine analog EdU. () Apoptosis of CD4+ T cells in 24-hour cocultures established as in , assessed as the frequency of cells positive for propidium iodide (PI) and annexin V (AnnV). () Expression of Foxp3 in 4-day cocultures as in , assessed by cytofluorometry as the frequency of CD4+Foxp3+ cells. Histograms and plots for – are in Supplementary Figures 2–4. () Proliferation of CD4+CD25− T cells cultured as in in various numbers (horizontal axis), in the presence of soluble anti-CD3 and irradiated populations of splenocytes depleted of T cells. () Cytokines in supernatants of 4-d! ay cocultures as in . NC, siRNA with a scrambled sequence (negative control). () TGF-β1 expression on pDCs from wild-type C57BL/6 mice (WT) or Ido1−/− mice after 24 h of treatment with TGF-β or medium alone (Control). Numbers in top right quadrants indicate percent TGF-β+ cells positive for the antibody 120G8 (to pDCs). Compiled results (mean ± s.d.): TGF-β, 4.5 ± 1.5; wild-type control, 1.2 ± 0.4 (P = 0.011 (Student's t-test)). *P < 0.01 and **P < 0.001 (Student's t-test). Data are from three independent experiments (mean and s.d. in –,) or are from one experiment representative of three (,; mean and s.d. of triplicates in ). * Figure 2: The catalytic activity of IDO is not required for IDO-dependent, immunoregulatory effects induced in vivo by pDCs conditioned with TGF-β. () In vivo suppression of the activity of HY-pulsed C57BL/6 CD8− DCs transferred into recipient mice coexpressing enhanced green fluorescent protein and Foxp3, either alone (None) or in combination with a minority fraction (5%) of pDCs with no conditioning (pDCs) or conditioned with IFN-γ (pDCs + IFN-γ) or TGF-β (pDCs + TGF-β) that were left untransfected (Control), transfected with siRNA with a scrambled sequence (negative control (NC)) or targeting Ido1 or Ido2, or treated with 1-MT, or treated with anti-TGF-β at the time of DC sensitization; analysis of skin reactivity of recipient mice to the eliciting peptide at 15 d is presented as change in footpad weight. () Histology of sections of footpads from mice sensitized for 15 d with pDCs (left untreated (No treatment) or treated with IFN-γ or TGF-β), then injected in the footpad with peptide (Experimental footpad) or vehicle alone (Control footpad); footpads removed 24 h later were stained with hematoxylin and eosi! n for analysis of leukocyte infiltration. Bottom, photographs of footpads. Scale bars, 100 μm. () Production of IFN-γ by leukocytes from mice (n = 6 per group) sensitized with peptide-pulsed DCs (left untreated (None) or treated with IFN-γ or TGF-β), treated as in (key), and challenged with HY 15 d later in the footpad; leukocytes collected 24 h later from popliteal lymph nodes (draining left hind footpads) were restimulated for 48 h in vitro with HY, followed by analysis of IFN-γ in culture supernatants. () Expression of Foxp3 by cells from popliteal lymph nodes obtained as in , assessed by cytofluorometry as the frequency of CD4+Foxp3+ cells. *P < 0.01 (Student's t-test). Data are representative of four (,), two () or three () experiments (mean and s.d. in ,,). * Figure 3: TGF-β induces the formation of IDO–SHP-1–SHP-2 complexes and activation of SHP-1 phosphatase activity in pDCs. () Kinetics of the phosphorylation of IDO ITIM2 in pDCs conditioned with TGF-β or IFN-γ, analyzed by immunoblot sequentially with antibody to phosphorylated ITIM2 (p-IDO), anti-IDO and anti-β-tubulin. () Real-time PCR analysis of Ptpn6, Ptpn11 and Inpp5d transcripts in pDCs treated as in , normalized to the expression of Gapdh (encoding glyceraldehyde phosphate dehydrogenase) and presented relative to results in untreated cells (dotted line, onefold). *P < 0.01 (Student's t-test). () Precipitation (Ppt) of SHP-1, SHP-2 and SHIP from lysates of untreated P1 cells and TGF-β-treated pDCs with unphosphorylated IDO peptides (ITIM1 and ITIM2) or tyrosine-phosphorylated IDO peptides (p-ITIM1 and p-ITIM2), analyzed by immunoblot sequentially with anti-SHP-1, anti-SHP-2 and anti-SHIP. WCL (below), immunoblot analysis of whole-cell lysates as above. () Immunoprecipitation (IP) of proteins from lysates from pDCs (left untreated (−) or treated for 24 h with TGF-β) with anti-IDO, ! analyzed by immunoblot sequentially with anti-SHP-1, anti-SHP-2 and anti-IDO. WCL (right), immunoblot analysis of whole-cell lysates (one-tenth of the full volume), as a loading control. () Phosphatase activity in anti-IDO immunoprecipitates of pDCs treated as in alone (−) or also transfected with negative control siRNA (NC) or siRNA targeting Ido1, Ptpn6 or Ptpn11 or treated with stibogluconate (Stibo); results are presented as free phosphate released from samples with a volume of 50 μl. *P < 0.01 and **P < 0.001 (Student's t-test). () Phosphatase activity (top) and kynurenine production (bottom) by P1 cells left untransfected (−), mock-transfected (Control), or transfected with constructs for wild-type IDO (IDO), IDO2, or IDO mutants lacking the histidine residue required for catalytic activity (IDO(H350A)) or the ITIM1 tyrosine (IDO(Y115F)), the ITIM2 tyrosine (IDO(Y253F)) or both (Y115F,Y253F)), then left untreated (open bars) or treated (filled bars) with stiboglu! conate (top) or 4 μM 1-MT (bottom). *P < 0.05 and **P < 0.005! (top) or **P < 0.001 (bottom; all Student's t-test). ND, not detectable. () Intracellular immunofluorescence analysis of the colocalization of IDO and SHP-1 in P1 cells transfected as in , treated for 10 min with Na3VO4 and fixed with formaldehyde; Alexa Fluor 488–labeled anti-IDO was used in combination with anti-SHP-1 and indocarbocyanine-conjugated antibody to mouse immunoglobulin, and nuclei were stained with the DNA-intercalating dye DAPI (blue). Data are from one experiment representative of three (,,,,) or are from three experiments (,; mean and s.d. in ,,). * Figure 4: IDO phosphorylation requires PI(3)K-dependent but Smad-independent TGF-β signaling events and is mediated by Fyn but not Syk. () Phosphorylation of IDO ITIM2 in pDCs pretreated for 1 h with SIS3 or LY294002 before incubation for 60 min with TGF-β, assessed by sequential immunoblot analysis with antibody to phosphorylated ITIM2 (p-IDO), anti-IDO and anti-β-tubulin. () Real-time PCR analysis of Ptpn6 transcripts in pDCs treated for 16 h with TGF-β with or without SIS3 or LY294002, normalized (as in Fig. 3b) and presented relative to results in untreated cells. *P < 0.001 (Student's t-test). () Distribution of absolute expression of genes encoding mouse Src kinases (horizontal axis) in untreated mouse pDCs (n = 17), based on data derived from publicly available gene expression data sets. Each symbol represents an individual pDC gene; small horizontal lines indicate the mean. () Heat map of standardized expression of genes as in (all values relative to an arbitrary unit scale) in untreated mouse pDCs (n = 17) derived from six different sets of samples from the Gene Expression Omnibus (numbers above ! lanes, accession codes for the 17 samples). () Immunoblot analysis of Fyn in lysates of pDCs left untreated (−) or treated for 24 h with TGF-β (+). () Phosphorylation of IDO ITIM2 (as in ) in pDCs treated for 1 h with PP2, PP3 or a Syk inhibitor before incubation for 60 min with TGF-β. Data are from one experiment representative of two (,,) or three (; error bars, s.d.) or are from one metanalysis (,). * Figure 5: IDO and SHP proteins drive a signaling pathway in pDCs that involves activation of the noncanonical NF-κB pathway and production of type I interferon. () Activation of IKKα and IKKβ in pDCs treated for 0–180 min (above lanes) with TGF-β or IFN-γ; lysates were sequentially blotted with antibody to phosphorylated IKKα or IKKβ, anti-IKKα and anti-IKKβ (labels between blots indicate expected migration). () Enzyme-linked immunosorbent assay of the activation of p65, p52 and RelB in nuclear extracts of pDCs not treated with cytokine (None) or treated with cytokine for 30 or 60 min as in (bottom), and not transfected or pretreated (None) or pretreated for 1 h with 1-MT or transfected with negative control siRNA or siRNA targeting Ido1, Ido2, Ptpn6 and/or Ptpn11 (horizontal axis); results are presented as absorbance at 450 nm (A450). *P < 0.05 and **P < 0.01, cytokine-treated versus untreated (Student's t-test). () Enzyme-linked immunosorbent assay of IFN-α in supernatants of BALB/c, C57BL/6 or Ido1−/− pDCs left untreated or treated for 24 h with various combinations of IFN-γ or TGF-β and 1-MT (key), plus no siRNA! (None), negative control siRNA or siRNA targeting various genes (horizontal axis; two separate siRNAs for Ido1 siRNA (as in Fig. 1a) and two for Irak1). *P < 0.01 and **P < 0.001 (Student's t-test). () Coimmunoprecipitation of IRAK1 and IDO with SHP proteins (with a mixture of anti-SHP-1 and anti-SHP-2) from lysates of pDCs transfected with the Ido1-containing construct, left untreated or treated for 24 h with TGF-β, followed by sequential immunoblot analysis with anti-IDO, anti-IRAK1, anti-SHP-1 and anti-SHP-2. Data are from one experiment representative of two () or three () or are from three experiments (,; mean and s.d.). * Figure 6: IDO-dependent immunoregulatory effects of pDCs conditioned with TGF-β are mediated by Fyn, SHP proteins, the noncanonical NF-κB pathway and type I interferon signaling. () Frequency of CD4+Foxp3+ cells in a CD4+CD25− T cell population cultured for 4 d together with BALB/c wild-type or 129/Sv wild-type or Ifnar−/− pDCs transfected with siRNA (horizontal axis) and treated with TGF-β. *P < 0.01 (Student's t-test). () IL-6 in supernatants of cocultures as in . () Real-time PCR analysis of Tgfb1 transcripts in pDCs transfected with siRNA (horizontal axis) and treated for 16 h with TGF-β; normalized results (as in Fig. 3b) are presented relative to results in untreated cells (dashed line, onefold). () Skin-test assay (as in Fig. 2a) of the in vivo suppressive activity of TGF-β-treated BALB/c pDCs pretreated for 1 h with PP2 or PP3 (key) or transfected with siRNA (horizontal axis), and TGF-β-treated untransfected 129/Sv wild-type or Ifnar−/− pDCs loaded with IGRP. *P < 0.001 (Student's t-test). Data are from three experiments (mean and s.d.). * Figure 7: TGF-β induces long-term expression of IDO in pDCs, but IFN-γ does not. () Chromatin-immunoprecipitation assay of the binding of p52-RelB, IRF3, IRF4 (negative control), IRF7 and IRF8 to the mouse Ido1 promoter in pDCs treated for 3 h (p52-RelB) or 16 h (IRFs) with TGF-β, quantified by real-time PCR with primers composed of the putative noncanonical (NC) NF-κB-, ISRE-1- or ISRE-2-binding region; normalized results (as in Fig. 3b) are presented relative to results in untreated cells (dashed line, onefold). () Time course of activation of the Ido1 promoter in pDCs transfected with a firefly luciferase construct of the Ido1 promoter plus noncoding sequence in Ido1 exon 1 and incubated for 3–48 h with IFN-γ or TGF-β; results are normalized to the activity of a cotransfected constitutive reporter and are presented relative to those in untreated cells (dashed line, onefold). () Ido1 promoter activity in wild-type BALB/c, wild-type (129/Sv) or Ifnar−/− 129/Sv, wild-type (C57BL/6) or Ido1−/− pDCs left untransfected (None) or transfected wi! th negative control siRNA or gene-specific siRNA (horizontal axis), then incubated for 24 h with TGF-β; luciferase activity was assessed as in . () Immunoblot analysis of the time course of IDO protein expression in pDCs either incubated for 0, 24 or 48 h (above lanes) with TGF-β or IFN-γ, or incubated for 24 h with TGF-β or IFN-γ, washed extensively and incubated for 24 h with medium alone (w); lysates were probed sequentially with anti-IDO and anti-β-tubulin. () Time-course of IDO catalytic activity in pDCs left untreated (None; time 0) or treated for 16, 24 or 48 h with IFN-γ or TGF-β for 16–48 h, or treated with washing as in (Wash), assessed as kynurenine in culture supernatants. *P < 0.01 (Student's t-test). Data are from three experiments (–,; mean and s.d.) or are from one experiment representative of three (). * Figure 8: Long-term immunoregulatory effects in vivo of pDCs conditioned by TGF-β but not those conditioned by IFN-γ. () Long-term skin-test assay of mice coexpressing enhanced green fluorescent protein and Foxp3 under the control of an endogenous promoter (B6.Cg-Foxp3 mice; given C57BL/6 pDCs) or C57BL/6 mice (given Ido1−/− pDCs) given transfer of HY-pulsed pDCs left untreated (None) or pretreated for 24 h with IFN-γ or TGF-β (C57BL/6 or Ido1−/−) and combined with HY-pulsed CD8− DCs, assessed by reactivity to a skin test 3 months later. *P < 0.001 (Student's t-test). () Cytofluorometry of the frequency of CD4+Foxp3+ cells among leukocytes from the popliteal lymph nodes (draining the left hind footpads) of mice (n = 6 per group) sensitized as in with peptide-pulsed DCs 3 months earlier and challenged in the footpad with peptide 24 h before analysis. () IFN-γ in leukocytes from popliteal lymph nodes collected as in , assessed after restimulation for 48 h in vitro with HY. () Cytofluorometry of the frequency of CD4+Foxp3+ cells (numbers in top right quadrants) among CD4+CD25− T! cells cultured for 4 d together with TGF-β-treated pDCs, plus no antibody (No Ab; left)), anti-TGF-β (middle) or isotype-matched control antibody (isotype; right) added at the beginning of culture (time 0) or at 48 h (right margin). *P < 0.01 and (Student's t-test). Data are from three experiments (–; mean and s.d.) or are from one experiment representative of two (). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * 15930 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Francesca Fallarino, * Paolo Puccetti & * Ursula Grohmann Affiliations * Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy. * Maria T Pallotta, * Ciriana Orabona, * Claudia Volpi, * Carmine Vacca, * Maria L Belladonna, * Roberta Bianchi, * Mario Calvitti, * Maria C Fioretti, * Francesca Fallarino, * Paolo Puccetti & * Ursula Grohmann * Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy. * Giuseppe Servillo & * Cinzia Brunacci * Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy. * Silvio Bicciato & * Emilia M C Mazza * Bioceros, Utrecht, The Netherlands. * Louis Boon * Institute for Research in Biomedicine, Bellinzona, Switzerland. * Fabio Grassi Contributions M.T.P. designed and did experiments; C.O., C.Vo. C.Va., M.L.B., R.B., C.B., M.C. and E.M.C.M. did experiments; G.S., S.B. and M.C.F. contributed to experimental design; L.B. and F.G. provided reagents; F.F. designed experiments and supervised research; P.P. supervised research; and U.G. designed experiments, supervised research and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ursula Grohmann Author Details * Maria T Pallotta Search for this author in: * NPG journals * PubMed * Google Scholar * Ciriana Orabona Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Volpi Search for this author in: * NPG journals * PubMed * Google Scholar * Carmine Vacca Search for this author in: * NPG journals * PubMed * Google Scholar * Maria L Belladonna Search for this author in: * NPG journals * PubMed * Google Scholar * Roberta Bianchi Search for this author in: * NPG journals * PubMed * Google Scholar * Giuseppe Servillo Search for this author in: * NPG journals * PubMed * Google Scholar * Cinzia Brunacci Search for this author in: * NPG journals * PubMed * Google Scholar * Mario Calvitti Search for this author in: * NPG journals * PubMed * Google Scholar * Silvio Bicciato Search for this author in: * NPG journals * PubMed * Google Scholar * Emilia M C Mazza Search for this author in: * NPG journals * PubMed * Google Scholar * Louis Boon Search for this author in: * NPG journals * PubMed * Google Scholar * Fabio Grassi Search for this author in: * NPG journals * PubMed * Google Scholar * Maria C Fioretti Search for this author in: * NPG journals * PubMed * Google Scholar * Francesca Fallarino Search for this author in: * NPG journals * PubMed * Google Scholar * Paolo Puccetti Search for this author in: * NPG journals * PubMed * Google Scholar * Ursula Grohmann Contact Ursula Grohmann Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (823K) Supplementary Figures 1–12 Additional data - Afferent lymph–derived T cells and DCs use different chemokine receptor CCR7–dependent routes for entry into the lymph node and intranodal migration
- Nat Immunol 12(9):879-887 (2011)
Nature Immunology | Article Afferent lymph–derived T cells and DCs use different chemokine receptor CCR7–dependent routes for entry into the lymph node and intranodal migration * Asolina Braun1 * Tim Worbs1 * G Leandros Moschovakis1 * Stephan Halle1 * Katharina Hoffmann1 * Jasmin Bölter1 * Anika Münk1 * Reinhold Förster1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:879–887Year published:(2011)DOI:doi:10.1038/ni.2085Received26 May 2011Accepted07 July 2011Published online14 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system. View full text Figures at a glance * Figure 1: Ccr7−/− BMDCs do not reach the paracortical TCZ after intralymphatic injection. () Expression of CD11c and major histocompatibility complex class II (MHCII) by DCs generated in vitro from the bone marrow of wild-type (WT) and Ccr7−/− (KO) mice. Numbers adjacent to outlined areas indicate percent CD11c+ DCs positive for major histocompatibility complex class II. () Retrieval of wild-type and Ccr7−/− BMDCs labeled with DDAO (wild-type) or TAMRA (Ccr7−/−), mixed at a ratio of 1:1 and injected intralymphatically (2.5 × 105 total cells); draining popliteal lymph nodes digested 2 h later were analyzed by flow cytometry. Numbers adjacent to outlined areas indicate percent DDAO+ cells (wild-type) and TAMRA+ cells (Ccr7−/−). () Quantification of BMDCs retrieved from draining popliteal lymph nodes (n = 11) as described in , presented as the ratio of wild-type cells to Ccr7−/− cells. () Microscopy of popliteal lymph nodes (popLN) obtained from mice 2–48 h (top right corners) after intralymphatic injection of eGFP-expressing wild-type BMDCs a! nd TAMRA-labeled Ccr7−/− BMDCs (1:1 mixture; 1 × 105 cells in 5 μl PBS). IgD, immunoglobulin D; M, orientation of the central medulla-hilus region. Repeat experiments with eGFP-expressing Ccr7−/− and TAMRA-labeled wild-type BMDCs yielded similar results (data not shown). () Microscopic analysis of the positioning of wild-type and Ccr7−/− BMDCs in a popliteal lymph node explanted 12 h after intralymphatic injection of cells (as in ) and sectioned completely; composite images acquired by fluorescence microscopy were assembled for three-dimensional reconstruction (Supplementary Video 1). () Microscopy of intralymphatically injected BMDCs in iliac lymph nodes (iLN) from the mice in . Scale bars, 100 μm (,) or 200 μm (). Data are representative of at least three independent experiments (–; mean and s.d. in ), two to five independent experiments with 4–12 lymph nodes per time point (,) or one experiment (). * Figure 2: Intranodal migratory activity of intralymphatically injected DCs in popliteal lymph nodes, as visualized by two-photon microscopy. () Microcopy of intranodal DC positioning in draining popliteal lymph nodes explanted 40 min after intralymphatic injection of 3 × 103 TAMRA-labeled wild-type BMDCs (red) and 3 × 103 eGFP-expressing Ccr7−/− BMDCs (green), imaged ex vivo at 1 h, ~4 h and ~8 h after cell transfer. Blue indicates the second-harmonics generation (SHG) signal of collagen fibers of the lymph-node capsule. () Ex vivo time-lapse imaging of a lymph node (0 min, top left) beginning 3 h after intralymphatic injection of 2 × 103 eGFP-expressing wild-type BMDCs (green). Colors of scale bars (bottom right corners) indicate time scale of DC tracks (right), from the start (blue) to the end (yellow) of imaging. () Ex vivo time-lapse imaging of a lymph node (as in ) beginning 4 h 20 min after intralymphatic injection of 2.5 × 103 eGFP-expressing Ccr7−/− BMDCs (green). (,) Ex vivo time-lapse imaging of a lymph node (as in ) beginning 1.5 h () or 3 h 45 min () after intralymphatic injection of 2 × ! 103 wild-type BMDCs () or 2.5 × 103Ccr7−/− BMDCs (), showing the cellular morphology of eGFP-expressing BMDCs (green) during intranodal migration. SHG signal (blue) at top indicates SCS position. () Ex vivo time-lapse imaging of a lymph node (as in ) beginning 6 h 30 min (wild-type) or 6 h (Ccr7−/−) after intralymphatic injection of 1.5 × 103 to 3.5 × 103 wild-type or Ccr7−/− BMDCs, analyzed after directional migration of wild-type BMDCs had ceased (additional images, Supplementary Video 5). () Ex vivo time-lapse imaging of a lymph node (as in ) beginning 2 h after intralymphatic injection of 3.5 × 103 CD11c-YFP DCs (green) that emigrated from the skin (additional images, Supplementary Video 6). SHG signal (blue) as in . () Statistical analysis of intranodal DC migration. For track speed and straightness, each symbol represents an individual cell track and red horizontal bars indicate the median; for the motility coefficient (MCoeff), each symbol represents ! a single time-lapse recording of a different imaging day and r! ed horizontal bars indicate the mean (colors in key match colors in plots). Mean displacement plots were calculated with one time-lapse recording per group. 2-P-M, two-photon microscopy. Time-lapse recordings are in Supplementary Video 2 (for ), Supplementary Video 3 (for ) or Supplementary Video 4 (for ). Scale bars, 50 μm (–,), 10 μm (,) or 5 μm (). Data are representative of four or more independent experiments with 7–19 lymph nodes (–), six independent experiments with six to eight lymph nodes (), three independent experiments with six lymph nodes () or three independent experiments (). * Figure 3: Intranodal positioning of afferent lymph–derived DCs contributes to T cell proliferation but not lymph-node shutdown. () Total living cells in the draining popliteal lymph node 72 h after intralymphatic (IL) or subcutaneous (SQ) injection of wild-type or Ccr7−/− BMDCs (5 × 103 cells per afferent lymph vessel or 1 × 105 cells per footpad) or PBS (control (Ctrl)). NS, not significant; *P < 0.01 and **P < 0.001 (unpaired two-tailed Student's t-test). () CFSE profiles of CD8+Ly5.1+Vα2+Vβ5+ lymphocytes isolated from draining popliteal lymph nodes (black lines) from bm1 recipients given adoptive transfer of 1.5 × 107 CSFE-labeled OT-I Ly5.1+ T cells by intravenous injection and, 1 d later, given intralymphatic injection of 5 × 103 SIINFEKL-pulsed wild-type BMDCs (top) or Ccr7−/− BMDCs (bottom), followed by flow cytometry 65 h later. Shaded curves, CFSE profile of the brachial lymph node of the same mouse (control). () Proliferation index of the cells in ; cell cycle number was calculated with FlowJo software. Each symbol represents an individual node; small horizontal bars indicate ! the mean. *P < 0.001 (unpaired two-tailed Student's t-test). Data are from three to four independent experiments with 8–15 mice (; mean and s.d.) or three independent experiments with 12 mice per group (,). * Figure 4: CD4+ T cells enter lymph-node parenchyma in peripheral medullary areas after intralymphatic injection and require CCR7 signals for migration into the paracortical TCZ. (,) Immunohistological analysis of draining popliteal lymph nodes after intralymphatic injection of 2 × 104 TAMRA-labeled wild-type (red) and 2 × 104 eGFP-expressing Ccr7−/− (green) polyclonal CD4+ T cells. Similar results were obtained with opposite labeling (data not shown). () Overview composite images at 2 h and 4 h after injection. Immunoglobulin D staining (blue) marks B cell follicles as well as medullary B cells; M indicates the orientation of the central medulla-hilus region. () LYVE-1 staining (blue) of lymphatic endothelium indicates sinus structures. White arrow indicates peripheral medullary sinus lumen; white arrowheads indicate medullary cords; white, DAPI nuclear staining. () Microscopy of LYVE-1+ peripheral medullay sinus structures (red) 15 min after intralymphatic injection of 2 × 104 inert 6-μm latex beads (green). () Microscopy of draining popliteal lymph nodes after intralymphatic injection of 2 × 104 TAMRA-labeled Ccr7−/− CD4+ T cells (red! ), followed by injection of 5 μl fixable fluorescein isothiocyanate–conjugated 500-kilodalton dextran (green) into the same afferent lymph vessel 15 min later and isolation and fixation with paraformaldehyde 5 min after that. LYVE-1 staining (blue) of lymphatic endothelium indicates sinus structures. Scale bars, 100 μm (), 20 μm () or 50 μm (,). Data are representative of five (,) or two (,) independent experiments (n = 15 lymph nodes per time point (,); n = 6 (); n = 12 ()). * Figure 5: Two-photon imaging of the entry into the lymph node of intralymphatically injected CD4+ T cells in peripheral medullary areas. (,) Ex vivo time-lapse imaging of a lymph node beginning 1 h 20 min () or 35 min () after intralymphatic injection of 1.6 × 104 to 2 × 104 polyclonal eGFP-expressing wild-type CD4+ T cells, showing entry into peripheral medullary cords by transmigration through LYVE-1+ endothelium (red, cyan outline) of the capsule-lining sinus floor (additional images, Supplementary Video 7). Blue indicates SHG signal of the lymph-node capsule; lines indicate tracks of individual cells. () Ex vivo time-lapse imaging of a lymph node beginning 2 h after intralymphatic injection of 1.6 × 104 wild-type CD4+ T cells (green), showing entry of cells into peripheral medullary cords by transmigration through LYVE-1+ endothelium (red) after extended migration in the lumen of the peripheral medullary sinus system (additional images, Supplementary Video 8). () Ex vivo time-lapse imaging of a lymph node beginning 1 h after intralymphatic injection of 1.6 × 104 polyclonal wild-type CD4+ T cells, show! ing intranodal migratory activity (tracks in red) in peripheral medullary areas directly after exit from the capsule-lining sinus lumen (white arrowhead). Grey three-dimensional structure represents the LYVE-1+ lymphatic endothelium and delineates the border between the peripheral medullary sinus lumen (S) and the parenchyma of peripheral medullary cords (P). Additional images, Supplementary Video 9. () Statistical analysis of the migration of wild-type CD4+ T cells (n = 202) in peripheral medullary areas 0.5–2 h after intralymphatic injection (three time lapse recordings; duration, 40–60 min). () Dwell index in the parenchyma versus sinus lumen (P/S), relative to compartment volume. () Transmigration events (compartment changes by migration through a LYVE-1+ lymphatic endothelial layer) per cell track. () Localization and direction of transmigration, presented as the frequency of cell tracks that remained in the lymph-node parenchyma (P) or the lumen of the peripheral ! medullary sinus (S) without transmigration or started in the l! umen of the peripheral medullary sinus and ended in the peripheral medullary cords (P) or vice versa (S). Scale bars, 10 μm (–) or 20 μm (). Data are representative of three independent experiments (–) or are from three independent experiments (–; mean and s.d.). * Figure 6: CCR7-mediated signals are required for the progression of T cells from peripheral medullary areas into the paracortical TCZ. () Ex vivo time-lapse imaging (0 min, far left) of a lymph node beginning 45 min after intralymphatic injection of 2.5 × 104 total CD4+ T cells (1:1 mixture of wild-type cells (red) and Ccr7−/− cells (green)), showing intranodal migration in peripheral medullary areas (additional images, Supplementary Video 10). Far right, cell track overlay; blue, SHG signal of the lymph-node capsule. () Statistical analysis of intranodal T cell migration, derived from time-lapse recordings starting 45 min to 3.5 h after intralymphatic injection of cells as in (presented as in Fig. 2h). *P < 0.05 (unpaired two-tailed Student's t-test). () Immunohistological analysis of CCL21 expression (red) in peripheral medullary areas. Blue, LYVE-1+ lymphatic endothelium; white dots outline a B cell follicle (B) devoid of CCL21 signal. Right, topological quantification of the LYVE-1 signals (blue) and CCL21 signals (red) in the area demarcated by the yellow lines at left. Scale bars, 50 μm (,). Dat! a are representative of three experiments (,) or are from three independent experiments (). * Figure 7: Transmigration of activated DCs changes the morphology of the SCS floor on the afferent side and allows direct entry of coinjected T lymphocytes into lymph-node parenchyma. () Ex vivo time-lapse imaging (0 min, left) of a lymph node beginning 30 min after intralymphatic injection of 2 × 105 wild-type CD4+ cells. Blue, SHG signal of the lymph-node capsule. () Ex vivo time-lapse imaging (0 min, left) of a lymph node after intralymphatic injection of 1.6 × 104 wild-type CD4+ cells and 3 × 103 TAMRA-labeled wild-type BMDCs (red) 40 min before injection of wild-type CD4+ T cells (green) into the same afferent lymph vessel, followed by imaging 1 h 20 min later. SHG signal (blue) as in . Right, overlay of wild-type CD4+ T cell tracks (green). (,) Cellular composition and morphology of the SCS after intralymphatic injection of 6-μm beads (green; ) or wild-type CD4+ T cells (green; ). Red, LYVE-1+ lymphatic endothelial cells; blue, CD169+ SCS-lining macrophages. () Microscopy of the transmigration of intralymphatically injected activated DCs (eGFP+ wild-type BMDCs; green) in the SCS. Blue, SCS-lining CD169+ macrophages (blue); red, LYVE-1-staining (! red). Below (–), enlargement of areas outlined in main images above. Scale bars, 50 μm (,) or 100 μm (–); inset size, 200 μm × 200 μm. Data are representative of three independent experiments with four to six lymph nodes (,) or two to four independent experiments with six to thirteen lymph nodes (–). Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Immunology, Hannover Medical School, Hannover, Germany. * Asolina Braun, * Tim Worbs, * G Leandros Moschovakis, * Stephan Halle, * Katharina Hoffmann, * Jasmin Bölter, * Anika Münk & * Reinhold Förster Contributions R.F., A.B. and T.W. designed experiments, analyzed data and wrote the paper; A.B. did experiments, including intralymphatic injection; T.W. contributed to Figure 1, two-photon imaging and data analysis; S.H. contributed to Figure 7 and read and commented on the manuscript; G.L.M. contributed to Supplementary Figure 2 and read and commented on manuscript; and A.M., K.H. and J.B. did histology, flow cytometry staining and DC cultures. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Reinhold Förster Author Details * Asolina Braun Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Worbs Search for this author in: * NPG journals * PubMed * Google Scholar * G Leandros Moschovakis Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Halle Search for this author in: * NPG journals * PubMed * Google Scholar * Katharina Hoffmann Search for this author in: * NPG journals * PubMed * Google Scholar * Jasmin Bölter Search for this author in: * NPG journals * PubMed * Google Scholar * Anika Münk Search for this author in: * NPG journals * PubMed * Google Scholar * Reinhold Förster Contact Reinhold Förster Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (7M) Ccr7-/- BMDCs do not reach the paracortical TCZ after intralymphatic application. * Supplementary Video 2 (10M) Long-term two-photon microscopical imaging of intranodal DC migration behavior. * Supplementary Video 3 (4M) Directional displacement and uropod formation of wt BMDCs is not reflected in Ccr7-/- BMDCs. * Supplementary Video 4 (1M) Close-up view of cellular morphology during intranodal migration. * Supplementary Video 5 (3M) Wt and Ccr7-/- BMDCs exhibit comparable dendrite movement and sweeping behavior. * Supplementary Video 6 (10M) After i.l. application, primary skin-derived wt DC display an intranodal migration comparable to wt BMDCs. * Supplementary Video 7 (7M) Transmigration of wt CD4 through lymphatic endothelium of the capsule-lining sinus floor. * Supplementary Video 8 (2M) Transmigration of wt CD4 through medullar sinus endothelium. * Supplementary Video 9 (10M) Upon exiting the capsule-lining sinus, wt CD4 preferably migrate within peripheral MC. * Supplementary Video 10 (4M) Intralymphatically administered CD4 T cells enter LN parenchyma in a CCR7-dependent manner primarily within peripheral medullary areas. * Supplementary Video 11 (8M) Immigration of DC allows CD4 T cells to enter the LN at SCS. * Supplementary Video 12 (1M) LPS-treated CD4 cells fail to enter popLN via SCS. PDF files * Supplementary Text and Figures (459K) Supplementary Figures 1–5 Additional data - The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function
- Nat Immunol 12(9):888-897 (2011)
Nature Immunology | Article The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function * Kai Yang1 * Geoffrey Neale2 * Douglas R Green1 * Weifeng He1 * Hongbo Chi1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:888–897Year published:(2011)DOI:doi:10.1038/ni.2068Received03 March 2011Accepted06 June 2011Published online17 July 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The mechanisms that regulate T cell quiescence are poorly understood. We report that the tumor suppressor Tsc1 established a quiescence program in naive T cells by controlling cell size, cell cycle entry and responses to stimulation of the T cell antigen receptor. Abrogation of quiescence predisposed Tsc1-deficient T cells to apoptosis that resulted in loss of conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells had more activity of the serine-threonine kinase complex mTORC1 but less mTORC2 activity, and activation of mTORC1 was essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in actively maintaining the quiescence of naive T cells to facilitate adaptive immune function. View full text Figures at a glance * Figure 1: Tsc1 deficiency leads to disrupted homeostasis of peripheral T cell populations. () Flow cytometry of thymocytes in wild-type (WT) and Tsc1−/− mice (left). Numbers adjacent to outlined areas indicate percent CD8+CD4− cells (top left), CD8+CD4+ cells (top right) or CD8−CD4+ cells (bottom right). Right, number of CD4+ single-positive (CD4SP) and CD8+ single-positive (CD8SP) thymocytes (n = 4 mice per group). Each symbol represents an individual mouse throughout; small horizontal lines indicate mean (± s.e.m.). () Flow cytometry of splenocytes from wild-type and Tsc1−/− mice (top). Numbers in quadrants indicate percent cells in each. Below, proportion (middle) and absolute number (bottom) of CD4+ and CD8+ T cells in the spleens of wild-type and Tsc1−/− mice (n = 4–6 per group). () Expression of CD62L and CD44 on wild-type and Tsc1−/− splenic T cells. Numbers in quadrants indicate percent cells in each. () Analysis of iNKT cells in the spleens of wild-type and Tsc1−/− mice (n ≥ 5 per group). Numbers adjacent to outlined areas ind! icate percent CD1d-PBS57+TCRβ+ cells (iNKT cells; top right) or CD1d-PBS57−TCRβ+ cells (conventional T cells; bottom right). Below, frequency (middle) and absolute number (bottom) of iNKT cells. () Flow cytometry of splenocytes from mixed–bone marrow chimeras generated by the transfer of a mixture (1:1) of bone marrow stem cells from wild-type (CD45.1+) and wild-type or Tsc1−/− (CD45.2+) mice into sublethally irradiated mice deficient in recombination-activating gene 1, followed by analysis at 8 weeks after reconstitution. Numbers in quadrants indicate percent cells in each. NS, not significant; *P < 0.005, **P < 0.0005 and ***P < 0.0001 (Student's t-test). Data are representative of four (–) or two () independent experiments. * Figure 2: Deletion of Tsc1 results in excessive apoptosis of T cells. () Caspase activity in freshly isolated wild-type and Tsc1−/− T cells, assessed by staining with fluorescein isothiocyanate–conjugated z-VAD-fmk (left). Numbers above bracketed lines indicate percent caspase-positive (Casp+) cells. Right, frequency of caspase-positive T cells (n = 7–8 mice per group). *P < 0.005 and **P < 0.0005 (Student's t-test). () Flow cytometry of spleen cells (SPL) and peripheral lymph node cells (PLN) in recipient (CD45.2+) mice 6 d after adoptive transfer of equal numbers of CD45.1+ (spike) cells and CD45.2+ wild-type or Tsc1−/− donor cells (CFSE labeled). Numbers adjacent to outlined areas indicate percent CSFE+CD45.1− cells (top left) or CSFE−CD45.1+ cells (bottom right). () Annexin V and 7-amino-actinomycin D (7-AAD) staining of naive wild-type or Tsc1−/− T cells cultured for 3 d in vitro with IL-7 (left), and expression of the IL-7 receptor α-chain (IL-7Rα) on freshly isolated cells (right). Numbers adjacent to outlined area! s (left) indicate percent 7-AAD+ annexin V–positive cells (top right) or 7-AAD− annexin V–negative cells (bottom left). () Propidium iodide (PI) staining of CD8+ T cells stimulated for 16 h with anti-CD3 alone (α-CD3), anti-CD3 plus anti-CD28 (α-CD3 + α-CD28) or PMA plus ionomycin (PMA + iono). Numbers above bracketed lines indicate percent propidium iodide–positive (apoptotic) cells. () Kinetics of apoptosis of CD4+ and CD8+ T cells stimulated with anti-CD3 plus anti-CD28, assessed as frequency of propidium iodide–positive (apoptotic) cells. () Propidium iodide staining of wild-type and Tsc1−/− CD8+ T cells pretreated for 1 h with vehicle (0) or 25 or 50 μM z-VAD-fmk, then activated for 10 h with anti-CD3 plus anti-CD28. Numbers above bracketed lines as in . Results similar to those in , were obtained with CD4+ T cells (data not shown). Data are representative of four (,), two (,), five () or three () independent experiments. * Figure 3: Tsc1-deficient T cells die via the Bcl-2 family–dependent intrinsic apoptotic pathway. () Intracellular Bcl-2 expression in wild-type and Tsc1−/− T cells. () Flow cytometry of splenocytes from wild-type, Tsc1−/−, Bcl2-transgenic (Bcl2-TG) and Tsc1−/−Bcl2-transgenic mice. Numbers in quadrants indicate percent cells in each. () Proportion (left) and absolute number (right) of CD4+ and CD8+ T cells in wild-type, Tsc1−/−, Bcl2-TG, and Tsc1−/−Bcl2-transgenic spleens (n = 3–6 mice per group). *P < 0.05, **P < 0.005 and ***P < 0.0001 (analysis of variance). (,) Propidium iodide staining of CD4+ cells () or CD8+ cells () stimulated for 16 h with anti-CD3 and anti-CD28. Numbers above bracketed lines indicate percent propidium iodide–positive (apoptotic) cells. () Expression of the Bim isoforms BimEL (extra-long) and BimL (long) in wild-type and Tsc1−/− CD4+ T cells left unstimulated (naive; 0) or activated for 3–10 h with anti-CD3 and anti-CD28. Numbers above lanes (results for BimEL) and below lanes (results for BimL) indicate band intens! ity relative to that of β-actin (loading control). () ROS production by freshly isolated wild-type and Tsc1−/− T cells. Data are representative of three (,–) or four (,) independent experiments. * Figure 4: Tsc1 deficiency causes cell-autonomous loss of quiescence in vivo and hyperactive responses to TCR stimulation. () Size of freshly isolated wild-type and Tsc1−/− T cells. FSC, forward scatter. () BrdU staining of wild-type and Tsc1−/− splenocytes 16 h after injection of BrdU (n = 3 mice per group). Numbers adjacent to outlined areas (left) indicate percent BrdU+ cells. (,) Expression of CD122 and CD44 on gated CD8+ splenocytes of wild-type and Tsc1−/− mice (; n = 5–8 per group) or mixed–bone marrow chimeras (; n = 3 per group (generated as in Fig. 1e)). Numbers in quadrants indicate percent cells in each. Right, ratio of CD122+ cells to CD122− cells among CD44hi populations () or CD44hi populations of CD45.2+ cells (). () Size of CD45.2+ cells in the mixed–bone marrow chimeras in . () Flow cytometry of wild-type and Tsc1−/− naive T cells stimulated for 16 h with anti-CD3 and anti-CD28, for analysis of cell size (top) and ROS production (bottom). () BrdU staining of naive T cells activated for 20 h with anti-CD3 and anti-CD28, followed by 90 min of pulsing with ! BrdU. Numbers adjacent to outlined areas indicate percent BrdU+ cells. () Expression of activation markers by wild-type and Tsc1−/− naive T cells stimulated for 16 h with anti-CD3 and anti-CD28. *P < 0.05, **P < 0.01 and ***P < 0.0001 (Student's t-test). Data are representative of five (,,), three (,,,) or four () independent experiments. * Figure 5: Tsc1-dependent gene expression programs. () Genes differently regulated in wild-type and Tsc1−/− CD4+ T cells left unstimulated (0 h) or activated for 4 h with anti-CD3 and anti-CD28 (difference of twofold or more with a false-discovery rate of less than 0.1; n = 3 mice per group). () Real-time PCR analysis of genes in wild-type and Tsc1−/− CD4+ T cells activated for various times as in ; results are presented relative to expression in wild-type unstimulated cells. Data are representative of two independent experiments (error bars, s.d.). * Figure 6: Loss of T cell quiescence results from inducible deletion of Tsc1 and is independent of cell survival. () Size of freshly isolated T cells from wild-type and Tsc1-CreER mice at 2 weeks after tamoxifen injection. () BrdU staining of splenocytes from mice in at 16 h after injection of BrdU. Numbers adjacent to outlined areas indicate percent BrdU+ cells. () Splenic T cell populations in the mice in . Numbers in quadrants indicate percent cells in each. () Propidium iodide staining of naive T cells obtained from mice treated as in and stimulated for 16 h with anti-CD3 and anti-CD28. Numbers above bracketed lines indicate percent propidium iodide–positive (apoptotic) cells. () Expression of CD122 and CD44 on gated CD8+ splenocytes from Bcl2-transgenic mice and Tsc1−/−Bcl2-transgenic mice; numbers in quadrants indicate percent cells in each. Middle, proportion of total CD44hi populations; below, ratio of CD122+ cells to CD122− cells among the CD44hi population (n = 4–6 mice per group). () Size of freshly isolated Bcl2-transgenic and Tsc1−/−Bcl2-transgenic T cells. ()! BrdU staining of Bcl2-transgenic and Tsc1−/−Bcl2-transgenic splenocytes at 16 h after injection of BrdU. () Flow cytometry of naive Bcl2-transgenic and Tsc1−/−Bcl2-transgenic T cells stimulation for 16 h with anti-CD3 and anti-CD28, for analysis of cell size (top left), ROS production (top right) and expression of CD25 and CD69 (bottom). *P = 0.0001 (Student's t-test). Data are representative of two (–,), four (,) or three () independent experiments. * Figure 7: Tsc1 regulates the activity of mTORC1 and mTORC2, and mTORC1 activation is essential for the disruption of immune quiescence and homeostasis. (,) Phosphorylation (p-) of S6K1, S6 and 4E-BP1 () and of Akt (Ser473), Foxo1 and Foxo3 () in wild-type and Tsc1−/− naive CD4+ T cells stimulated for various times (above lanes) with anti-CD3 and anti-CD28. *, nonspecific bands. Numbers below lanes indicate band intensity relative to that of β-actin (loading control). () Size of freshly isolated splenocytes from wild-type and Tsc1−/− mice given mock injection (Mock) or treated by daily injection of rapamycin for a total of 3 d or 5 d. () Flow cytometry of splenocytes from recipient (CD45.2+) mice 6 d after adoptive transfer of equal numbers of CD45.1+ (spike) cells and CD45.2+ wild-type or Tsc1−/− donor cells (CFSE labeled) purified from mock- or rapamycin-treated mice. Numbers adjacent to outlined areas indicate percent CSFE+CD45.1− cells (top left) or CSFE−CD45.1+ cells (bottom right). Similar results were obtained for recipient lymph nodes (data not shown). (,) Apoptosis () and size () of naive T cells ob! tained from mock- or rapamycin-treated wild-type and Tsc1−/− mice, then stimulated overnight with anti-CD3 and anti-CD28. Numbers above bracketed lines () indicate percent propidium iodide–positive (apoptotic) cells. Data are representative of five (,) or three (–) independent experiments. * Figure 8: Tsc1 deficiency dampens antibacterial immune response in vivo. () Flow cytometry of CD8+ T cells from wild-type, Tsc1−/− and Tsc1−/−Bcl2-transgenic mice infected with OVA-expressing L. monocytogenes. Numbers adjacent to outlined areas indicate percent cells positive for the tetramer of H-2Kb and OVA peptide (amino acids 257–264; H-2Kb–SIINFEKL). () Proportion and absolute number of OVA-reactive tetramer-positive CD8+ T cells from the mice in . (,) Flow cytometry () and proportion and absolute number () of OVA-reactive interferon-γ (IFN-γ)-positive CD8+ T cells from mice infected with OVA-expressing L. monocytogenes, detected after OVA stimulation and intracellular cytokine staining. Numbers adjacent to outlined areas () indicate percent interferon-γ-positive cells. *P < 0.005, **P < 0.001 and ***P < 0.0001 (analysis of variance). Data are representative of three independent experiments (n = 8–10 mice per group). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE29797 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Kai Yang, * Douglas R Green, * Weifeng He & * Hongbo Chi * Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Geoffrey Neale Contributions K.Y. designed and did cellular, molecular and biochemical experiments and contributed to the writing of the manuscript; G.N. did bioinformatic analyses; D.R.G. contributed genetic models and conceptual insights; W.H. contributed to cell purification; and H.C. designed experiments, wrote the manuscript and provided overall direction. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hongbo Chi Author Details * Kai Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey Neale Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas R Green Search for this author in: * NPG journals * PubMed * Google Scholar * Weifeng He Search for this author in: * NPG journals * PubMed * Google Scholar * Hongbo Chi Contact Hongbo Chi Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–15 and Tables 1–2 Additional data - Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation
- Nat Immunol 12(9):898-907 (2011)
Nature Immunology | Article Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation * Marc Beyer1 * Yasser Thabet1 * Roman-Ulrich Müller2, 3 * Timothy Sadlon4 * Sabine Classen1 * Katharina Lahl5 * Samik Basu6 * Xuyu Zhou7 * Samantha L Bailey-Bucktrout7 * Wolfgang Krebs1 * Eva A Schönfeld1 * Jan Böttcher8 * Tatiana Golovina6 * Christian T Mayer5 * Andrea Hofmann1 * Daniel Sommer1 * Svenja Debey-Pascher1 * Elmar Endl8 * Andreas Limmer8 * Keli L Hippen9 * Bruce R Blazar9 * Robert Balderas10 * Thomas Quast11 * Andreas Waha12 * Günter Mayer13 * Michael Famulok13 * Percy A Knolle8 * Claudia Wickenhauser14 * Waldemar Kolanus11 * Bernhard Schermer2, 3 * Jeffrey A Bluestone7 * Simon C Barry4 * Tim Sparwasser5 * James L Riley6 * Joachim L Schultze1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:898–907Year published:(2011)DOI:doi:10.1038/ni.2084Received24 May 2011Accepted07 July 2011Published online14 August 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Regulatory T cells (Treg cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (Teff cell) function and gain of suppressive activity by Treg cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of Treg cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3′ untranslated region. Release of SATB1 from the control of Foxp3 in Treg cells caused loss of suppressive function, establishment of transcriptional Teff cell programs and induction of Teff cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining Treg cell functionality. View full text Figures at a glance * Figure 1: Foxp3-dependent repression of SATB1 expression in human Treg cells. () Microarray analysis of SATB1 mRNA expression in resting (Rest), activated (Act), transforming growth factor-β–treated (TGF) or population-expanded (Exp) human Tconv cells and Treg cells. () SATB1 mRNA expression in human Treg cells and Tconv cells, presented relative to β2-microglobulin expression. () Immunoblot analysis of SATB1 and β-actin in human Treg cells and Tconv cells (left), and densitometric quantification of those results (right), presented as the ratio of SATB1 to β-actin. () Flow cytometry analysis of SATB1 expression in human Treg cells and Tconv cells (left), and quantification of those results (right), presented as mean fluorescence intensity (MFI). () Flow cytometry analysis of SATB1 expression in human Treg cells and Tconv cells left unstimulated (resting cells (Rest)) or stimulated for 2 d with anti-CD3 and IL-2 (α-CD3 + IL-2) or anti-CD3 and anti-CD28 (α-CD3 + α-CD28; left), and quantification of those results (right), presented as normalized! results relative to those in resting Tconv cells. () SATB1 mRNA expression in human iTreg cells and T cells stimulated with anti-CD3 and anti-CD28 (Stim T) and unstimulated T cells (Unstim T) on day 5, presented as in . () Flow cytometry analysis of SATB1 expression in cells as in (left), and quantification of those results (right), presented as in . () Cytometric bead assay of IL-4 and IFN-γ in the supernatants of cells as in . () Expression of Foxp3 and SATB1 mRNA in Tconv cells transduced with lentivirus containing a vector encoding Foxp3 (Foxp3 vector) or control vector (Ctrl vector), then allowed to 'rest' for 3 d; results presented as in . () Expression of IL-5 and IFN-γ mRNA, assessed and presented as in . Numbers in plots (,) indicate mean fluorescence intensity. *P < 0.05 (Student's t-test). Data are representative of at least eight experiments per group (), or five (,,,), six (,), three () or eleven () experiments (mean and s.d.) or three independent experiment! s (; mean and s.d. of triplicate wells), each with cells deriv! ed from a different donor. * Figure 2: Restoration of SATB1 expression after silencing of Foxp3 in Treg cells. () Flow cytometry analysis of Foxp3 expression (left) in human Treg cells transfected for 48 h with siRNA targeting Foxp3 (Foxp3 siRNA) or nontargeting (control) siRNA (Ctrl siRNA), and quantification of those results (right), presented relative to expression in control siRNA–transfected Treg cells. () Suppression of allogeneic CD4+ T cells labeled with the cytosolic dye CFSE (responding T cells (Tresp)) by human Treg cells transfected with siRNA as in , presented as CFSE dilution in responding T cells cultured at a ratio of 1:1 with Treg cells plus beads coated with anti-CD3 and anti-CD28, or without Treg cells (Tresp only). () Expression of SATB1 mRNA in human Treg cells transfected with siRNA as in , then cultivated for 48 h without stimulation (Rest) or in the presence of anti-CD3 and IL-2 or beads coated with anti-CD3 and anti-CD28; results are presented relative to β2-microglobulin expression. () Expression of IL-5 and IFN-γ mRNA in Treg cells transfected with siRN! A as in , then stimulated for 48 h with anti-CD3 and IL-2; results presented as in . () Cytometric bead assay of IL-4 and IFN-γ in supernatants of siRNA-treated Treg cells stimulated as in . () Expression of IL-5 and IFN-γ mRNA in Treg cells transfected with nontargeting siRNA or siRNA targeting Foxp3 alone (Foxp3 siRNA) or both Foxp3 and SATB1 (Foxp3 + SATB1 siRNA), followed by stimulation for 48 h with beads coated with anti-CD3 and anti-CD28; results presented as in . *P < 0.05 (Student's t-test (,–) or one-way analysis of variance with Fisher's least-significant-difference test ()). Data are representative of six (,) or four (,) experiments (mean and s.d.) or three independent experiments (,; mean and s.d. of triplicate wells in ), each with cells derived from a different donor. * Figure 3: Foxp3-dependent repression of SATB1 expression in mouse Treg cells. () Flow cytometry analysis of SATB1 in Treg cells and Tconv cells freshly isolated from the spleens of male DEREG mice (presented as in Fig. 1d). () Immunoblot analysis of SATB1 and the kinases Erk1 and Erk2 in mouse Treg cells and Tconv cells. () Immunofluorescence analysis (z projection) of SATB1 (red) and GFP (green) in thymocytes from male DEREG mice; nuclei are stained with the DNA-intercalating dye DAPI (blue). White arrows indicate Treg cells. Original magnification, ×50. () SATB1 mRNA expression in CD4+GFP+ Treg cells and CD4+GFP− Tconv cells from male DEREG or DEREG × scurfy mice, presented relative to β-actin expression. () Expression of IL-6 and IFN-γ mRNA in CD4+GFP+ Treg cells from male DEREG or DEREG × scurfy mice, presented as in . ND, not detectable. () Confocal microscopy of SATB1 (red) and Foxp3 (green) in thymic CD4+GFP+Foxp3− Treg cells (Foxp3 (sf)) or CD4+GFP+Foxp3+ Treg cells (Foxp3 (WT)) from female DEREG mice heterozygous for the scurfy mutat! ion (n = 25 cells), counterstained with DAPI (blue). Original magnification, ×240. () Flow cytometry analysis of SATB1 expression in thymic Treg cells as in (presented as in Fig. 1d). Isotype, isotype-matched control antibody. *P < 0.05 (Student's t-test). Data are representative of three independent experiments (,; mean and s.d.) or two independent experiments (–,; mean and s.d. in and mean and s.d. of triplicate wells in ,). * Figure 4: Direct suppression of SATB1 transcription by Foxp3. () Foxp3 ChIP tiling array data from expanded populations of human cord blood–derived Treg cells (blue; top), analyzed by model-based analysis of tiling array and overlaid onto the SATB1 locus for identification of binding regions (1–13 (magenta); P < 10−5; false-discovery rate < 0.5%). Chr3, chromosome 3. () Foxp3-binding regions (BR1–BR16) in the human genomic SATB1 locus, identified by in silico prediction in the regions identified in . () ChIP analysis of expanded populations of human cord blood–derived Treg cells with a Foxp3-specific antibody and PCR primers specific for Foxp3-binding regions; results are presented relative to input, normalized to immunoglobulin G. −15 kb (far left), region 15 kb upstream of the transcription start site (negative control). *P < 0.05, each binding region versus the negative control (horizontal lines at top; Student's t-test). () Luciferase assay of the binding of Foxp3 to binding regions in the SATB1 locus in HEK293T cells t! ransfected with luciferase constructs containing wild-type (WT) or mutated (Mut) binding regions BR9–BR14 of the SATB1 locus (with mutation of the Foxp3-binding motifs) or control vector (Ctrl), together with a Foxp3 expression vector; results are presented in arbitrary units (AU) relative to those obtained with control vector. Numbers below graph indicate total Foxp3-binding motifs in each region. *P < 0.05, experimental versus control (top) or wild-type versus mutated (directly above bars; Student's t-test). () Filter-retention analysis of the binding of Foxp3 to a wild-type or mutated Foxp3-binding motif in binding region 9 or 10 of the SATB1 locus; numbers in plots indicate dissociation constant (Kd). Data are representative of two () or three (,) independent experiments with cells derived from different donors (mean and s.d. in and mean and s.d. of triplicates in ) or one experiment representative of two (; mean and s.d. of triplicate wells). * Figure 5: SATB1 expression in human Treg cells reprograms them into Teff cells. () Suppression of CD8+ T cells (labeled with the cytosolic dye CFSE) by human Treg cells transduced with lentivirus containing vector expressing SATB1 (Treg (SATB1); blue) or control vector (Treg (Ctrl); red), presented as CFSE dilution (left) in responding T cells cultured at a ratio of 1:1 with Treg cells plus anti-CD3-coated beads or without Treg cells (CD8+ T only), and as the frequency of proliferating CD8+ T cells plotted against the ratio of Treg cells to CD8+ T cells (right). () Cytometric bead assay of IL-4 and IFN-γ in supernatants of human Treg cells transduced with lentivirus as in , assessed 4 and 16 h after stimulation with beads coated with anti-CD3 and anti-CD28. (–) Expression of IL-5 mRNA (), IFN-γ mRNA () and IL-17A mRNA () in human Tconv cells and Treg cells transduced with lentivirus as in and activated for 16 h with beads as in ; results are presented relative to β2-microglobulin expression. *P < 0.05 (Student's t-test). Data are representative of ! three independent experiments (; mean and s.d.) or are from one experiment representative of two (–; mean and s.d. of triplicate wells), with cells derived from different donors. * Figure 6: Induction of transcriptional Teff cell programs in SATB1-expressing Treg cells. () Microarray analysis of human Treg cells transduced with lentivirus containing vector expressing SATB1 (blue) or control vector (red) and stimulated for 16 h with beads coated with anti-CD3 and anti-CD28, presented as a heat map of the z-scores of genes with differences in expression. () Cross-annotation analysis of four classes of genes: those associated with Tconv cells but not Treg cells (Tconv cell–dependent); those associated with T cell activation (Activation-dependent); common T cell genes (Common); and SATB1-induced genes (SATB1-induced). Numbers in chart indicate percent cells in each group; numbers below labels (n) indicate total genes in each group. () Expression of genes associated with TH1, TH2 or TH17 differentiation, presented as a heat map of z-scores. Helper T cell–specific enrichment: TH1, P = 3.24 × 10−6; TH2, P = 9.03 × 10−15; TH17, P = 1.16 × 10−6, versus complete data set (χ2 test). () Change in the expression of genes associated with th! e human Treg cell signature in Treg cells left untransduced (red) or transduced as in (blue), presented as gene expression in Treg cells versus Tconv cells or in SATB1-expressing versus control vector–transduced Treg cells, plotted against ranking by change in expression in Treg cells versus Tconv cells. Data are from three independent experiments with cells derived from different donors. * Figure 7: Repression of SATB1 expression by miRNA in Treg cells. () Human genomic SATB1 3′ UTR and conserved miRNA-binding sites. () Expression of miR-155, miR-21, miR-7, miR-34a and miR-18a human Treg cells and Tconv cells, presented relative to expression of the ubiquitously expressed U6 small nuclear RNA. *P < 0.05 (Student's t-test). () Correlation of miRNA expression with SATB1 mRNA expression, plotted against change in miRNA expression in Treg cells versus Tconv cells, for all 735 miRNAs assessed by microarray (colors for miR-155, miR-21, miR-7, miR-34a and miR-18a correspond to those in ,). () Foxp3 ChIP tiling array data (blue) for miR-155, miR-21 and miR-7-1 and miR-7-2 from expanded populations of human cord blood–derived Treg cells, analyzed by model-based analysis of tiling array and overlaid onto the miRNA locus for identification of binding regions (P < 10−5; false-discovery rate <0.5%). () ChIP analysis of expanded populations of human cord blood–derived Treg cells with a Foxp3-specific antibody and PCR primers spec! ific for miR-155, miR-21 or miR-7-1; results are presented relative to input, normalized to immunoglobulin G. AFM (far left), locus encoding α-albumin (negative control). *P < 0.05, each miRNA compared with negative control (horizontal lines at top; Student's t-test). () Dual-luciferase assay of HEK293T cells transfected with luciferase constructs containing a wild-type or mutated SATB1 3′ UTR (with mutation of the miRNA-responsive elements), together with synthetic mature miRNA or a synthetic control miRNA (Ctrl), presented in arbitrary units (AU) relative to results obtained for control miRNA. *P < 0.05 experimental versus control (top) or wild-type versus mutated (directly above bars; Student's t-test). () Immunoblot analysis of SATB1 and Erk1 and Erk2 in Treg cells from mice with complete Treg cell–specific loss of Dicer (Dicer1−/−) and mice heterozygous for loxP-flanked Dicer (Dicer1fl/−). Data are representative of five () or three () experiments or two () ! or three () independent experiments with cells derived from di! fferent donors, or three () or two () independent experiments (mean and s.d. in ,,). * Figure 8: Ectopic expression of SATB1 in Treg cells results in less suppressive function in vivo. () Suppression of CD4+ T cells (labeled with the cytosolic dye eFluor 670 (Tconv cells) and cultured with beads coated with anti-CD3 and anti-CD28) by expanded populations of mouse Treg cells transduced with lentivirus containing vector expressing SATB1 or control vector, presented as the frequency of proliferating Tconv cells plotted against the ratio of Treg cells to Tconv cells. () Expression of IL-5 and IFN-γ mRNA in mouse Treg cells transduced with lentivirus as in , presented relative to β-actin expression. () Hematoxylin and eosin staining of colon sections from Rag2−/− mice at 9 weeks after the transfer of CD4+CD45RBhi naive T cells (Naive T) alone (top) or in combination with Treg cells transduced with lentivirus as in . Scale bars, 100 μm. () Histology scores of colon sections of Rag2−/− mice at 9 weeks after the cell transfer in . () Body weight of Rag2−/− mice at 9 weeks after the cell transfer in , presented relative to initial body weight. (,) Re! covery of Tconv cells () and Treg cells () from spleens, mesenteric lymph nodes (mLN) and peripheral lymph nodes (pLN) of Rag2−/− mice at 9 weeks after the cell transfer in . NS, not significant; *P < 0.05 (Student's t-test). Data are representative of two () or three () independent experiments (mean and s.e.m. of triplicate cultures () or mean and s.d. ()) or are pooled from two independent experiments (–; mean and s.d. of four or five recipient mice). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE6681 * GSE11775 * GSE15390 * GSE20995 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Life and Medical Sciences Institute, Laboratory for Genomics and Immunoregulation, University of Bonn, Bonn, Germany. * Marc Beyer, * Yasser Thabet, * Sabine Classen, * Wolfgang Krebs, * Eva A Schönfeld, * Andrea Hofmann, * Daniel Sommer, * Svenja Debey-Pascher & * Joachim L Schultze * Renal Division, Department of Medicine and Centre for Molecular Medicine, University of Cologne, Cologne, Germany. * Roman-Ulrich Müller & * Bernhard Schermer * Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. * Roman-Ulrich Müller & * Bernhard Schermer * Discipline of Paediatrics, Women's and Children's Health Research Institute, University of Adelaide, North Adelaide, Australia. * Timothy Sadlon & * Simon C Barry * Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany. * Katharina Lahl, * Christian T Mayer & * Tim Sparwasser * Department of Microbiology, Translational Research Program, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Philadelphia, USA. * Samik Basu, * Tatiana Golovina & * James L Riley * Diabetes Center and the Department of Medicine, University of California, San Francisco, California, USA. * Xuyu Zhou, * Samantha L Bailey-Bucktrout & * Jeffrey A Bluestone * Institutes of Molecular Medicine and Experimental Immunology, University Hospital Bonn, Bonn, Germany. * Jan Böttcher, * Elmar Endl, * Andreas Limmer & * Percy A Knolle * Cancer Center and Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA. * Keli L Hippen & * Bruce R Blazar * BD Biosciences–Pharmingen, San Diego, California, USA. * Robert Balderas * Life and Medical Sciences Institute, Laboratory for Molecular Immunology, University of Bonn, Bonn, Germany. * Thomas Quast & * Waldemar Kolanus * Department of Neuropathology, University Hospital Bonn, Bonn, Germany. * Andreas Waha * Life and Medical Sciences Institute, Laboratory of Chemical Biology, University of Bonn, Bonn, Germany. * Günter Mayer & * Michael Famulok * Department for Diagnostic, Institute for Pathology, University Hospital Leipzig, Leipzig, Germany. * Claudia Wickenhauser Contributions M.B. designed, did and supervised experiments, analyzed data and wrote the manuscript; Y.T. did quantitative PCR, cytometric bead assay, immunoblot analysis, overexpression experiments and filter-retention analysis and analyzed data; R.-U.M. designed and did reporter assays; S.C. did experiments and analyzed data; T.S. did ChIP experiments and analyzed data; K.L. and C.T.M. did experiments with DEREG mice; S.B. and T.G. did overexpression experiments; E.A.S. did and analyzed immunofluorescence experiments; W.K. did histone-methylation studies, S.L.B.-B. and X.Z. did experiments with mice with loxP-flanked Dicer1 alleles; A.H. did bioinformatics analysis; D.S. generated lentivirus contructs; S.D.-P. did microarray experiments; E.E. did flow cytometry sorting; J.B. and A.L. did experiments with Rag2−/− mice; P.A.K. was involved in study design; K.L.H. and B.R.B. provided vital analytical tools; R.B. provided vital analytical tools; T.Q. supervised and analyzed immunofluore! scence experiments; C.W. did immunohistochemistry; A.W. did, designed and supervised DNA-methylation experiments; G.M. and M.F. designed and supervised filter-retention experiments; W.K. designed and supervised experiments and wrote the manuscript; B.S. designed and analyzed reporter assays; S.C.B. designed and supervised ChIP experiments; T.S. designed and supervised experiments with DEREG mice and provided vital analytical tools; J.A.B. designed and supervised experiments with mice with loxP-flanked Dicer1 alleles; J.L.R. designed and supervised SATB1-overexpression experiments and wrote the manuscript; J.L.S. designed, supervised and analyzed experiments and wrote the manuscript; and all authors discussed the results and commented on the manuscript. Competing financial interests Research support to J.L.S. and M.B. has been provided in part by Becton Dickinson; R.B. is employed by Becton Dickinson; S.C. is employed by Miltenyi Biotech; and J.L.S., M.B. and R.B. have applied for several US and international patents on Treg cell biology. Corresponding author Correspondence to: * Joachim L Schultze Author Details * Marc Beyer Search for this author in: * NPG journals * PubMed * Google Scholar * Yasser Thabet Search for this author in: * NPG journals * PubMed * Google Scholar * Roman-Ulrich Müller Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy Sadlon Search for this author in: * NPG journals * PubMed * Google Scholar * Sabine Classen Search for this author in: * NPG journals * PubMed * Google Scholar * Katharina Lahl Search for this author in: * NPG journals * PubMed * Google Scholar * Samik Basu Search for this author in: * NPG journals * PubMed * Google Scholar * Xuyu Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Samantha L Bailey-Bucktrout Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Krebs Search for this author in: * NPG journals * PubMed * Google Scholar * Eva A Schönfeld Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Böttcher Search for this author in: * NPG journals * PubMed * Google Scholar * Tatiana Golovina Search for this author in: * NPG journals * PubMed * Google Scholar * Christian T Mayer Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea Hofmann Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Sommer Search for this author in: * NPG journals * PubMed * Google Scholar * Svenja Debey-Pascher Search for this author in: * NPG journals * PubMed * Google Scholar * Elmar Endl Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Limmer Search for this author in: * NPG journals * PubMed * Google Scholar * Keli L Hippen Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce R Blazar Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Balderas Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Quast Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Waha Search for this author in: * NPG journals * PubMed * Google Scholar * Günter Mayer Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Famulok Search for this author in: * NPG journals * PubMed * Google Scholar * Percy A Knolle Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Wickenhauser Search for this author in: * NPG journals * PubMed * Google Scholar * Waldemar Kolanus Search for this author in: * NPG journals * PubMed * Google Scholar * Bernhard Schermer Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey A Bluestone Search for this author in: * NPG journals * PubMed * Google Scholar * Simon C Barry Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Sparwasser Search for this author in: * NPG journals * PubMed * Google Scholar * James L Riley Search for this author in: * NPG journals * PubMed * Google Scholar * Joachim L Schultze Contact Joachim L Schultze Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–17, Tables 1–17 and Methods Additional data - Autocrine IL-2 is required for secondary population expansion of CD8+ memory T cells
- Nat Immunol 12(9):908-913 (2011)
Nature Immunology | Article Autocrine IL-2 is required for secondary population expansion of CD8+ memory T cells * Sonia Feau1 * Ramon Arens1, 2 * Susan Togher1 * Stephen P Schoenberger1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:908–913Year published:(2011)DOI:doi:10.1038/ni.2079Received21 April 2011Accepted23 June 2011Published online31 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Two competing theories have been put forward to explain the role of CD4+ T cells in priming CD8+ memory T cells: one proposes paracrine secretion of interleukin 2 (IL-2); the other proposes the activation of antigen-presenting cells (APCs) via the costimulatory molecule CD40 and its ligand CD40L. We investigated the requirement for IL-2 by the relevant three cell types in vivo and found that CD8+ T cells, rather than CD4+ T cells or dendritic cells (DCs), produced the IL-2 necessary for CD8+ T cell memory. Il2−/− CD4+ T cells were able to provide help only if their ability to transmit signals via CD40L was intact. Our findings reconcile contradictory elements implicit in each model noted above by showing that CD4+ T cells activate APCs through a CD40L-dependent mechanism to enable autocrine production of IL-2 in CD8+ memory T cells. View full text Figures at a glance * Figure 1: CD4+ T cells require CD40L, not IL-2, to provide help to CD8+ T cells. () Flow cytometry of splenic CD8+ T cells from C57BL/6J mice that had undergone thymectomy and were first depleted of CD4+ T cells, then given injection of PBS alone (No OT-II) or 5 × 104 OT-II cells (OT-II) or OT-II Il2−/− cells (OT-II Il2−/−), then primed 1 d later with 5 × 106 Act-mOVA H-2Kb-deficient splenocytes and challenged 30 d later with 5 × 106 plaque-forming units (PFU) of vaccinia virus–OVA. Numbers adjacent to outlined areas indicate percent H-2Kb–OVA tetramer–positive CD8+ T cells (mean ± s.e.m. of six mice). (,) Frequency () and absolute number () of IFN-γ+ CD8+ T cells among total splenocytes from the mice in (n = 6 per group), assessed by intracellular staining at day 7 (primary) or at day 35 in mice that had (secondary) or had not (memory) been challenged with vaccinia virus–OVA 5 d earlier. Each symbol represents an individual mouse; small horizontal lines indicate the mean (). () Flow cytometry of CD8+ T cells in blood from the mice i! n that were given no antibody treatment (far left) or treated with control immunoglobulin (control Ig) or blocking antibody to CD40L (α-CD40L), assessed at day 7 (primary), day 30 (memory) or day 35 (secondary) after mice were challenged with vaccinia virus–OVA 5 d earlier. Numbers adjacent to outlined areas indicate percent OVA(257–264)–H-2Kb tetramer–specific CD8+ T cells (mean ± s.e.m. of six mice). NS, not significant; *P < 0.05 (two-tailed unpaired t-test). Data are representative of three (,) or two (,) independent experiments (mean ± s.e.m.). * Figure 2: DC-derived IL-2 is not required for the secondary population expansion of CD8+ T cells. () Flow cytometry of splenocytes from H-2Kbm1 mice given injection of 5 × 104 OT-I H-2Kbm1 CD45.1+ cells, then primed 1 d later with 7 × 104 wild-type (WT) or Il2−/− Act-mOVA DCs, followed by analysis of the primary CD8+ T cell response after 7 d (top row) or boosted 139 d later with 7 × 105 wild-type or Il2−/− Act-mOVA DCs, followed by analysis of the OT-I response in both primed and boosted groups 5 d later. Numbers adjacent to outlined areas indicate percent OT-I CD8+ T cells among total splenocytes (mean ± s.e.m.). () Absolute number of OT-I CD8+ T cells among total splenocytes during the memory and secondary response in . Data are representative of two experiments (mean ± s.e.m. of three to four mice in ). * Figure 3: CD8+T cells require autocrine IL-2 for a memory response to a replicating immunogen. (,) Frequency () and absolute number () of OT-I (CD45.1+) CD8+ T cells among total splenocytes from intact C57BL/6J (CD45.2+) mice (+ help) or C57BL/6J (CD45.2+) mice depleted of CD4+ T cells (– help; n = 3–4 per group) given 50 wild-type or Il2−/− (CD45.1+) OT-I CD8+ T cells, then infected 1 d later with 1 × 106 PFU vaccinia virus–OVA and, for some groups (secondary), challenged 40 d later with 0.6 half-maximal lethal dose of L. monocytogenes–OVA, followed by analysis at day 7 (primary), or at day 45 for mice that had (secondary) or had not (memory) been challenged with L. monocytogenes–OVA 5 d earlier. Each symbol represents an individual mouse; small horizontal lines indicate the average (). *P < 0.05 (two-tailed unpaired t-test). Data are representative of three experiments (error bars (), s.e.m.). () IFN-γ production by the endogenous (CD45.1−) and OT-I (CD45.1+) CD8+ T cells during the primary, memory and secondary responses in ,. Numbers in quadrants! indicate percent cells in each (mean ± s.e.m.). Data are representative of three experiments with 8–12 mice. () Population expansion of the OT-I CD8+ T cells in ,. Data are representative of three experiments. * Figure 4: CD8+T cells require autocrine IL-2 for a memory response to a nonreplicating immunogen. (,) Frequency () and absolute number () of OT-I (CD45.1+) CD8+ T cells among total splenocytes from C57BL/6 (CD45.2+) mice (n = 3–4 per group) given 50 wild-type or Il2−/− (CD45.1+) OT-I CD8+ T cells, then immunized 1 d later with 5 × 106 Act-mOVA H-2Kb-deficient splenocytes and, for some groups (secondary), challenged 30 d later with 5 × 106 PFU vaccinia virus–OVA, followed by analysis at day 7 (primary), or at day 45 in mice that had (secondary) or had not (memory) been challenged with vaccinia virus–OVA 5 d (presented as in Fig. 3a,b). () IFN-γ production by endogenous (CD45.1−) and OT-I (CD45.1+) CD8+ T cells during the primary, memory and secondary responses in (presented as in Fig. 3c; n = 8–12 mice). Data are representative of two experiments (mean ± s.e.m. in ,). * Figure 5: Functional and phenotypic comparison of wild-type and IL-2-deficient OT-I CD8+ T cells. () Frequency of OT-I CD8+ T cells producing IFN-γ alone (IFN-γ+) or both IFN-γ and tumor necrosis factor (IFN-γ+TNF+) among total splenocytes from C57BL/6J (CD45.2+) mice (n = 3–4 per group) given wild-type (OT-I) or Il2−/− (OT-I Il2−/−) CD45.1+ OT-I CD8+ T cells and infected 1 d later with 1 × 106 PFU vaccinia virus–OVA, analyzed by intracellular staining of CD45.1+ cells at day 7 (primary), or at day 45 in mice that had (secondary) or had not (memory) been challenged with 0.6 half-maximal lethal dose of L. monocytogenes–OVA 5 d earlier. *P < 0.05 and **P < 0.005 (two-tailed unpaired t-test). () Frequency of OT-I cells with positive or negative expression of the activation and memory markers KLRG-1 and CD127 (IL-7 receptor α-chain) on CD45.1+ cells in the blood from the mice in at day 7 (primary). () Frequency of OT-I CD8+ T cells with the surface phenotype of central memory cells (CD127+CD62L+) or effector memory cells (CD127+CD62L−) at day 40 (memory! ) in the blood of the mice in . () In vivo cytotoxicity of wild-type and Il2−/− OT-I cells 6 d after immunization with vaccinia virus–OVA, presented as the frequency of tetramer-positive OT-I cells in wild-type mice (numbers above outlined areas; left) and frequency of target cells loaded with various concentrations of the cytosolic dye CSFE and pulsed with the peptide epitope of adenovirus early 1B protein amino acids 192–200 (E1B) or OVA(257–264) (OVA) 16 h after adoptive transfer into the immunized mice (right; numbers in plots indicate percent CSFE+ cells). () Expression of granzyme B by wild-type and Il2−/− OT-I cells at days 0–5 after immunization of the mice in with vaccinia virus–OVA. IgG (key), staining with isotype-matched control antibody. Data are representative of three (,) or two (,,) experiments (error bars (–), s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Cellular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA. * Sonia Feau, * Ramon Arens, * Susan Togher & * Stephen P Schoenberger * Present address: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. * Ramon Arens Contributions S.F., R.A. and S.P.S. designed the experiments; S.F. and R.A. did the experiments with assistance from S.T.; and S.F. and S.P.S. analyzed the data and wrote the manuscript with contributions from R.A. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sonia Feau Author Details * Sonia Feau Contact Sonia Feau Search for this author in: * NPG journals * PubMed * Google Scholar * Ramon Arens Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Togher Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen P Schoenberger Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (795K) Supplementary Figures 1–2 Additional data
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