Latest Articles Include:
- Funding the future in UK science
- Nat Immunol 12(1):1 (2011)
Nature Immunology | Editorial Funding the future in UK science Journal name:Nature ImmunologyVolume: 12,Page:1Year published:(2011)DOI:doi:10.1038/ni0111-1Published online17 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Against a backdrop of some of the most savage spending cuts in the developed world, the UK science budget has emerged relatively unscathed, but funding priorities may yet prove problematic. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Control of T cell activation by vitamin D
- Nat Immunol 12(1):3 (2011)
Nature Immunology | Correspondence Control of T cell activation by vitamin D * Joost Smolders1 Contact Joost Smolders Search for this author in: * NPG journals * PubMed * Google Scholar * Mariëlle Thewissen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Damoiseaux3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Page:3Year published:(2011)DOI:doi:10.1038/ni0111-3aPublished online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. To the Editor: We read with great interest the paper by von Essen et al. published in the April 2010 issue of Nature Immunology1. The authors show a critical difference in the expression of the T cell antigen receptor (TCR) pathway signaling molecule PLC-γ1 between naive CD4+ T cells and in vitro–primed CD4+ T cells. However, the suggested role of vitamin D in the induction of PLC-γ1 expression is disputable. View full text 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 Affiliations * School for Mental Health and Neuroscience/Department of Internal Medicine, Division of Clinical and Experimental Immunology, Maastricht University Medical Center, Maastricht, The Netherlands. * Joost Smolders * Cardiovascular Research Institute Maastricht–Department of Internal Medicine, Division of Clinical and Experimental Immunology, Maastricht University Medical Center, Maastricht, The Netherlands. * Mariëlle Thewissen * Laboratory for Clinical Immunology, Maastricht University Medical Center, Maastricht, The Netherlands. * Jan Damoiseaux Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joost Smolders Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Reply to "Control of T cell activation by vitamin D"
- Nat Immunol 12(1):3-4 (2011)
Nature Immunology | Correspondence Reply to "Control of T cell activation by vitamin D" * Carsten Geisler1 Contact Carsten Geisler Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 12,Pages:3–4Year published:(2011)DOI:doi:10.1038/ni0111-3bPublished online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Geisler replies: The induction of VDR and PLC-γ1 takes place in both CD4+ T cells and CD8+ T cells after TCR stimulation1 and not only in CD4+ cells, as stated by Smolders et al. above. As for the availability of 25(OH)D3 and 1,25(OH)2D3 in the T cells in our experimental setup, it is known that the concentrations of 25(OH)D3 and 1,25(OH)2D3 in serum are around 100 nM and 80 pM, respectively. Most 25(OH)D3 and 1,25(OH)2D3 in serum is bound to the vitamin D–binding protein DBP2. We now have data that demonstrate endocytosis of DBP by T cells (data not shown). The intracellular concentrations of 25(OH)D3 and 1,25(OH)2D3 in freshly isolated T cells are therefore most likely equal to their concentrations in serum. In addition, according to the manufacturer, X-VIVO 15 medium contains human serum albumin and thus most probably also contains DBP3; both of these are sources of 25(OH)D3 and 1,25(OH)2D3. View full text 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 Affiliations * Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. * Carsten Geisler Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Carsten Geisler Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Diet, gut microbiota and immune responses
- Nat Immunol 12(1):5-9 (2011)
Nature Immunology | Commentary Diet, gut microbiota and immune responses * Kendle M Maslowski1 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles R Mackay2 Contact Charles R Mackay Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:5–9Year published:(2011)DOI:doi:10.1038/ni0111-5Published online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. The fields of immunology, microbiology, nutrition and metabolism are rapidly converging. Here we expand on a diet-microbiota model as the basis for the greater incidence of asthma and autoimmunity in developed countries. View full text Figures at a glance * Figure 1: Diet, microbial composition and regulation of the immune system. Diet and other environmental and host factors have a major effect on gut microbial composition. Our model would suggest that balanced microbial composition results in symbiosis; this provides regulation of immune and inflammatory responses through anti-inflammatory and/or immunomodulatory products such as SCFA, polysaccharide A (PSA) and PTGN, which helps maintain homeostasis. Dysbiosis would lead to dysregulation of the immune system through lack of beneficial microbial products and an increase in virulence factors, which could leave the host susceptible to inflammation. Dysbiosis could occur through the consumption of a Western diet, as well as through changes induced by factors such as host genetics, maternal transfer and antibiotic use. * Figure 2: Diet, fatty acids and the actions of anti-inflammatory GPCRs. SCFA (derived from complex plant polysaccharides) and ω-3 fatty acids regulate inflammation through GPR43 and GPR120, respectively. SCFA are produced by the gut microbiota as a byproduct of fermentation of dietary fiber and have several beneficial effects. In the colonic epithelium, butyrate is the main energy source of colonic epithelial cells and is transported into cells via monocarboxylate transporters (such as MCT1 and SLC5A8). SCFA are important for maintaining epithelial barrier function, regulating proliferation and tumor suppression. SCFA also diminish oxidative DNA damage and regulate cytokine production. The effects of SCFA on epithelial cells relate mostly to their role as an energy source and also their inhibition of histone deacetylases. SCFA could also operate through GPR41, GPR43 and GPR109A. In the immune system, SCFA have several anti-inflammatory effects but are also important for stimulating immune function, and their role therefore seems to be important! for the regulation of timely immune responses and in resolution of inflammation. Acetate enhances the production of reactive oxygen species (ROS) and phagocytosis but also induces apoptosis and modulates neutrophil recruitment. Many of these anti-inflammatory effects are mediated through GPR43 (ref. 4). The ω-3 fatty acids have anti-inflammatory and antidiabetic effects through their binding of GPR120 expressed on macrophages28. 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 Affiliations * Kendle M. Maslowski is with the Garvan Institute of Medical Research, Sydney, Australia, and the Cooperative Research Centre for Asthma and Airways, Sydney, Australia, and is affiliated with the Department of Medicine, St Vincent's Clinical School, University of New South Wales, Sydney, Australia. * Charles R. Mackay is with the Department of Immunology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia, and the Cooperative Research Centre for Asthma and Airways, Sydney, Australia. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Charles R Mackay Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - ZAPS electrifies RIG-I signaling
- Nat Immunol 12(1):11-12 (2011)
Nature Immunology | News and Views ZAPS electrifies RIG-I signaling * Helene Minyi Liu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Gale Jr1 Contact Michael Gale Jr Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:11–12Year published:(2011)DOI:doi:10.1038/ni0111-11Published online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. ZAPS, a member of the poly(ADP-ribose) polymerase family, modulates innate antiviral immunity by boosting signaling of the RNA helicase RIG-I. View full text 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 Affiliations * Helene Minyi Liu and Michael Gale Jr. are in the Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Gale Jr Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Fine tuning NF-κB: new openings for PKC-ζ
- Nat Immunol 12(1):12-14 (2011)
Nature Immunology | News and Views Fine tuning NF-κB: new openings for PKC-ζ * Jorge Moscat1 Contact Jorge Moscat Search for this author in: * NPG journals * PubMed * Google Scholar * Maria T Diaz-Meco1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:12–14Year published:(2011)DOI:doi:10.1038/ni0111-12Published online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. NF-κB is a critical transcription factor that is regulated by several post-transcriptional modifications. The characterization of their roles would help in the design of new therapeutic targets in cancer and inflammation. View full text 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 Affiliations * Jorge Moscat and Maria T. Diaz-Meco are at the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jorge Moscat Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Cooperative regulatory events and Foxp3 expression
- Nat Immunol 12(1):14-16 (2011)
Nature Immunology | News and Views Cooperative regulatory events and Foxp3 expression * Masahide Tone1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark I Greene2 Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:Nature ImmunologyVolume: 12,Pages:14–16Year published:(2011)DOI:doi:10.1038/ni0111-14Published online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. The molecular mechanisms that control Treg and TH17 development and the precise role of TGF-β in this process are complex and imperfectly understood. New findings indicate that the helix-loop-helix proteins E2A and Id3 are also critically involved in some of these processes. View full text 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 Affiliations * Masahide Tone is in the Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, USA. * Mark I. Greene is in the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Masahide Tone Competing financial interests The authors declare no competing financial interests. Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Explaining discordant coordination
- Nat Immunol 12(1):16-17 (2011)
Nature Immunology | News and Views Explaining discordant coordination * Melanie Van Stry1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Bix1 Contact Mark Bix Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:16–17Year published:(2011)DOI:doi:10.1038/ni0111-16Published online17 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. TH2 cells control immune responses to helminth infection and contribute to the development of allergic asthma. A single intronic enhancer element in Il4 can regulate TH2 differentiation and susceptibility to allergic asthma via interaction with the transcription factor GATA-3. View full text 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 Affiliations * Melanie Van Stry and Mark Bix are in the Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mark Bix Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Research Highlights
- Nat Immunol 12(1):19 (2011)
Nature Immunology | Research Highlights Research Highlights Journal name:Nature ImmunologyVolume: 12,Page:19Year published:(2011)DOI:doi:10.1038/ni0111-19Published online17 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. TRIMing infections with antibodies Antibodies are typically thought to act in the extracellular space by specifically binding pathogens and triggering effector functions. In the Proceedings of the National Academy of Sciences, James and colleagues delineate a mechanism by which antibodies can still mediate pathogen neutralization even in the cytosol of a cell. In an adenovirus infection model in vitro, they observe that virus bound by antibody extracellularly still remains bound in the cytosol after infection of the cell. In the cytoplasm, the antibody invariant region then rapidly recruits the cytosolic immunoglobulin receptor TRIM21. Not only does TRIM21 bind with an affinity higher than that of any other immunoglobulin receptor, its E3 ubiquitin ligase activity targets the virus-antibody complex for degradation via the proteasome. TRIM21 binds to IgG and, with a lower affinity, to IgM, which suggests that this mechanism is applicable to various isotypes and multiple infection stages. Intracellular degradat! ion via TRIM21 thus represents the last line of defense in antibody-mediated pathogen neutralization. ZF Proc. Natl. Acad. Sci. USA107, 19985–19990 (2010) View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling
- Nat Immunol 12(1):21-27 (2011)
Nature Immunology | Review The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling * Hergen Spits1 Contact Hergen Spits Search for this author in: * NPG journals * PubMed * Google Scholar * James P Di Santo2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:21–27Year published:(2011)DOI:doi:10.1038/ni.1962Published online28 November 2010 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 Research has identified what can be considered a family of innate lymphoid cells (ILCs) that includes not only natural killer (NK) cells and lymphoid tissue–inducer (LTi) cells but also cells that produce interleukin 5 (IL-5), IL-13, IL-17 and/or IL-22. These ILC subsets are developmentally related, requiring expression of the transcriptional repressor Id2 and cytokine signals through the common γ-chain of the IL-2 receptor. The functional differentiation of ILC subsets is orchestrated by distinct transcription factors. Analogous to helper T cell subsets, these evolutionarily conserved yet distinct ILCs seem to have important roles in protective immunity, and their dysregulation can promote immune pathology. View full text Figures at a glance * Figure 1: The expanding family of ILCs. Distinct ILC subsets develop from hematopoietic precursors in an Id2-dependent way in a process orchestrated by transcription factors (Fig. 2). ILCs can be grouped into three branches: NK, helper and RORγt. The IL-15-dependent NK branch includes conventional NK (cNK) cells, which have spontaneous cytotoxicity, and the IFN-γ+ ILC1 subset, which includes thymus and IL-7-dependent NK cells in mice and a cytokine-polarized subset of CD56hi cells in humans. ILC1 cells function to protect against infection by viruses and intracellular pathogens, and their activity is promoted through IL-12 and IL-18. The helper branch contains the ILC2 subset (including nuocytes and NH cells) that produce abundant IL-13 under the influence of IL-25 and IL-33. ILC2 cells are critical in the control of extracellular parasites. The RORγt branch includes LTi cells, ILC17 cells and ILC22 cells. All of these ILC subsets express and depend on RORγt and require IL-7 for their development. Signals that! trigger secretion of cytokines from these cells vary; the RANK ligand triggers LTi cells to express cell surface heterotrimers of LT-α and LT-β, whereas IL-23 or IL-1β triggers the production of IL-17 and IL-22 from ILC17 and ILC22 cells, respectively. Dysregulation of the different ILC subsets may be associated with disease as follows: ILC1, inflammation; ILC2, allergy; ILC17, autoimmunity; and ILC22, autoimmunity. ILCP, Id2-expressing ILC precursor; IBD, inflammatory bowel disease. * Figure 2: Transcription factors regulate the differentiation of distinct ILC subsets. Different transcription factors are essential for the development of different ILC subsets, including bone marrow NK precursors (bmNKP), thymic NK precursors (thyNKP), ILC2 precursors (ILC2P), LTi precursors (LTiP), ILC17 precursors (ILC17P) and ILC22 precursors (ILC22P), from Id2-expressing ILC precursors (ILCP). Author information * Abstract * Author information Affiliations * Tytgat Institute for Liver and Intestinal Research, Academic Medical Centre, Amsterdam, The Netherlands. * Hergen Spits * Innate Immunity Unit, Institut Pasteur, Paris, France. * James P Di Santo * Institut National de la Santé et de la Recherche Médicale U668, Paris, France. * James P Di Santo Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hergen Spits Additional data - Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling
- Nat Immunol 12(1):29-36 (2011)
Nature Immunology | Article Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling * Dan Levy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alex J Kuo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yanqi Chang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Uwe Schaefer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher Kitson4 Search for this author in: * NPG journals * PubMed * Google Scholar * Peggie Cheung1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexsandra Espejo5 Search for this author in: * NPG journals * PubMed * Google Scholar * Barry M Zee6 Search for this author in: * NPG journals * PubMed * Google Scholar * Chih Long Liu1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie Tangsombatvisit7 Search for this author in: * NPG journals * PubMed * Google Scholar * Ruth I Tennen8 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Y Kuo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Song Tanjing9 Search for this author in: * NPG journals * PubMed * Google Scholar * Regina Cheung7 Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin F Chua8, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul J Utz7 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaobing Shi9 Search for this author in: * NPG journals * PubMed * Google Scholar * Rab K Prinjha4 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Lee4 Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin A Garcia6 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark T Bedford5 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Tarakhovsky3 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaodong Cheng2 Search for this author in: * NPG journals * PubMed * Google Scholar * Or Gozani1 Contact Or Gozani Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:29–36Year published:(2011)DOI:doi:10.1038/ni.1968Received15 October 2010Accepted09 November 2010Published online05 December 2010 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 Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation–linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the ! target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs. View full text Figures at a glance * Figure 1: SETD6 monomethylates RelA at Lys310. () Methylation reactions (3H autoradiogram; left) with recombinant RelA(1–431) as substrate and recombinant PKMT enzymes (full-length or SET domains only; above lanes) and Coomassie staining (right) of recombinant enzymes (left margin, molecular size in kilodaltons (kDa)). Top band, automethylated SETD6. () SETD6-catalyzed methylation assay (autoradiogram; top) with wild-type or mutant RelA(1–431) (above lanes), and Coomassie staining (below) of proteins used. () Mass spectrometry analysis of methylation assays of RelA peptide (amino acids 300–320; RelA(300–320)) with (right) or without (left) SETD6; results are presented relative to those of the most abundant ion, set as 100%. Numbers above peaks indicate the mass/charge (m/z) ratio. () Immunoblot analysis of methylation reactions with (+) or without (−) SETD6 on wild-type (WT) RelA(1–431) or RelA(1–431) with the K310R substitution. () Immunoblot analysis of whole-cell extracts (WCE; 5% of total) of 293T cells! transfected with Flag-tagged SETD6 and either RelA or RelA(K310R), probed with antibodies to various molecules (left margin). () In vitro methylation reaction (top) with wild-type SETD6 or SETD6(Y285A) on RelA(1–431) and Coomassie staining (below) of recombinant proteins used. () Immunoblot analysis of immunoprecipitated RelA (IP) or WCE (5% of total) from 293T cells transfected with wild-type SETD6 or SETD6(Y285A). () Immunoblot analysis (as in ) of 293T cells treated with control (C) or SETD6-specific siRNA (two independent siRNAs: 1 and 2). () Immunoblot analysis of RelA or control immunoglobulin G (IgG) protein-protein ChIP, probed with antibodies to various molecules (left margin). Input, 5% of total. () Immunoblot analysis of 293T cells biochemically separated into cytoplasmic (Cyt), nucleoplasmic (Nuc) or chromatin-enriched (Chrom) fractions; tubulin and H3 signals serve as controls for fractionation integrity. () Immunoblot analysis of cytoplasmic and chromatin-e! nriched fractions (as in ) from 293T and U2OS cells with (+) o! r without (−) treatment with TNF (10 ng/ml for 1 h). Data are representative of three (,,,,) or two (,,,–) independent experiments. * Figure 2: Monomethylation of RelA by SETD6 inhibits the transactivation activity of RelA. () Occupancy of RelAK310me1 at the promoters of IL8, IL1a, MYC, CCND1 and GAPDH (control) in U2OS cells treated with control or SETD6 siRNA, assessed by real-time PCR analysis of ChIP samples; enrichment is presented as (ChIP/input) × 100. () ChIP assays (as in ) of U2OS cells (left) and THP-1 cells (right) with (+) or without (−) TNF stimulation (20 ng/ml for 1 h). ChIP with negative control antibody (,), Supplementary Figure 9. () Activity of a κB-Luc luciferase reporter (top) 24 h after transfection of U2OS cells with increasing amounts (wedges) of wild-type SETD6 or SETD6(Y285A); results are normalized to those of renilla luciferase and are presented relative to those of cells transfected with control vector. Below, immunoblot analysis of SETD6 and SETD6(Y285A). () Luciferase activity (as in ) in 293T cells transfected with control or SETD6-specific siRNA with (right) or without (far left) TNF treatment (10 ng/ml for 1 h). () Real-time PCR analysis of the efficiency ! of knockdown of SETD6 mRNA by SETD6-specific siRNA in U2OS cells, THP-1 cells and mouse BMDMs (mBMDM), presented relative to its expression in cells transfected with control siRNA (C). (–) Real-time PCR analysis of various mRNAs (vertical axes) in U2OS cells (), THP-1 cells () and primary mouse BMDMs () transfected with control or SETD6-specific siRNA with or without TNF (20 ng/ml for 1 h) or LPS (100 ng/ml for 1 h). Data are from at least three experiments (error bars, s.e.m.). * Figure 3: SETD6 attenuates RelA-driven cell proliferation. () Growth of U2OS cells treated with control shRNA (Ctrl sh) or SETD6-specific shRNA (SETD6sh) and/or RelA-specific shRNA (RelAsh), assessed daily for 7 d. () Colonies of U2OS cells, treated as in , in soft agar. () Growth of Rela+/+ and Rela−/− MEFs treated and assessed as in (right), and immunoblot analysis of WCE of Rela+/+ and Rela−/− 3T3 fibroblasts left untreated (−) or treated with SETD6-specific shRNA (+). (,) Growth curves (right) and immunoblot analysis (of WCE; left) of U2OS cells () and 3T3 fibroblasts () treated with control or SETD6-specific shRNA and reconstituted (Recon vec) with SETD6 or SETD6(Y285A) or not reconstituted (C). Data are from at least three independent experiments (error bars, s.e.m.). * Figure 4: SETD6 attenuates RelA-driven inflammatory responses. () SETD6 expression in patients with rheumatoid arthritis (RA; n = 8) and healthy controls (n = 15), presented as the normalized log2 ratio of the sample compared with a common reference (left); and SETD6 expression in patients with septic shock (n = 30) and healthy controls (n = 15), presented relative to the median of the results obtained with healthy controls (right). Each symbol (in boxes) represents an individual sample. *P = 0.0015 and **P = 0.00036 (two-tailed t-test). () Enzyme-linked immunosorbent assay of cytokines in supernatants of THP-1 cells transfected with control siRNA or SETD6-specific siRNA with or without TNF (20 ng/ml). () Real-time PCR analysis of Il1a and Tnf mRNA in mouse BMDMs transfected with control siRNA (Ctrl si) or SETD6-specific siRNA (SETD6si). () Multiplex enzyme-linked immunosorbent assay of RelA-regulated cytokines in supernatants of primary mouse BMDMs treated for 2 h as in , presented as (SETD6 siRNA / control siRNA – 1) × 100. () Real! -time PCR analysis of SETD6 mRNA in primary human monocyte-derived dendritic cells (hMDDC; n = 3 donors) transfected with control siRNA (C) or SETD6-specific siRNA (1,2). () Secretion of TNF and IL-6 by human monocyte-derived dendritic cells transduced with siRNA as in and treated for 6 or 24 h with LPS (left; n = 3 donors) or at 24 h after treatment with 10 or 100 ng/ml of LPS (right; n = 1 donor). Data are an analysis of published studies (; error bars indicate minimum and maximum values within 1.5 interquartile range of the lower and upper quartile, respectively), are from at least three independent experiments (,,,; error bars, s.e.m.) or are representative of two independent experiments (). * Figure 5: GLP(ANK) binds specifically to RelAK310me1. () CADOR microarray analysis of the binding of proteins with 268 unique domains (Supplementary Fig. 17) to RelA(300–320) and RelAK310me1. () Peptide-binding assay of the precipitation of various biotinylated peptides (above lanes) with glutathione S-transferase-linked GLP(ANK). () Anti-RelA immunoprecipitation of RelA(1-431), either mock-methylated (−) or methylated by SETD6 (+), then incubated with GLP(ANK), analyzed by immunoblot with anti-GLP or anti-RelA. Below, immunoblot analysis of input (10% of starting material). () Immunoprecipitation (with anti-Flag) of proteins from 293T cells transfected with plasmids encoding Flag-tagged GLP and wild-type RelA or RelA(K310R), followed by immunoblot analysis of immunoprecipitates and WCE (10% of total). () Immunoprecipitation (with anti-RelA) of proteins from 293T cells left untransfected (−) or transfected with plasmid encoding SETD6, followed by immunoblot analysis of immunoprecipitates and WCE (10% of total). () Immunop! recipitation and immunoblot analysis as in of 293T cells treated with control or SETD6-specific siRNA. () Immunoprecipitation and immunoblot analysis as in of U2OS cells transfected with plasmid encoding GLP, with or without TNF treatment (10 ng/ml). () Occupancy of GLP and H3K9me2 at the IL8 and MYC promoters in U2OS cells treated with control siRNA (C) or SETD6-specific siRNA (1,2), with or without TNF treatment (20 ng/ml), assessed as in Figure 2a (ChIP with negative control antibody, Supplementary Fig. 19). Data are representative of three (,) or two (–) independent experiments or are from at least three experiments (; error bars, s.e.m.). * Figure 6: Phosphorylation of RelA at S311 by PKC-ζ blocks GLP recognition of RelAK310me1. () Model of the mechanism by which a methylation-phosphorylation switch at RelA Lys310 and Ser311 (bolded residues) regulates the recognition of RelAK310me1 by GLP. () Peptide-binding assay of the precipitation of various biotinylated peptides (above lanes) with glutathione S-transferase–linked GLP(ANK). () Immunoprecipitation (with anti-RelA) of proteins from 293T cells left untransfected (−) or transfected with plasmid encoding PKC-ζ(ca), followed by immunoblot analysis of immunoprecipitates and WCE (5% of total). Bottom row, blot probed antibody to V5-tagged PKC-ζ(ca). () Immunoblot analysis of chromatin-enriched fractions (Chrom) isolated from 293T cells with or without treatment with TNF or transfection of V5-tagged PKC-ζ(ca) (above lanes). () Dot-blot analysis of in vitro kinase reactions with recombinant PKC-ζ plus various peptides (left margin), spotted at a concentration of 0.25 μg/μl followed by 5× serial dilutions (wedges), probed with anti-RelAS311ph, ! anti-RelAK310me1, anti-PKC-ζ or horseradish peroxidase (HRP)-conjugated streptavidin (loading control). () Immunoblot analysis of RelA immunoprecipitated from 293T cells transfected with V5-tagged PKC-ζ(ca) and left untreated (−) or treated for 1 h (+) with calf intestinal alkaline phosphatase (CIP). Data are representative of two (,,,) or three () independent experiments. * Figure 7: RelA methylation-phosphorylation switch at chromatin regulates NF-κB signaling. () ChIP assay of RelAK310me1, GLP and H3K9me2 at the promoters of Il6, Tnf, Ccnd1 and Myc in Prkcz+/+ (WT) and Prkcz−/− MEFs23 with or without TNF treatment (20 ng/ml), presented as in Figure 2a (ChIP with negative control antibody, Supplementary Fig. 24). () Real-time PCR analysis (below) of Il1a and Il6 mRNA in Prkcz+/+ and Prkcz−/− cells with or without TNF treatment (20 ng/ml), and immunoblot analysis (above) of PKC-ζ in Prkcz+/+ and Prkcz−/− MEFs23. Data are from at least three experiments (error bars, s.e.m.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GDS3628 * GSE8121 * GSE13501 * GSE13849 Protein Data Bank * 2H2E * 2H2E Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biology, Stanford University, Stanford, California, USA. * Dan Levy, * Alex J Kuo, * Peggie Cheung, * Chih Long Liu, * Andrew Y Kuo & * Or Gozani * Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA. * Yanqi Chang & * Xiaodong Cheng * Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, New York, USA. * Uwe Schaefer & * Alexander Tarakhovsky * EpiNova DPU, Immuno-Inflammation group, GlaxoSmithKline, Stevenage, UK. * Christopher Kitson, * Rab K Prinjha & * Kevin Lee * Department of Carcinogenesis, M.D. Anderson Cancer Center, Smithville, Texas, USA. * Alexsandra Espejo & * Mark T Bedford * Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA. * Barry M Zee & * Benjamin A Garcia * Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California, USA. * Chih Long Liu, * Stephanie Tangsombatvisit, * Regina Cheung & * Paul J Utz * Department of Endocrinology, Gerontology, and Metabolism Medicine, Stanford University School of Medicine, Stanford, California, USA. * Ruth I Tennen & * Katrin F Chua * Center for Cancer Epigenetics, University of Texas M.D. Anderson, Houston, Texas, USA. * Song Tanjing & * Xiaobing Shi * Geriatric Research, Education, and Clinical Center, VA Palo Alto Health Care System, Palo Alto, California, USA. * Katrin F Chua Contributions D.L. did most of the molecular biology and cellular studies; Y.C. did binding affinity studies and modeling; A.J.K., P.C. and X.S. generated the PKMT library; A.J.K. identified and initially characterized the activity of SETD6 on RelA Lys310; B.Z. did mass spectrometry analysis; U.S. and C.K. did the primary cells experiments; A.E. did CADOR array experiments; C.L.L. analyzed gene expression data sets; R.I.T., S.T., A.Y.K., R.C. and S.T. provided technical support; X.S., P.J.U., K.C., B.G., R.P., M.B., A.T., X.C. and O.G. discussed studies; D.L. and O.G. designed studies, analyzed data, and wrote the paper; D.L. and A.J.K. contributed independently to the work; and all authors discussed and commented on the manuscript. Competing financial interests C.K., R.K.P. and K.L. are employees of GlaxoSmithKline. Corresponding author Correspondence to: * Or Gozani Supplementary information * Abstract * Accession codes * Author information * Supplementary information Excel files * Supplementary Data (468K) Expression array data set. PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–25, Tables 1–3 and Data Additional data - ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses
- Nat Immunol 12(1):37-44 (2011)
Nature Immunology | Article ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses * Sumio Hayakawa1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Souichi Shiratori1, 2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroaki Yamato1, 3, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Takeshi Kameyama1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chihiro Kitatsuji1 Search for this author in: * NPG journals * PubMed * Google Scholar * Fumi Kashigi1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Showhey Goto1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Shoichiro Kameoka1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Daisuke Fujikura4 Search for this author in: * NPG journals * PubMed * Google Scholar * Taisho Yamada1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tatsuaki Mizutani5 Search for this author in: * NPG journals * PubMed * Google Scholar * Mika Kazumata1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maiko Sato1 Search for this author in: * NPG journals * PubMed * Google Scholar * Junji Tanaka2 Search for this author in: * NPG journals * PubMed * Google Scholar * Masahiro Asaka3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yusuke Ohba5 Search for this author in: * NPG journals * PubMed * Google Scholar * Tadaaki Miyazaki4 Search for this author in: * NPG journals * PubMed * Google Scholar * Masahiro Imamura2 Search for this author in: * NPG journals * PubMed * Google Scholar * Akinori Takaoka1, 6 Contact Akinori Takaoka Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:37–44Year published:(2011)DOI:doi:10.1038/ni.1963Received04 August 2010Accepted28 October 2010Published online21 November 2010Corrected online08 December 2010 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 poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5′-triphosphate–modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-β and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control. View full text Figures at a glance * Figure 1: Involvement of PARP-superfamily members in interferon responses to cytosolic nucleic acids. () Quantitative RT-PCR analysis (top) of the induction of human IFN-β mRNA by stimulation with 3pRNA, poly(rI:rC) or poly(dA:dT) (1 μg/ml) in HEK293T cells transfected with control vector or expression vectors for various members of the mouse PARP family; results are presented in relative expression units (RE), relative to the expression of ACTB (encoding β-actin). Below, immunoblot analysis of PARP expression with antibody to hemagglutinin (anti-HA) or anti-β-actin (lanes correspond to bars above). NS, nonspecific band. () Quantitative RT-PCR analysis (middle) of IFN-β mRNA after 8 h of no stimulation (−) or stimulation with 3pRNA (+), and luciferase activity (right) of a p-125Luc firefly luciferase reporter plasmid at 48 h after transfection of HEK293T cells with increasing doses (wedges) of control vector (C) or expression vector for HA-tagged ZAPS or ZAP. Results are presented relative to ACTB expression (middle) or the activity of renilla luciferase (right). Left! , immunoblot analysis of the expression of HA-tagged ZAPS and ZAP; far left lane, HEK293T cells transfected with control vector. () Quantitative RT-PCR analysis of ZAPS and ZAP mRNA in HEK293T cells stimulated for 0–12 h with 3pRNA (left) or IFN-α (500 U/ml; right); results are presented relative to expression at 0 h. () Quantitative RT-PCR analysis of ZAPS and ZAP mRNA induced by 3pRNA in human primary CD14+ monocytes purified from peripheral blood mononuclear cells; results are presented relative to expression at 0 h. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from one representative of at least two independent experiments (mean and s.d. of triplicate (,) or duplicate (,) samples). * Figure 2: ZAPS is a potent stimulator of RIG-I-mediated type I interferon responses activated by 3pRNA. () Quantitative RT-PCR analysis of IFN-α1, IFN-α4 and IFN-β mRNA after 0–12 h of stimulation with 3pRNA in HEK293T cells transfected with control vector (open bars) or expression vector for ZAPS (filled bars). (,) Quantitative RT-PCR analysis of IFN-β mRNA after 8 h of 3pRNA stimulation () and luciferase activity of a p-125Luc reporter plasmid after 16 h of 3pRNA stimulation () in HEK293T cells transfected with increasing doses of control vector or ZAPS expression vector. () Quantitative RT-PCR analysis of IFN-β mRNA after 8 h of 3pRNA stimulation (left) and ELISA of IFN-β 24 h after 3pRNA stimulation (right) in HEK293T cells transfected with control siRNA (siControl) or with siZAPS1, siZAPS2 or siZAPS3. ND, not detected. () Quantitative RT-PCR analysis of IFN-β mRNA after 8 h of 3pRNA stimulation in HEK293T cells cotransfected with control vector or expression vector for mouse ZAPS (mZAPS) or PARP-1, plus siControl or siZAPS3. () Quantitative RT-PCR analysis of IFN! -α1 and IFN-β mRNA after 8 h of stimulation with viral RNA derived from influenza virus (Flu vRNA) in A549 cells treated with siControl or siZAPS3. (,) Quantitative RT-PCR analysis of type I interferon mRNA after 8 h of stimulation with 3pRNA in human primary CD14+ monocytes () and MRC-5 fibroblasts () treated for 48 h with siControl or siZAPS3. RT-PCR results (,,–) are presented relative to ACTB expression; luciferase results are presented relative to the activity of renilla luciferase (). *P < 0.05 and **P < 0.01 (Student's t-test). Data are from one representative of at least two independent experiments (mean and s.d. of triplicate (–,) or duplicate () samples). * Figure 3: ZAPS activates both the NF-κB and IRF3 transcriptional pathways in a RIG-I- and MAVS-dependent manner. () Quantitative RT-PCR analysis of TNF, IL-6 and CXCL10 mRNA after 8 h of 3pRNA stimulation in A549 cells treated with siControl or siZAPS3; results are presented relative to ACTB expression. () Luciferase activity of an NF-κB–luciferase reporter plasmid after 16 h of 3pRNA stimulation in HEK293T cells transfected with increasing doses of control vector or ZAPS expression vector, presented relative to the activity of renilla luciferase. () Luciferase activity of an NF-κB–luciferase reporter plasmid in TLR8-expressing HEK293T cells, measured 16 h after stimulation with 3pRNA or R-848, presented relative to the activity of renilla luciferase. () Native PAGE and immunoblot analysis (top) of 3pRNA-induced dimerization of endogenous IRF3 in HEK293T cells treated with siControl (open bars) or siZAPS3 (filled bars); right, lysates from HEK293T cells transfected with control or MAVS expression vector (negative or positive control, respectively). Below, band intensity of the IR! F3 dimer relative to that of the IRF3 monomer, assessed by densitometry. () Electrophoretic mobility-shift assay of NF-κB activation induced by 3pRNA stimulation in HEK293T cells treated with siControl or siZAPS3; far right (Cm), competition by cold probe. () Quantitative RT-PCR analysis of IFN-β mRNA 48 h after cotransfection of HEK293T cells with siControl or siRNA targeting STING, MAVS or RIG-I, along with ZAPS expression vector; results are presented relative to ACTB expression. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from one representative of at least two independent experiments (mean and s.d. of triplicate (–) or duplicate () samples). * Figure 4: ZAPS interacts with RIG-I to positively modulate the RIG-I activity. () Fluorescence confocal microscopy of HeLa cells cotransfected with YFP-tagged ZAPS and Flag-tagged RIG-I (secondarily visualized with Alexa Fluor 594), before (−) and after (+) 4 h of 3pRNA stimulation. Top right, enlargement of area outlined at left (bottom row); below, line scan of fluorescence intensity of the white line (–) above. AU, arbitrary units. Original magnification, ×60. () Intermolecular FRET (top) of the interaction between YFP-tagged ZAPS and cyan fluorescent protein (CFP)-tagged RIG-I in HeLa cells with (+) or without (−) 3pRNA stimulation, presented as the ratio of corrected FRET (FRETc) to CFP (small horizontal bars, mean). Below, fluorescence images of corrected FRET (pseudocolor mode). () Immunoprecipitation (IP; with anti-Flag) of HA-tagged ZAPS together with Flag-tagged RIG-I in lysates of HEK293T cells after mock treatment or treatment with 3pRNA (left) or infection with NDV (middle), followed by immunoblot analysis with anti-HA or anti-Flag.! Right, coimmunoprecipitation of endogenous ZAPS and RIG-I in MRC-5 cells after 6 h of stimulation with 3pRNA; anti-ZCCHV, rabbit polyclonal antibody that can detect ZAPS as well as ZAP. Bottom, immunoblot analysis of whole-cell lysates (WCL) with anti-HA (left, middle) or anti-RIG-I (right). () Interaction of HA-tagged ZAPS with Flag-tagged full-length RIG-I or deletion mutants of RIG-I in HEK293T cells after 6 h of stimulation with 3pRNA, assessed as described in . C-RIG-I, carboxy-terminal region of RIG-I; N-RIG-I, amino-terminal CARDs of RIG-I. () Interaction of Flag-tagged carboxy-terminal region of RIG-I with HA-tagged ZAPS or its mutants (N1-ZAPS, N2-ZAPS or ZAPSΔzf; right) in HEK293T cells, analyzed as described in . () Native PAGE and immunoblot analysis (top) of the oligomerization of Flag-tagged RIG-I induced by NDV infection (16 h) in HEK293T cells treated with siControl or siZAPS3. Below, band intensity of the RIG-I oligomer relative to that of the RIG-I monom! er. () ATPase activity of recombinant RIG-I protein after the ! addition of 3pRNA in the presence of increasing doses of recombinant glutathione S-transferase–tagged ZAPS or ZAPSΔzf. *P < 0.01 (Student's t-test). () Association of Flag-tagged RIG-I with HA-tagged MAVS after 6 h of 3pRNA stimulation in HEK293T cells treated with siControl or siZAPS3, assessed as described in . Data are from one representative of at least two independent experiments (mean and s.d. of triplicate samples in ). * Figure 5: ZAPS is a key regulator of RIG-I-mediated induction of type I interferons and antiviral innate defense. (,) Quantitative RT-PCR analysis of the expression of IFN-β, IFN-α1, IL-6, TNF and CXCL10 mRNA () and ELISA of IFN-β protein () in A549 cells treated with siControl or siZAPS3 after infection for 0 or 6 h () or for 0 or 24 h () with influenza virus (strain A/X-31; multiplicity of infection, 1.0). () Quantitative RT-PCR analysis of nucleoprotein gene expression (Flu NP) in A549 cells treated as described in and (72 h of infection). () Quantitative RT-PCR analysis (left) of IFN-β mRNA induced by infection with influenza virus (multiplicity of infection, 1.0) in HEK293T cells transfected with control or ZAPS expression vector. Right, plaque-forming assay of viral titers after 48 h of infection (PFU, plaque-forming units). (,) Quantitative RT-PCR analysis of the expression of IFN-β and IFN-α1 mRNA () and ELISA of IFN-β () in A549 cells treated with siControl or siZAPS3 after infection for 0 or 12 h () or for 0 or 24 h () with NDV. () Quantitative RT-PCR analysis of the ex! pression of the gene encoding NDV F protein in cells treated as described in and (72 h of infection). RT-PCR results (,–,) are presented relative to the expression of GAPDH (encoding glyceraldehyde phosphate dehydrogenase). *P < 0.05 and **P < 0.01 (Student's t-test). Data are from one representative of at least two independent experiments (mean and s.d. of triplicate (–,–) or duplicate () samples). * Figure 6: Crucial role for ZAPS in the induction of cytokine genes by NDV infection. () Strategy for site-specific gene disruption for the generation of human ZC3HAV1-knockout cells, showing the ZFN-targeting site in the exon 3; ZFN-L and ZFN-R are recognition sites of the designed ZFN pairs. () Immunoprecipitation and immunoblot analysis of ZAPS expression in parental HEK293T cells (WT) and ZC3HAV1-knockout clones 32 and 89, probed with anti-ZCCHV. Bottom, immunoblot analysis of whole-cell lysates (loading control). () Quantitative RT-PCR analysis of the induction of IFN-β, TNF and CXCL10 mRNA in response to NDV infection (8 h) in parental HEK293T cells (open bars) and ZC3HAV1-knockout clones 32 and 89 (filled bars). () Quantitative RT-PCR analysis of IFN-β mRNA after 8 h of 3pRNA stimulation in parental HEK293T cells and ZC3HAV1-knockout clones 32 and 89, transfected with control vector (C; open bars) or ZAPS expression vector (Z; filled bars). RT-PCR results are presented relative to ACTB expression. *P < 0.05 and **P < 0.01 (Student's t-test). Data are! from one representative of at least two independent experiments (mean and s.d. of triplicate samples in ,). Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Entrez Nucleotide * NM_024625.3 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrigendum 08 December 2010In the version of this article initially published online, the affiliation of F. Kashigi, S. Goto and S. Kameoka with the Department of Chemistry, Graduate School of Science, Hokkaido University Sapporo, Japan, was omitted. 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. * Sumio Hayakawa, * Souichi Shiratori & * Hiroaki Yamato Affiliations * Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan. * Sumio Hayakawa, * Souichi Shiratori, * Hiroaki Yamato, * Takeshi Kameyama, * Chihiro Kitatsuji, * Fumi Kashigi, * Showhey Goto, * Shoichiro Kameoka, * Taisho Yamada, * Mika Kazumata, * Maiko Sato & * Akinori Takaoka * Department of Hematology and Oncology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. * Souichi Shiratori, * Junji Tanaka & * Masahiro Imamura * Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. * Hiroaki Yamato & * Masahiro Asaka * Department of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan. * Daisuke Fujikura & * Tadaaki Miyazaki * Laboratory of Pathophysiology and Signal Transduction, Hokkaido University Graduate School of Medicine, Sapporo, Japan. * Tatsuaki Mizutani & * Yusuke Ohba * Research Center of Infection-Associated Cancer, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan. * Akinori Takaoka * Department of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Japan. * Fumi Kashigi, * Showhey Goto & * Shoichiro Kameoka Contributions S.H., S.S., H.Y., T.K., C.K., F.K., S.G., S.K., T.Y., M.K., M.S., J.T., M.A. and M.I. planned studies, did experiments and analyzed data; D.F. and T. Miyazaki contributed to viral infection experiments and helped with data analyses; T. Mizutani and Y.O. did fluorescence microscopy experiments and FRET analysis; and A.T. supervised the project, designed experiments and wrote the manuscript with comments from the coauthors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Akinori Takaoka Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (616K) Supplementary Figures 1–5 and Tables 1–2 Additional data - Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes
- Nat Immunol 12(1):45-53 (2011)
Nature Immunology | Article Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes * Jan H Kessler1 Contact Jan H Kessler Search for this author in: * NPG journals * PubMed * Google Scholar * Selina Khan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ulrike Seifert2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sylvie Le Gall3 Search for this author in: * NPG journals * PubMed * Google Scholar * K Martin Chow4 Search for this author in: * NPG journals * PubMed * Google Scholar * Annette Paschen5 Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra A Bres-Vloemans1 Search for this author in: * NPG journals * PubMed * Google Scholar * Arnoud de Ru1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nadine van Montfoort1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kees L M C Franken1 Search for this author in: * NPG journals * PubMed * Google Scholar * Willemien E Benckhuijsen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jill M Brooks6 Search for this author in: * NPG journals * PubMed * Google Scholar * Thorbald van Hall7 Search for this author in: * NPG journals * PubMed * Google Scholar * Kallol Ray4 Search for this author in: * NPG journals * PubMed * Google Scholar * Arend Mulder1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ilias I N Doxiadis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul F van Swieten8 Search for this author in: * NPG journals * PubMed * Google Scholar * Hermen S Overkleeft8 Search for this author in: * NPG journals * PubMed * Google Scholar * Annik Prat9 Search for this author in: * NPG journals * PubMed * Google Scholar * Birgitta Tomkinson10 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacques Neefjes11 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter M Kloetzel2 Search for this author in: * NPG journals * PubMed * Google Scholar * David W Rodgers4 Search for this author in: * NPG journals * PubMed * Google Scholar * Louis B Hersh4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jan W Drijfhout1 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter A van Veelen1, 12 Search for this author in: * NPG journals * PubMed * Google Scholar * Ferry Ossendorp1, 12 Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelis J M Melief1, 12 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:45–53Year published:(2011)DOI:doi:10.1038/ni.1974Received14 June 2010Accepted17 November 2010Published online12 December 2010 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 Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer. View full text Figures at a glance * Figure 1: PRA(190–198) is an HLA-A3-presented CTL epitope with a proteasome-independent C terminus. () Proteasomal digestion site (arrow) in the 27-residue peptide PRA(182–208) (ELFSYLIEK; bold). () Specific lysis of K562-A3 cells, the renal cell carcinoma cell lines MZ1257 and 94.15 (both PRAME+HLA-A3+) and K562-A2 cells (K562 cells transfected with HLA-A2 (HLA-A2+)) by a CTL clone (anti-ELFSYLIEK CTL) raised against the PRA(190–198) peptide (ELFSYLIEK) exogenously loaded on HLA-A3. E:T, effector/target ratio. Data are from one experiment representative of five (mean and s.e.m. of triplicate wells). () Recognition of the 25-residue peptide PRA(190–214) (ELFSYLIEKVKRKKNVLRLCCKKLK; epitope at N terminus) digested for 1 h without enzyme (Mock digest (background)) or with immunoproteasomes (I-prot) or constitutive proteasomes (C-prot), then diluted and loaded at two different concentrations (horizontal axis), onto EKR cells (HLA-A3+PRAME−) and coincubated with the CTL clone in , then analyzed by enzyme-linked immunosorbent assay (ELISA) of the production of interferon! -γ (IFN-γ) by the CTL clone (to assess recognition). 9-mer, nine-residue peptide ELFSYLIEK loaded at the same concentrations (positive control). Data are from one experiment representative of three (mean and s.e.m. of triplicate wells). * Figure 2: Nardilysin produces precursors of PRA(190–198) with C-terminal extension. () Recognition, by the anti-ELFSYLIEK CTL clone, of K562-A3 cells treated with various inhibitors (horizontal axis), assessed as intracellular IFN-γ production and presented relative to results obtained with untreated K562-A3 cells, set as 100%. Data are from three experiments (mean and s.e.m.). () Fluorescence production by the fluorogenic substrate ELFSYL(-dab)IEKVKRC(-FL)KN after digestion for 30 min by cytosolic fractions of K562-A3 cells (KCl gradient, right vertical axis) with (+ phenan) or without (FL-pep) 1 mM phenanthroline, measured at 460 nm and presented in arbitrary units (AU). The same substrate with D-amino acids at the first positions had only slightly less fluorescence (data not shown). Right, separation of fraction 37 (Fr 37) by SDS-PAGE; nardilysin was identified in band 5 (Supplementary Fig. 4). Data are representative of two experiments. () Nardilysin digestion sites (top) in the 25-residue peptide PRA(190–214) (epitope, bold); arrows indicate cleavag! e that generates the 11-, 12- and 13-residue peptides. Below, recognition of PRA(190–214) by the anti-ELFSYLIEK CTL clone after digestion for 30 min with purified nardilysin, immunoproteasomes or constitutive proteasomes and loading onto EKR cells, assessed by ELISA of IFN-γ. Data are from one experiment representative of four (s.e.m. of triplicates). () Recognition, by the anti-ELFSYLIEK CTL clone, of K562-A3 and HeLa-A3 cells expressing control siRNA (si-Ctrl) or nardilysin-specific siRNA (si-NRD), assessed by ELISA of IFN-γ (bottom); peptide-loading control experiments, Supplementary Figure 5a. Suppression of nardilysin did not compromise proteasome-mediated processing (Supplementary Fig. 6). Above, immunoblot analysis of nardilysin and GAPDH (loading control) in K562-A3 cells expressing control or nardilysin-specific siRNA. Data are from one experiment representative of five (s.e.m. of triplicates). * Figure 3: TOP produces the C terminus of the PRA(190–198) epitope. () Recognition, by the anti-ELFSYLIEK CTL clone, of K562-A3 cells transfected with siRNA pools targeting TOP (si-TOP), neurolysin (si-neurolys) or insulin-degrading enzyme (si-IDE) or of K562-A2 cells alone (left), or of K562-A3 cells stably expressing TOP-specific or control siRNA (top right), assessed as ELISA of IFN-γ and presented relative to results obtained with K562-A3 cells transfected with nontargeting control siRNA, set as 100% (peptide-loading control experiments, Supplementary Fig. 5a). TOP suppression did not compromise proteasome-mediated processing (Supplementary Fig. 6). Bottom right, immunoblot analysis of TOP and GAPDH in K562-A3 cells stably transfected with control or TOP-specific siRNA. Data are representative of three or four experiments (error bars, s.e.m.). () Recognition, by the anti-ELFSYLIEK CTL clone, of the 12-residue peptide PRA(190–201) (ELFSYLIEKVKR) after digestion for 10 min (12-mer (10 min)) or 1 h (12-mer (1 h)) with TOP or without TOP ! (12-mer (mock)), followed by 'titration' and loading onto EKR cells, assessed by ELISA of IFN-γ. 9-mer, nine-residue peptide ELFSYLIEK (positive control). Data are from one experiment representative of three (s.e.m. of triplicate wells). * Figure 4: Role of nardilysin in HLA class I antigen processing. (,) Digestion sites of purified nardilysin in long peptides encompassing published ligands for HLA-A3, HLA-A11 and HLA-B27 (from the SYFPEITHI database). Bolding indicates dibasic motifs; arrows indicate cleavage present in >5% (bold arrows) or ≤5% (debold arrows) of the fragments after 30 min of digestion with nardilysin. () Top, HIV-1 gag p17 amino acids 8–37 containing two overlapping HLA-A3-presented epitopes (below; underlined, with amino acids in parentheses). Bottom, thymosin-β amino acids 1–30 containing the HLA-A11 ligand ASFDKAKLK (underlined). Proteasomes failed to produce the cleavages noted here (data not shown). () Peptides encompassing HLA-B2705 ligands (underlined). The top four ligands are reportedly insensitive to treatment with proteasome inhibitor15 (details, Supplementary Table 2). () Recognition, by a CTL clone directed against EBNA3C(258–266), of RT cells (HLA-B2705+EBNA3C+) and of K562 cells (K562-B27-mini) that express HLA-B2705 and MFLRGKWQ! RRYRRIYDLIEL (EBNA3C(247–266); epitope underlined), transfected expressing control or nardilysin-specific or siRNA, assessed by ELISA of IFN-γ (exogenous peptide-loading control experiments, Supplementary Fig. 5b). Above, immunoblot analysis of nardilysin in RT cells transfected expressing control or nardilysin-specific siRNA. Data are from one experiment representative of four (s.e.m. of triplicate wells). * Figure 5: The epitope-generating trimming capacity of TOP. () Digestion by purified TOP of variants of the 13-residue peptide ELFSYLIEKVKRK (PRA(190–202)) with systematic substitution of P1 and P1′ residues surrounding the TOP cleavage site. Downward arrow (above) and bold V or K (horizontal axes) indicate the wild-type peptide (included twice). Above, epitope production after 10 min or 30 min of digestion, presented as percentage of total summed fragment intensities (ELFSYLIEK or ELFSYLIEX for P1′ or P1 substitution, respectively, where X is mutated); substitutions in the order of their efficiency of epitope production at 10 min. Below, epitope destruction through a subsequent cleavage in the middle of the epitope, presented as percentage of peptides in the digestion constituting fragments of the epitope after 10 min or 30 min of digestion. () Recognition, by the anti-ELFSYLIEK CTL clone, of TOP-digested P1′-substitution variants loaded onto EKR cells (HLA-A3+), assessed by ELISA of IFN-γ. The P1′-proline variant, which ! was not digested, serves as a negative control. Data are representative of two () or three () experiments with similar results. * Figure 6: TOP-dependent presentation by HLA-A2 of the MART-1 CTL epitope. () Recognition, by a CTL clone directed against MART-1(26–35) (EAAGIGILTV), of UKRV-Mel-15a melanoma cells (HLA-A2+MART-1+) transfected with TOP-specific siRNA (si-TOP-1 or si-TOP-2), assessed by enzyme-linked immunospot assay of IFN-γ and presented relative to results obtained with cells transfected with control siRNA, set as 100% (left; peptide-loading control experiments, Supplementary Fig. 5c). Right, RT-PCR analysis of TOP and GAPDH in the UKRV-Mel-15a cells. () Digestion sites of purified TOP (arrows) in the 13-, 14-, 15-, 16- and 17-residue substrates (top to bottom) MART-1(26–38), MART-1(26–39), MART-1(26–40), MART-1(26–41) and MART-1(26–42); the EAAGIGILTV epitope is underlined. () Specific lysis of JY cells (HLA-A2+MART-1−) loaded with various MART-1 peptides (10 μM; horizontal axis: amino acids (left to right) 26–35 (EAAGIGILTV), 26–34, 27–34 or 27–37) or the HLA-A2-restricted PRA(100–108) epitope20 (VLDGLDVLL; negative control) by the CT! L clone in , after coculture at various effector/target ratios (E:T; key). () Recognition by the CTL clone in of JY cells loaded with various concentrations (key) of the peptides in , assessed as intracellular IFN-γ. Data are representative of two experiments (; mean and s.e.m.) or are from one experiment representative of three (,). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * Hs.584782 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Peter A van Veelen, * Ferry Ossendorp & * Cornelis J M Melief Affiliations * Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. * Jan H Kessler, * Selina Khan, * Sandra A Bres-Vloemans, * Arnoud de Ru, * Nadine van Montfoort, * Kees L M C Franken, * Willemien E Benckhuijsen, * Arend Mulder, * Ilias I N Doxiadis, * Jan W Drijfhout, * Peter A van Veelen, * Ferry Ossendorp & * Cornelis J M Melief * Institut für Biochemie-Charité, Medical Faculty of the Humboldt University Berlin, Berlin, Germany. * Ulrike Seifert & * Peter M Kloetzel * Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts, USA. * Sylvie Le Gall * Department of Molecular and Cellular Biochemistry, and Center for Structural Biology, University of Kentucky, Lexington, Kentucky, USA. * K Martin Chow, * Kallol Ray, * David W Rodgers & * Louis B Hersh * Department of Dermatology, University Clinics Essen, Essen, Germany. * Annette Paschen * Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK. * Jill M Brooks * Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands. * Thorbald van Hall * Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands. * Paul F van Swieten & * Hermen S Overkleeft * Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Canada. * Annik Prat * Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. * Birgitta Tomkinson * Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. * Jacques Neefjes Contributions J.H.K. conceived of the study, coordinated the work, designed, did and analyzed most experiments and wrote the manuscript with major input from C.J.M.M. and minor input from other authors; C.J.M.M., F.O. and P.A.v.V. provided intellectual input; S.K. did the experiments and analyses in Figure 2b and immunoblot analysis; K.M.C., L.B.H. and A. Prat contributed to the experiments about nardilysin; D.W.R., K.R., U.S. and A. Paschen contributed to the experiments about TOP; P.M.K., U.S. and B.T. contributed to the experiments about TPPII; H.S.O. and P.F.v.S. contributed to the experiments about the proteasome; J.N. and T.v.H. contributed to the experiments about TAP; N.v.M., U.S., A. Paschen, S.L.G. and J.M.B. contributed to CTL experiments; J.W.D., F.O., J.N., S.K. and W.E.B. contributed to substrate design and synthesis; S.A.B.-V. and K.L.M.C.F. contributed to the molecular biology; A.M. and I.I.N.D. made HLA class I mAbs and cell lines expressing a single HLA class I allele; a! nd P.A.v.V. and A.d.R. did mass spectrometry. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jan H Kessler Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–12 and Supplementary Tables 1–2 Additional data - HLA-DM captures partially empty HLA-DR molecules for catalyzed removal of peptide
- Nat Immunol 12(1):54-61 (2011)
Nature Immunology | Article HLA-DM captures partially empty HLA-DR molecules for catalyzed removal of peptide * Anne-Kathrin Anders1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Melissa J Call1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Monika-Sarah E D Schulze1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin D Fowler4 Search for this author in: * NPG journals * PubMed * Google Scholar * David A Schubert1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nilufer P Seth1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric J Sundberg5 Search for this author in: * NPG journals * PubMed * Google Scholar * Kai W Wucherpfennig1, 2 Contact Kai W Wucherpfennig Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:54–61Year published:(2011)DOI:doi:10.1038/ni.1967Received26 August 2010Accepted09 November 2010Published online05 December 2010 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 The mechanisms of HLA-DM-catalyzed peptide exchange remain uncertain. Here we found that all stages of the interaction of HLA-DM with HLA-DR were dependent on the occupancy state of the peptide-binding groove. High-affinity peptides were protected from removal by HLA-DM through two mechanisms: peptide binding induced the dissociation of a long-lived complex of empty HLA-DR and HLA-DM, and high-affinity HLA-DR–peptide complexes bound HLA-DM only very slowly. Nonbinding covalent HLA-DR–peptide complexes were converted into efficient HLA-DM binders after truncation of an N-terminal peptide segment that emptied the P1 pocket and disrupted conserved hydrogen bonds to HLA-DR. HLA-DM thus binds only to HLA-DR conformers in which a critical part of the binding site is already vacant because of spontaneous peptide motion. View full text Figures at a glance * Figure 1: Peptide disrupts the long-lived complex of empty HLA-DR and HLA-DM. (–) Specificity of the SPR assay. () Effect of wild-type HLA-DM (DM WT), HLA-DMα R98A (DM Mut1) and HLA-DMα R98A-R194A (DM Mut2) on the binding of Alexa Fluor 488–labeled MBP(85–99) (30 nM) to HLA-DR2 (150 nM), measured by FP and presented in millipolarization units (mP). () SPR analysis of the binding of HLA-DR2–CLIP (2 μM) by wild-type and mutant HLA-DM (pH 5.35, 25 μl/min, 30 °C) injected for 5 min (stage 1), followed by buffer (stage 2) and 1 μM MBP(85–99) (stage 3), presented in resonance units (RU) as HLA-DM flow cell – reference flow cell (reference is streptavidin here). () Binding of labeled MBP(85–99) to wild-type HLA-DR2 (DR2 WT) or the HLA-DRα S53D mutant (150 nM; DR2 Mut) with (+ DM) or without HLA-DM (25 nM). () Binding of HLA-DM by wild-type or mutant HLA-DR2–CLIP (1 μM) injected for 5 min, followed by buffer and 10 μM CLIP peptide of amino acids 87–101 (CLIP(87–101)). () Dissociation of the HLA-DM–HLA-DR2 complex by peptide, ass! essed by injection of high-affinity MBP(85–99) (MBP) or the MBP P4D analog (1 μM). Data are representative of two (,,) or more than three (,) independent experiments. * Figure 2: Rate of HLA-DM–HLA-DR complex dissociation is determined by peptide affinity. (,) Effect of CLIP and mutant CLIP on the dissociation of HLA-DM–HLA-DR, assessed by injection of 5 μM HLA-DR2–CLIP (5 min, pH 5.35, 15 μl/min, 30 °C), followed by buffer and wild-type or mutant CLIP (10 μM). () Competition assay of the binding of CLIP peptides (threefold dilution: 10 μM to 14 nM) to HLA-DR2 (100 nM) versus Alexa Fluor 488–labeled MBP(85–99) (10 nM); the median inhibitory concentration (IC50) is plotted against the rate of peptide-induced dissociation of HLA-DM–HLA-DR (from ,). () Effect of CLIP and two CLIP mutants, injected in stage 3 (0.5–100 μM), on the rate of dissociation of HLA-DR from HLA-DM. () Effect of MBP(85–99) and mutants on HLA-DM–HLA-DR dissociation, injected in stage 3 as in () Effect of MBP peptide affinity on the HLA-DM–HLA-DR dissociation rate, presented as the median inhibitory concentration determined by competition assay (as in ) versus dissociation rate (from ). Data are representative of two independent experi! ments (–) and more than three additional independent experiments under similar conditions (-,,; triplicate samples in ,). * Figure 3: High-affinity HLA-DR–peptide complexes interact slowly with HLA-DM. () SPR assay of the binding of HLA-DM by HLA-DR2–CLIP (5 μM), with (Cut DR2-CLIP) or without (Uncut DR2-CLIP) cleavage of the peptide linker by thrombin, injected for 5 min, followed by buffer (stage 2) and 20 μM CLIP(87–101) (stage 3). () SPR assay of the binding of HLA-DM by HLA-DR1–CLIPlow (5 μM), with (Cut DR1-CLIPlow) or without (Uncut DR1-CLIPlow) cleavage by the 3C protease, injected for 2 min, followed by buffer and HA(306–318) (50 μM). () SPR assay of the binding of HLA-DM by HLA-DR2 preloaded with CLIP or MBP (2 μM) and injected for 5–7 min, followed by buffer and 10 μM CLIP(87–101). () SPR assay of the binding of HLA-DM by HLA-DR2 preloaded with high-affinity MBP(85–99) and injected for 10 min, followed by buffer (stage 2) and 5 μM MBP(85–99) (stage 3). () SPR assay of the binding of HLA-DM by HLA-DR1 preloaded with CLIP or HA (10 μM) and injected for 5–7 min, followed by buffer and 50 μM HA(306–318). () SPR assay of the binding of HLA! -DM by HLA-DR1 preloaded with high-affinity HA(306–318) and injected for 10 min, followed by buffer (stage 2) and 50 μM HA(306–318) (stage 3). () SPR assay of the binding of HLA-DM by HLA-DR4 preloaded with CLIP or HA (2 μM) and injected for 5–7 min, followed by buffer and 50 μM HA(306–318). Assays were done at 25 °C (,) or 37 °C (–) and at a pH of 5.35 and a rate of 15 μl/min. Data are representative of three (,,) or two (,,,) independent experiments. * Figure 4: HLA-DM binds with fast kinetics to HLA-DR–peptide complexes without an engaged peptide N terminus. (,) Hydrogen-bonding network between HLA-DR1 and full-length, covalently linked HA(306–318) () or a linked HA mutant peptide lacking three N-terminal residues (). Numbers along peptide 'backbone' (P–2 to P11; between HLA-DR helices) indicate peptide position. The valine at position 65 in the HLA-DRα chain was replaced with cysteine to enable the formation of a disulfide bond with a cysteine at position P6 of HA (HA6). () Space-filling model of the empty HLA-DR1 groove showing residues contacted by two N-terminal peptide residues (red) and the P1 anchor (green). (–) Binding to HLA-DM by HLA-DR1 with linked HA6 peptides (5 μM), injected for 2 min (pH 5.35, 15 μl/min, 25 °C), followed by injection of buffer. Data are representative of at least three (,) or two () independent experiments. * Figure 5: Truncated peptides covalently linked through peptide position P3 also bind HLA-DM. (,) Binding of HLA-DM by HLA-DR1–peptide complexes (5 μM) in which the glycine at position 58 of the HLA-DRα chain was replaced with cysteine to permit the formation of disulfide bonds with HA peptides carrying a cysteine at the position P3 (HA3), assessed as in Figure 4. Data are representative of three () or two () independent experiments. * Figure 6: HLA-DRα Trp43 is important for interaction with HLA-DM. () HLA-DRα model showing the surface accessibility of Trp43 and its interaction with the P1 tyrosine of HA(306–318), as well as the interaction of hydrophobic residues in the HLA-DRα segment of positions 40–54 with the P1 anchor (Trp43, Ala52 and Phe54) and residues associated with the HLA-DM interaction (Glu40, Phe51 and Ser53). The segment of HA(306–318) from P–2 to P2 is presented as a stick model. () FP analysis of the dissociation of Alexa Fluor 488–labeled MBP(85–99) from wild-type or mutant (W43F) HLA-DR2 over a range of HLA-DM concentrations (horizontal axis). () Binding of HLA-DM by wild-type or mutant (W43F) HLA-DR2 with bound CLIP (1 μM; pH 5.35, 30 °C, 15 μl/min), followed by buffer and 5 μM CLIP(87–101). () FP analysis of the dissociation of Alexa Fluor 488–labeled MBP(85–99) from preloaded wild-type or mutant (W43F) HLA-DR2 complexes (100 nM) in presence of 10 μM unlabeled MBP(85–99) competitor peptide (pH 5.4, 25 °C). (,) FP assay (! ) and SPR assay () of four HLA-DRα chain mutants in the conditions in () and (). Data are representative of two independent experiments. * Figure 7: Large energy barrier for the binding of HLA-DR–peptide to HLA-DM. (,) Binding of HLA-DM by HLA-DR1–CLIPlow (1 μM, ) or HLA-DR1–HA6 P2-P11 (1 μM, ) injected at various temperatures (key; 15 μl/min, pH 5.35), followed by buffer and 50 μM HA(306–318). () Binding at 250–270 s of the association phase in , (and Supplementary Fig. 6a), plotted against the corresponding temperature. () Affinity of HLA-DM for HLA-DR1–HA6 P2–P11 injected at various concentrations (key) as in ; results were fitted with a 1:1 Langmuir binding model by BIAevaluation software. (,) Binding of HLA-DM by HLA-DR1–CLIPlow (5 μM; ) or HLA-DR1–HA6 P2-P11 (5 μM; ) injected for 2 min at 25 °C as in at various pH values (key). () Binding at 91–110 s of the association phase in , (and for HLA-DR2–CLIP; not presented in ,), plotted against pH, with binding at pH 5.0 set as 1.0. Data are representative of two (,) or three (,,) independent experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. * Anne-Kathrin Anders, * Melissa J Call, * Monika-Sarah E D Schulze, * David A Schubert, * Nilufer P Seth & * Kai W Wucherpfennig * Program in Immunology, Harvard Medical School, Boston, Massachusetts, USA. * Anne-Kathrin Anders & * Kai W Wucherpfennig * Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, Berlin, Germany. * Monika-Sarah E D Schulze * Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge, Massachusetts, USA. * Kevin D Fowler * Boston Biomedical Research Institute, Watertown, Massachusetts, USA. * Eric J Sundberg * Present addresses: The Walter and Eliza Hall Institute, Parkville, Victoria, Australia (M.J.C.) and Pfizer, Cambridge, Massachusetts, USA (N.P.S.). * Melissa J Call & * Nilufer P Seth Contributions A.-K.A., M.J.C. and K.W.W. conceived of the study, designed experiments and wrote the paper; A.-K.A. generated HLA-DR–peptide complexes and did most of the SPR experiments; M.-S.E.D.S. did a set of SPR experiments with high-affinity HLA-DR–peptide complexes; E.J.S. provided advice for SPR experiments; M.J.C. did most of the FP peptide-binding assays; K.D.F. did mathematical modeling of Biacore data; D.A.S. did functional assays of HLA-DR–peptide complexes; and N.P.S. generated Chinese hamster ovary cell lines producing HLA-DR–CLIP complexes. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kai W Wucherpfennig Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (988K) Supplementary Figures 1–10 Additional data - Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes
- Nat Immunol 12(1):62-69 (2011)
Nature Immunology | Article Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes * Arito Yamane1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Resch1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Nan Kuo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Kuchen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiyu Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hong-wei Sun2 Search for this author in: * NPG journals * PubMed * Google Scholar * Davide F Robbiani3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin McBride3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michel C Nussenzweig3 Contact Michel C Nussenzweig Search for this author in: * NPG journals * PubMed * Google Scholar * Rafael Casellas1, 4 Contact Rafael Casellas Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:62–69Year published:(2011)DOI:doi:10.1038/ni.1964Received02 August 2010Accepted02 November 2010Published online28 November 2010 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 cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID's promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA to the immunoglobulin loci was facilitated by phosphorylation of AID at Ser38 and Thr140. We propose that stalled polymerases recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome. Efficient hypermutation and switch recombination required AID phosphorylation and correlated with recruitment of RPA. Our findings provide a rationale for the oncogenic role of AID in B cell malignancy. View full text Figures at a glance * Figure 1: Extent of AID recruitment in activated B cells. () ChIP-seq analysis of the Igh locus (chromosome 12 (Chr12)) in B cells stimulated with LPS and IL-4, showing acetylation of histone H4 (H4Ac), as well as recruitment of PolII and AID; results are presented as sequence tags per million sequences (TPM) in 100-bp windows. Bottom row, specificity of AID immunoprecipitation, assessed in Aicda−/− cells with an AID-specific antibody (α-AID)22. Above, Igh locus, showing the S domains (light gray boxes) and C domains (black boxes) as well as the 5′ μ-chain enhancer (Eμ), 3′ α-chain enhancer (Eα) and insulator (I). () Total sequence tags of AID-recruiting genes (n = 5,910), showing the approximate positions of a subset of AID targets, including immunoglobulin genes (underlined). () Recruitment of AID at a domain of chromosome 7 ~500 kb in length containing sequence encoding the B cell–specific CD19 antigen, assessed in Aicda+/+ and Aicda−/− (control) cells. Data are representative of four independent experiments. * Figure 2: AID targets are somatically hypermutated. Mutation frequencies of genes that recruited AID (AID+; n = 9) or did not recruit AID (AID−; n = 11) in activated Igk-AID Ung−/− B cells and Aicda−/− B cells. Bottom, adjusted P values (q), calculated to control for false-discovery rate (Fisher's exact test). Data are representative of one experiment. * Figure 3: AID recruitment is biased toward actively transcribed genes associated with an open chromatin configuration. () Deep-sequencing analysis of mRNA for genes that recruited or did not recruit AID. Transcript abundance is presented as total mRNA sequences per gene length (in kb) per million aligned reads (RPKM), including the median (small horizontal lines), quartiles (vertical lines) and outliers (bolding). *P < 0.0005 (Wilcoxon rank-sum test). () Heat maps of ChIP-seq results for PolII, H3K4me3 and H3K27me3 at all (100%) genes that recruited or did not recruit AID, rank-ordered from least (top) to most (bottom) in terms of their association with PolII or chromatin modifications and centered on the TSS (arrow) of each gene ± 2 kb. P < 0.0005 (χ2 test). () Aligned 'read' density for H3K4me3, H3K27me3, PolII, mRNA and AID at Mycn and Myc loci; numbers in plots (top right) indicate total sequenced tags aligned in each genomic domain. Data are representative of one experiment. * Figure 4: Epigenetic signature of AID recruitment. () Hierarchical clustering of AID, PolII, CTCF, p300 and 36 chromatin modifications on AID islands, presented as log2-transformed data and normalized Euclidean distances: red, high density; blue, low density. Numbers in parenthesis (right) indicate enrichment distance for each variable relative to that of AID. () Composite (metagene) profiles of ChIP-seq for H3K4me3, H2BK5Ac, H3K79me2 and H3K36me3, presented as tags per million aligned sequences per gene per nucleotide (density) versus position relative to the TSS (–2 kb to +5 kb). Data are representative of one experiment. * Figure 5: Genome-wide correlation between AID and PolII occupancy. () Composite profiles of the density of PolII and AID at elongating genes (n = 352) that recruit AID (presented as in Fig. 4b). () Quantitative correlation of PolII and AID at the Mir142 and Cd79b loci. Bottom row, microsequencing background in Aicda−/− cells. () Composite profiles of the density of PolII and AID at stalled genes (n = 4,756) that recruit AID (presented as in Fig. 4b). () Quantitative correlation of PolII and AID at the Lyn and Atm loci (stalled genes). Data are representative of one experiment. * Figure 6: AID hypermutates basal promoters. () Alignment of sequences from small RNA cDNA libraries (n = 14.7 × 106) relative to gene TSS (± 2 kb) in activated B cells. Arrows indicate sense of transcription. () PolII density at all genes in B cells activated with LPS and IL-4. Dashed lines indicate the coincidence of the two stalling peaks with the TSSs of both sense and antisense RNA in . () AID-mediated hypermutation of the Pax5 promoter-proximal area in Igk-AID Ung−/− B cells (top) detected from four PCR products (cyan rectangles); black box indicates Pax5 exon 1, and arrow indicates TSS and direction of transcription. Middle, mutation frequency: segment size indicates the proportion of sequences with the number of mutations noted along circle margin; center, total sequences. Bottom, mutation frequency (calculated as total mutations per total base pairs sequenced) and P value (relative to Aicda−/− control). Data are representative of one experiment. * Figure 7: Recruitment of RPA to on-target sites of AID. () RPA occupancy at the immunoglobulin gene locus in B cells (genotype, top left) stimulated for 72 h with LPS and IL-4. AID(S38A) or AID(T140A), replacement of AID Ser38 or AID Thr140, respectively, with alanine. Numbers in plots (top right) indicate total sequence tags per million for the entire locus. () Background signals at the Mir142 locus after ChIP-seq analysis of RPA. Red asterisk indicates position of the microRNA in the microRNA transcript (arrow). () AID recruitment and activity in B lymphocytes. Top: AID accumulates predominantly at PolII pausing sites across the genome by interacting with the stalling factor Spt5. At proximal promoter areas, RPA is either absent or fails to accumulate in sufficient amounts to fully engage AID activity. This results in little hypermutation both upstream and downstream of TSSs. With few exceptions, hypermutation at these sites is efficiently erased by base excision–repair and mismatch-repair activity. Bottom: at on-target immun! oglobulin sites, RPA is recruited as a result of site-specific phosphorylation (P) of AID at Ser38 and Thr140. This activity enhances DNA deamination and the formation of double-stranded DNA breaks, particularly in the core of immunoglobulin S domains, where PolII frequently stalls. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24178 * GSE21630 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Arito Yamane & * Wolfgang Resch Affiliations * Genomics & Immunity, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Arito Yamane, * Wolfgang Resch, * Nan Kuo, * Stefan Kuchen, * Zhiyu Li & * Rafael Casellas * Biodata Mining and Discovery, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Hong-wei Sun * Laboratory of Molecular Immunology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA. * Davide F Robbiani, * Kevin McBride & * Michel C Nussenzweig * Center of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. * Rafael Casellas Contributions A.Y. did deep sequencing, cloning and conventional sequencing experiments; W.R. and H.-w.S. analyzed data; N.K. contributed data; Z.L. maintained the mouse colonies and cultured cells; D.F.R. contributed the Igk-AID mice; M.C.N. made suggestions for experiments and reviewed and wrote sections of the manuscript; R.C. designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Michel C Nussenzweig or * Rafael Casellas Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3.4M) Supplementary Figures 1–10, Supplementary Text and Supplementary Tables 1–8 Additional data - Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions
- Nat Immunol 12(1):70-76 (2011)
Nature Immunology | Article Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions * Robert W Maul1 Search for this author in: * NPG journals * PubMed * Google Scholar * Huseyin Saribasak1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stella A Martomo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rhonda L McClure1 Search for this author in: * NPG journals * PubMed * Google Scholar * William Yang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandra Vaisman2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hillary S Gramlich3 Search for this author in: * NPG journals * PubMed * Google Scholar * David G Schatz4 Search for this author in: * NPG journals * PubMed * Google Scholar * Roger Woodgate2 Search for this author in: * NPG journals * PubMed * Google Scholar * David M Wilson III1 Search for this author in: * NPG journals * PubMed * Google Scholar * Patricia J Gearhart1 Contact Patricia J Gearhart Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:70–76Year published:(2011)DOI:doi:10.1038/ni.1970Received29 July 2010Accepted12 November 2010Published online12 December 2010 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 Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA. View full text Figures at a glance * Figure 1: Strategy for identifying uracil residues in DNA. Genomic DNA is isolated from activated Ung−/− B cells and treated with UNG enzyme to remove the uracil bases. The DNA is then digested with APE1 enzyme to convert abasic sites into single-strand breaks. The fragmented DNA is measured in assays that detect either loss of intact DNA or gain of fragment ends. LM-PCR, ligation-mediated PCR. * Figure 2: Uracil residues in the rearranged Igl allele from UNG-deficient DT40 cells. () Unrearranged and rearranged alleles. Digestion with the restriction enzymes SacI (Sa) and SpeI (Sp) produces fragments of 6.4 and 4.6 kb. Dotted oval indicates the scope of SHM; gray bar shows the position of the Cλ probe; double-ended arrows indicate the area amplified for quantitative PCR analysis. () Southern blot analysis (top) of genomic DNA obtained from cells containing a transgene for overexpression of chicken AID (cAIDR) and from Aicda−/− clones, then treated with APE1 with (+) or without (−) UNG treatment, separated by electrophoresis though an alkaline gel and blotted with the Cλ probe. U, unrearranged; R, rearranged. Below, quantification of band intensity. *P = 0.0001 (two-tailed t-test). Data are representative of six experiments with individual isolation of DNA in each (error bars, s.e.m.). () Expression of human AID (top) by DT40 clones transfected with vector encoding wild-type human AID (hAIDR; n = 6 clones) or a catalytically inactive mutant of ! human AID (hAIDR(E58A); n = 4 clones) or empty vector (Aicda−/−; n = 5 clones), presented relative to the expression of chicken β-actin. ND, not detectable. Below, quantitative PCR analysis of the amplification of VJλ in genomic DNA from treated and untreated samples, presented relative to the amplification of Cλ. *P = 0.0001 (two-tailed t-test). Data are representative of three experiments (average and s.e.m.). * Figure 3: Uracil residues in VH and Vκ regions from mouse germinal center B cells. () Rearranged mouse Igh and Igk loci. Primers were designed to amplify 500-bp fragments spanning introns downsteam of V-gene segments rearranged to JH4 and Jκ2 gene segments, and genes encoding Cμ, and Cκ. Dotted ovals indicate the range of SHM on both loci. () Quantitative PCR analysis of intact DNA isolated from B220+GL7+ spleen cells of Ung−/− and Ung−/−Aicda−/− mice (immunized with KLH in adjuvant), then treated with UNG and APE1; results are presented as the difference in amplification of treated versus untreated DNA, relative to Gapdh amplification. *P = 0.008 and **P = 0.001 (two-tailed t-test). Data are from six experiments (error bars, s.e.m.). * Figure 4: Uracil residues in the Sμ region after ex vivo stimulation of mouse splenic B cells with LPS and IL-4. () Map of the Sμ region. Dotted oval indicates the scope of SHM; Sμ primers amplify 858 bp by quantitative PCR; gray bar indicates position of the probe to hybridize a 2.6-kb fragment generated by digestion with HindIII (H). () Immunoblot analysis (top) of AID expression in Ung−/− mouse splenic B cells treated with LPS and IL-4. Below, quantification of band intensity, presented relative to that of β-actin. Data are representative of three experiments (error bars, s.e.m.). () Quantitative PCR analysis of the amplification of Sμ in intact DNA from Ung−/− cells (presented as in Fig. 3b). *P = 0.02 (two-tailed t-test). Data are representative of four experiments (error bars, s.e.m.). (,) Southern blot analysis (top) of intact DNA isolated from Ung−/− cells () or Ung−/−Aicda−/− cells (), digested with HindIII, treated with APE1 with or without UNG, separated by electrophoresis through in alkaline agarose gels and blotted with probes to Sμ and Dhfr. Below! , quantification of uracil in treated samples, standardized to Dhfr results and presented relative to that of untreated samples. *P = 0.02 (two-tailed t-test). Data are from three independent experiments (error bars, s.e.m.). * Figure 5: DNA replication decreases the uracil content. () Model of the effect of DNA replication on uracil content, with the presumption that AID functions within the first 24 h of stimulation. The U:G mispair would then be replicated so that one daughter cell receives a U:A pair and the other receives a C:G pair, thus diminishing the uracil content by half. When replication is blocked by aphidicolin, uracil residues accumulate. () Quantitative PCR analysis of the amplification of Sμ in DNA from Ung−/− B cells (presented as in Fig. 3b). *P = 0.04 and **P = 0.000038 (two-tailed t-test). Data are from four independent experiments (error bars, s.e.m.). () Southern blot analysis (top) of DNA from Ung−/− B cells treated with APE1 with or without UNG and separated by electrophoresis through alkaline agarose gels. Below, quantification of uracil content in treated samples, standardized to Dhfr intensity and presented relative to that of untreated samples. *P = 0.02 and **P = 0.0001 (two-tailed t-test). Data are from at least t! wo independent experiments (error bars, s.e.m.). * Figure 6: Uracil residues are present on both DNA strands in Sμ after ex vivo stimulation. () Southern blot analysis of DNA obtained from Ung−/− splenic B cells and treated with APE1 with or without UNG, then separated by electrophoresis through alkaline agarose gels and hybridized to a double-stranded (ds) probe (both strands) and single-stranded (ss) probes (nontranscribed or transcribed strands); the 2.6 kb band (HindIII digestion) is presented here. () Quantification of the uracil content of treated samples in , presented relative to that of untreated samples. *P = 0.04, **P = 0.008 and ***P = 0.004 (two-tailed t-test). Data are from three independent experiments (error bars, s.e.m.). * Figure 7: Detection of uracil residues by ligation-mediated PCR. () Experimental design: genomic DNA (100 ng) is treated with UNG, APE1 and DNA polymerase β-lyase to create a blunt 5′ end, then is extended from a primer (arrow) with polymerase to produce a double strand. The product is ligated to a linker and amplified with primers (arrows) for the linker and JH4 intron. () Southern blot analysis of DNA obtained from C57BL/6, Ung−/−Aicda−/−, Ung−/− and Ung−/−Msh2−/− B cells, treated as in (below lanes), amplified by extension initiated ~400 bp downstream of the JH4 gene segment along the nontranscribed strand, separated by gel electrophoresis and hybridized to a JH4 probe. Below, ethidium bromide staining of Gapdh (control for the DNA input used for the initial extension). Data are representative of 50 experiments with amplification of DNA from six to nine mice per strain. * Figure 8: Uracil residues preferentially replace cytosine residues. () Linker sites identified after sequencing of the PCR products in Figure 7. Downward triangles, linker location; rectangles, JH4 gene segment; arrow, primer for extension. () Bases adjacent to the linker ligation position for Ung−/− cells (n = 69) and Ung−/−Msh2−/− cells (n = 48). Size of and numbers in circles indicate proportion and percent, respectively, of total linkers at each nucleotide; data are corrected for nucleotide composition on the nontranscribed strand (C = 16%, G = 27%, T = 31%, A = 26%). *P = 0.00016 for Ung−/− and 0.02 for Ung−/−Msh2−/− (linkers at C versus total cytosine content; Fisher's exact test). Data are derived from 50 experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA. * Robert W Maul, * Huseyin Saribasak, * Stella A Martomo, * Rhonda L McClure, * William Yang, * David M Wilson III & * Patricia J Gearhart * Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA. * Alexandra Vaisman & * Roger Woodgate * Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA. * Hillary S Gramlich * Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA. * David G Schatz Contributions R.W.M., H.S., S.A.M. and P.J.G. designed the study; R.W.M., H.S., R.L.M., W.Y., A.V. and H.S.G. did experiments; D.G.S., R.W. and D.M.W. provided reagents and suggestions; and R.W.M. and P.J.G. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Patricia J Gearhart Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (320K) Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Methods Additional data - The enhancer HS2 critically regulates GATA-3-mediated Il4 transcription in TH2 cells
- Nat Immunol 12(1):77-85 (2011)
Nature Immunology | Article The enhancer HS2 critically regulates GATA-3-mediated Il4 transcription in TH2 cells * Shinya Tanaka1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Yasutaka Motomura1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshie Suzuki1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ryoji Yagi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiromasa Inoue2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shoichiro Miyatake3 Search for this author in: * NPG journals * PubMed * Google Scholar * Masato Kubo1, 4 Contact Masato Kubo Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:77–85Year published:(2011)DOI:doi:10.1038/ni.1966Received23 June 2010Accepted08 November 2010Published online05 December 2010 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 GATA-3 is a master regulator of T helper type 2 (TH2) differentiation. However, the molecular basis of GATA-3-mediated TH2 lineage commitment is poorly understood. Here we identify the DNase I–hypersensitive site 2 (HS2) element located in the second intron of the interleukin 4 locus (Il4) as a critical enhancer strictly controlled by GATA-3 binding. Mice lacking HS2 showed substantial impairment in their asthmatic responses and their production of IL-4 but not of other TH2 cytokines. Overexpression of Gata3 in HS2-deficient T cells failed to restore Il4 expression. HS2 deletion impaired the trimethylation of histone H3 at Lys4 and acetylation of histone H3 at Lys9 and Lys14 in the Il4 locus. Our results indicate that HS2 is the target of GATA-3 in regulating chromosomal modification of the Il4 locus and is independent of the Il5 and Il13 loci. View full text Figures at a glance * Figure 1: Histone modification of the Il13 and Il4 loci and regulation of IL-4 by HS sites. () ChIP analysis of chromatin from C57BL/6 CD4+ T cells (left unstimulated (Naive) or stimulated under TH1- or TH2-polarizing conditions) immunoprecipitated with antibody to H3K9ac and H3K14ac, anti-H3K4me3, anti-H3K9me3 or anti-H3K27me3, then analyzed by real-time PCR of DNA from immunoprecipitated chromatin components or an equivalent mass of input DNA (purified from chromatin fractions before immunoprecipitation). Results are presented as the ratio of 2−CT(IP) to 2−CT(input), where 'IP' is immunoprecipitated chromatin and 'CT' indicates the cycling threshold. () ICS analysis of IL-4 and IFN-γ (top) and ELISA of IL-4 production (bottom) in naive CD4+ T cells isolated from mice with deletion (KO) of CGRE, CNS-1, HS2, 3′ UTR, HS4 or CNS-2 (above plots) and their wild-type littermates (WT), then stimulated for 7 d under TH2-polarizing conditions and then restimulated with monoclonal antibody (mAb) to TCRβ. Numbers in or below quadrants indicate percent cells in each. ! *P < 0.05 (Student's t-test). Data are representive of three independent experiments with similar results (error bars (), s.d.). * Figure 2: Antibody and asthmatic airway responses in HS2-deficient mice. () ELISA of total and OVA-specific serum IgG1, IgG2c and IgE in HS2-deficient mice (HS2-KO; n = 6) and their wild-type control littermates (HS2-WT; n = 8) immunized twice with OVA in alum and then boosted with OVA in PBS, assessed on days 0, 7 and 14 after immunization. () Airway pressure in the mice in , given inhaled OVA by the aerosol route after boosting and then treated with various concentrations (horizontal axis) of acetylcholine (ACh). () Hematoxylin and eosin staining of bronchial tissue sections from the mice in . Original magnification, ×100. () Cell content in bronchoalveolar lavage fluid isolated from the challenged mice in , assessed by microscopy after Wright-Giemsa staining. Tot, total cells; Mac; macrophage; Neu, neutrophil; Eos, eosinophil; CD4+ Lym, lymphocyte. *P < 0.05 (Student's t-test). Data are representative of three experiments (error bars (,,), s.d.). * Figure 3: HS2 is critical for the primary production of IL-4 by MP T cells and NKT cells. () ICS analysis of IFN-γ- and IL-4-producing cells among wild-type and HS2-deficient CD4+ T cells stimulated for 7 d under TH0 conditions, then restimulated for 6 h with mAb to TCRβ. Numbers in or below quadrants indicate percent cells in each. () ELISA of cytokine production by CD44hiCD62Llo (MP) and CD44loCD62Lhi (Naive) wild-type and HS2-deficient T cells stimulated for 48 h with mAb to TCRβ and mAb to CD28. () ELISA of IL-4, IL-13 and IFN-γ in splenic wild-type and HS2-deficient NKT cells stimulated for 48 h with α-galactosylceramide (10 ng/ml). *P < 0.05 (Student's t-test). Data are representative of three independent experiments (error bars (,), s.d.). * Figure 4: The HS2 enhancer regulates IL-4 production in TH2 cells. () ICS analysis of IL-4, IL-13 and IFN-γ in naive wild-type and HS2-deficient CD4+ T cells stimulated for 7 d under TH0- or TH2-polarizing conditions, then restimulated with mAb to TCRβ. Numbers in or below quadrants indicate percent cells in each. () ELISA of cytokines in the cells in at 24 h after restimulation. *P < 0.05 (Student's t-test). () ICS analysis of IL-4 and IL-13 in naive CGRE–wild-type (CGRE-WT) and CGRE-deficient (CGRE-KO) CD4+ T cells stimulated for 7 d under TH2-polarizing conditions, then restimulated with mAb to TCRβ. Numbers in or below quadrants (left) indicate percent cells in each. *P < 0.05 (Student's t-test). () ELISA of IL-4, IL-13 and IL-5 in naive CNS-2-deficient (CNS-2-KO) and control C57BL/6 (CNS-2-WT) CD4+ T cells stimulated for 7 d under TH2-polarizing conditions, then restimulated with mAb to TCRβ. *P < 0.05 (Student's t-test). Data are representative of three independent experiments with similar results (), three independent experimen! ts () or four experiments with four mice per group (,; error bars (–), s.d.). * Figure 5: The HS2 enhancer is required for GATA-3-mediated production of IL-4. () Production of IL-4 and IFN-γ in green fluorescent protein–positive (GFP+) cells among CD4+ T cells derived from wild-type mice (HS2-WT) and mice with heterozygous (HS2-HET) or homozygous (HS2-KO) deletion of HS2, then transduced with retroviral vector encoding GFP alone or GFP and GATA-3, cultured for 7 d under TH1-polarizing conditions and restimulated with mAb to TCRβ. () ELISA of cytokine production by cells transduced and restimulated as in . () Production of IL-4, IL-13 and IFN-γ in GFP+ cells among CD4+ T cells derived from wild-type and CGRE-deficient mice, then transduced for 7 d with retrovirus as in under TH1-polarizing conditions and then restimulated with mAb to TCRβ. Numbers in or below quadrants (,) indicate percent cells in each. Data are representative of three independent experiments with similar results (,) or are from three independent experiments (; error bars, s.d.). * Figure 6: Histone H3 modification and binding of GATA-3 to the Il13 and Il4 loci. () ChIP analysis of chromatin extracts obtained from C57BL/6 naive T cells and TH2 cells, immunoprecipitated with anti-GATA-3–conjugated Sepharose beads and analyzed by real-time PCR as in Figure 1a. () ChIP analysis of C57BL/6 naive T cells stimulated for 0 h, 24 h, 48 h, 72 h and 1 week under TH2 conditions, then collected for immunoprecipitation with anti-GATA-3. () ChIP analysis of C57BL/6 naive T cells stimulated for 24, 48 or 72 h under TH1- or TH2-polarizing conditions, followed by detection of H3K9ac-H3K14ac and H3K4me3 with the primers at top (Il13 and Il4 loci). Data are representative of three (,) or two () independent experiments (error bars (,), s.d.). * Figure 7: HS2-deficient T cells have a defect in histone acetylation and methylation. () ChIP plus microarray analysis of the binding of H3K9ac-H3K14ac and GATA-3 to regions in chromosome11 (53238000–53359000) including the Il4 and Il13 loci, in chromatin extracts obtained from wild-type and HS2-deficient TH2 cells, presented as the logarithmic ratio of the hybridization intensity of H3K9ac-H3K14ac or GATA-3 to that of the control, with colors assigned on the basis of the false-discovery rate score: red, ≤0.05; orange, 0.05 ≤ 0.1; yellow, 0.1 ≤ 0.2; gray, >0.2. Data are representative of genome-wide ChIP-plus-microarray analysis. () ChIP analysis of wild-type and HS2-deficient naive T cells stimulated for 24 h, 48 h, 72 h or 1 week under TH2 conditions, followed by detection of H3K9ac-H3K14ac and H3K4me3 with various primer sets (horizontal axis). Data are representative of three independent experiments. () ChIP analysis of H3K4me3 in wild-type and CGRE-deficient TH2 cells, detected with the primers above (in the Il13 and Il4 loci). Data are represent! ative of three experiments. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Shinya Tanaka & * Yasutaka Motomura Affiliations * Laboratory for Signal Network, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan. * Shinya Tanaka, * Yasutaka Motomura, * Yoshie Suzuki, * Ryoji Yagi & * Masato Kubo * Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. * Hiromasa Inoue * Cytokine Project, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. * Shoichiro Miyatake * Research Institute for Biological Sciences, Tokyo University of Science, Chiba, Japan. * Masato Kubo Contributions S.T. built the initial constructs, generated mouse lines and confirmed mouse lines in vivo; Y.M. did ChIP analysis; Y.S. screened mouse lines; R.Y. built the initial constructs; H.I. did airway hyper-responsiveness experiments; S.M. distributed materials; and M.K. designed experiments, supervised the project and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Masato Kubo Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (392K) Supplementary Figures 1–3 and Supplementary Table 1 Additional data - Control of the differentiation of regulatory T cells and TH17 cells by the DNA-binding inhibitor Id3
- Nat Immunol 12(1):86-95 (2011)
Nature Immunology | Article Control of the differentiation of regulatory T cells and TH17 cells by the DNA-binding inhibitor Id3 * Takashi Maruyama1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Li1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jose P Vaque2 Search for this author in: * NPG journals * PubMed * Google Scholar * Joanne E Konkel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Weifeng Wang3 Search for this author in: * NPG journals * PubMed * Google Scholar * Baojun Zhang4 Search for this author in: * NPG journals * PubMed * Google Scholar * Pin Zhang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brian F Zamarron1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dongyang Yu3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuntao Wu3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuan Zhuang4 Search for this author in: * NPG journals * PubMed * Google Scholar * J Silvio Gutkind2 Search for this author in: * NPG journals * PubMed * Google Scholar * WanJun Chen1 Contact WanJun Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:86–95Year published:(2011)DOI:doi:10.1038/ni.1965Received01 September 2010Accepted04 November 2010Published online05 December 2010 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 The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4+ T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3+ regulatory T cells (Treg cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3−/− CD4+ T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3−/− T cells showed greater differentiation into the TH17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of TH17 cells. View full text Figures at a glance * Figure 1: Id3 regulates the generation of Foxp3+ Treg cells. (,) Flow cytometry of CD4+CD8− T cells in the thymus (Thy) and spleen (Spl) of 3-week-old wild-type (Id3+/+) and Id3−/− mice. Numbers in quadrants indicate percent Foxp3+CD25− cells (top left) or Foxp3+CD25+ cells (top right; ), or percent Foxp3+Helios− cells (top left) or Foxp3+Helios+ cells (top right; ). (,) Frequency () and total number () of CD4+Foxp3+ Treg cells in the mice in ,. () Proliferation of wild-type CD4+CD25− responder T cells cultured together at various ratios (horizontal axis) with Id3+/+ or Id3−/− Treg cells (key), assessed as uptake of [3H]thymidine. () Flow cytometry of gated CD4+CD8− CD45.1− T cells in the thymus and spleen of Rag1−/− recipient mice 4 weeks after transfer of Id3+/+ or Id3−/− (CD45.2+) bone marrow mixed at a ratio of 1:2 with C57BL/6 (CD45.1+) bone marrow. Numbers in quadrants indicate percent Foxp3+Helios− cells (top left) or Foxp3+Helios+ cells (top right). (,) Frequency () and total number () of CD4+ Fox! p3+ T cells in the mice in . *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of more than three experiments with four mice per group (), three experiments (), three experiments with seven mice (,; mean and s.d.), four independent experiments (; mean ± s.d. of triplicate wells), two experiments with five mice per group () or two experiments (,; mean and s.d.). * Figure 2: Id3−/− T cells fails to generate Foxp3+ Treg cells in response to TGF-β1. () Quantitative analysis of Foxp3 mRNA expression in naive CD4+CD25− T cells after TCR stimulation overnight in the presence (TGF-β1) or absence (Med) of TGF-β1, presented relative to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase). () Flow cytometry of naive CD4+CD25− T cells cultured for 3 d with TCR stimulation with or without TGF-β1. Numbers in plots indicate percent Foxp3+ Treg cells. () Frequency of Foxp3+ Treg cells among the cells in . *P < 0.01 (Student's t-test). (,) Flow cytometry of CD4+CD25− T cells cultured for 3–5 d with TGF-β1 alone (left; ,) or with TGF-β1 plus IL-2 (right; ) or retinoic acid (RA; right; ). Numbers in quadrants indicate percent CD25+Foxp3+ Treg cells. Data are from one experiment representative of at least five (; mean ± s.d. of duplicate wells) or at least two (,), one experiment representative of five () or five experiments (; mean ± s.d.). * Figure 3: Enrichment of E2A at the Foxp3 promoter. () E-protein–binding sites (E-boxes) at the Foxp3 promoter. () ChIP-coupled quantitative PCR analysis of the enrichment of E2A at E-boxes at various positions along Foxp3 (horizontal axis) in CD4+CD25− T cells after TCR stimulation and treatment with TGF-β1, assessed with antibody to E47 (anti-E47) and presented relative to results obtained with control IgG. () ChIP-coupled quantitative PCR analysis of the enrichment of E2A in the region between positions +327 and +513 of the Foxp3 promoter in wild-type CD4+CD25− T cells 12–24 h after TCR stimulation with TGF-β1, assessed as in and presented relative to results obtained without TGF-β1 (Med), set as 1. () ChIP-coupled quantitative PCR analysis of the binding of E2A and TBP to the Foxp3 promoter in wild-type CD4+CD25− T cells as described in , presented as the results obtained with anti-E47 or anti-TBP relative to those obtained with control IgG. () ChIP-coupled quantitative PCR analysis of E2A enrichment as in in! purified CD4+CD25+ Treg cells (CD25+), presented relative to results obtained with purified CD4+CD25− Treg cells (CD25−), set as 1. *P < 0.01 (Student's t-test). Data are representative of four experiments (; error bars, s.d.), four independent experiments (; mean and s.d.) or one experiment representative of two (,; mean and s.d. of duplicate () or triplicate () wells). * Figure 4: Binding of E2A to the Foxp3 promoter is required for activation of Foxp3 by TGF-β1. () Foxp3 mRNA expression in EL4 LAF cells treated with E2A-specific short hairpin RNA (shRNA) or scrambled (control) shRNA (Scram), assessed 12 h after activation with anti-CD3 and anti-CD28, with or without TGF-β1, presented relative to Hprt1 expression. Inset, immunoblot analysis of E47 and GAPDH (glyceraldehyde phosphate dehydrogenase; loading control). () Luciferase assay of TGF-β1-induced Foxp3 activity (right) in EL4 LAF cells with wild-type (WT) or mutated (Mut) Foxp3 E-boxes (constructs, left), assessed after TCR stimulation with or without TGF-β1 and presented relative to. (,) Flow cytometry of splenic CD4+ T cells () and frequency of CD4+Foxp3+ Treg cells among splenic CD4+CD25− T cells () obtained from E2af/fHebf/fEr-Cre+ mice treated with sunflower seed oil (control (Ctrl)) or tamoxifen (Tamox) and cultured for 3 d with anti-CD3 plus anti-CD28, and TGF-β1 (0.2 ng/ml). Numbers in plots () indicate percent Foxp3+CD25+ cells; each symbol () represents an indiv! idual mouse. () Foxp3 mRNA (right) in naive CD4+CD25− T cells treated with E2A-specific siRNA (siRNA) or scrambled siRNA (Scram), assessed after TCR stimulation with or without TGF-β1 and presented relative to Hprt1 expression. Left, E2a mRNA in cells treated with E2A-specific or scrambled siRNA; results are presented relative to Hprt1 expression. () Enrichment of E2A at the Foxp3 promoter in Id3−/− CD4+CD25− T cells with or without TGF-β1. NS, not significant; *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of three experiments (; mean and s.d.), two independent experiments (,), two experiments (; mean and s.d. of four samples) or three independent experiments (; mean and s.d.) or are from one experiment representative of three (; mean ± s.d. of triplicate measurements). * Figure 5: Id3 deficiency increases GATA-3. () Gata3 mRNA expression in naive CD4+CD25− T cells 12 h after activation of the TCR and treatment with TGF-β1 alone or TGF-β1 and anti-IL-4 (α-IL-4), presented relative to Hprt1 expression. () Flow cytometry of CD4+CD25− T cells at 24 h after activation of the TCR and treatment with various combinations of TGF-β1 and anti-IL-4 (above plots). () Il4 mRNA in CD4+CD25− T cells at 2 h after activation and treatment (horizontal axis), presented relative to Hprt1 expression. () Flow cytometry of Tgfbr1f/fCd4-Cre+ and Tgfbr1f/+Cd4-Cre+ CD4+CD25− T cells cultured for 24 h (treatment, above plots). () Il4 mRNA expression in wild-type naive CD4+CD25− T cells treated with E2A-specific or scrambled siRNA, presented relative to Hprt1 expression. *P < 0.05 (Student's t-test). () Binding of GATA-3 to the Foxp3 promoter in Id3+/+ or Id3−/− CD4+CD25− T cells cultured for 24 h (treatment, horizontal axis), assessed with anti-GATA-3 and presented relative to results obtain! ed with control IgG. () Enrichment of E2A at the Foxp3 promoter in Id3+/+ or Id3−/− CD4+CD25− T cells cultured for 24 h with TCR simulation and treatment with TGF-β1 and anti-IL-4, presented relative to Hprt1 expression. Numbers in quadrants (,) indicate percent Foxp3+GATA-3− cells (top left), Foxp3+GATA-3+ cells (top right) or Foxp3−GATA3+ cells (bottom right). Data are from one experiment representative of at least three (–) or two (–); mean and s.d. of duplicate (,,,) or triplicate ()) measurements. * Figure 6: Id3−/− CD4+ T cells differentiate into TH17 cells in response to TGF-β1 in vitro. () Flow cytometry of intracellular IL-17 in CD4+ T cells from the lamina propia (LP) and Peyer's patches (PP) of 7- to 8-week-old Id3+/+ and Id3−/− mice. Numbers in plots (left) indicate percent IL-17+ cells; numbers below plots (right) indicate mean fluorescence intensity (M; bracketed lines above) of IL-17+ cells among gated CD4+ T cells. () Absolute number of IL-17+CD4+ cells in the mice in . () Flow cytometry of intracellular IL-17 in naive CD4+CD25− T cells obtained from mice 3–5 weeks of age and cultured for 3–5 d with TCR stimulation plus various combinations of TGF-β1 and IL-6 (above plots). Numbers in quadrants indicate percent IL-17+CD4+ cells. () Enzyme-linked immunosorbent assay of IL-17 in supernatants of CD4+CD25− T cells cultured for 72 h as in . () IL-17 production in naive CD4+ T cells cultured with TCR stimulation alone (Med) or with the addition of TGF-β1 alone or with TGF-β1 and anti-IL-4. () Quantitative RT-PCR analysis of Rorc mRNA in CD4! +CD25− T cells 48 h after activation of the TCR and treatment with various combinations of TGF-β1 and IL-6 (horizontal axis), presented relative to Hprt1 expression. *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three independent experiments with three mice (,; mean and s.d.), one experiment representative of more than five (), four independent experiments (; except IL-6 treatment (two experiments); mean ± s.d.) or two independent experiments (; mean ± s.d. of four samples), or are from one of three independent experiments (; mean ± s.d. of duplicate wells). * Figure 7: Id3 regulates TH17 cells in HDM-induced asthma. () Mucus in the airways and inflammation in the lungs of Id3+/+ and Id3−/− mice, assessed by periodic acid Schiff staining. Inset, enlargement of area indicated by yellow asterisk. Scale bars, 1,000 μm (main images) or 250 μm (insets). () Inflammatory cells in BAL fluid 4 d after the second of two intratracheal challenges of Id3+/+ and Id3−/− mice with HDM. PMN, polymorphonuclear neutrophils; Bas, basophils; Eos, eosinophils; Mac, macrophages; Lymph, lymphocytes. (,) Enzyme-linked immunosorbent assay of IgE in BAL fluid () or plasma () of Id3+/+ and Id3−/− mice. (–) Flow cytometry of the intracellular expression of IL-17 and IL-13 (,) or of IFN-γ and IL-4 (,) in CD4+ T cells in BAL fluid of Id3+/+ and Id3−/− mice. (,) Flow cytometry of intracellular Foxp3 and surface CD25 in gated CD4+ T cells from Id3+/+ and Id3−/− mice. Numbers in quadrants (,,) indicate percent cells in each; results at right indicate frequency of cytokine-positive cells (,) or CD! 4+Foxp3+ cells () among CD4+ T cells. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of three independent experiments with nine to ten mice per group (); four mice per group (; mean ± s.d.); four () or six to nine () mice per group (,; mean ± s.d.); ten Id3+/+ mice or nine Id3−/− mice (–; mean ± s.d. in ,); or nine to ten mice per group (,; mean ± s.d. in ). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Takashi Maruyama & * Jun Li Affiliations * Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA. * Takashi Maruyama, * Jun Li, * Joanne E Konkel, * Pin Zhang, * Brian F Zamarron & * WanJun Chen * Oral Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA. * Jose P Vaque & * J Silvio Gutkind * Department of Microbiology and Molecular Biology, George Mason University, Manassas, Virginia, USA. * Weifeng Wang, * Dongyang Yu & * Yuntao Wu * Department of Immunology, Duke University, Durham, North Carolina, USA. * Baojun Zhang & * Yuan Zhuang Contributions T.M. and J.L. designed and did experiments, analyzed data and contributed to the writing of the manuscript; J.P.V. designed and did ChIP experiments, analyzed data and contributed to the writing of the manuscript; J.E.K. did experiments, analyzed data and contributed to the writing of the manuscript; Y.W. designed and W.W. and D.Y. did the luciferase, E2A-knockdown and Id3 immunoblot experiments and analyzed data; B.Z. and Y.Z. generated and identified mice doubly deficient in E2A and HEB and provided critical input; P.Z. and B.F.Z. did experiments; J.S.G. supervised and designed the ChIP study and contributed to the writing of the manuscript; and W.C. conceived of the research, directed the study, designed experiments, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * WanJun Chen Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–12 Additional data - T-bet represses TH17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt
- Nat Immunol 12(1):96-104 (2011)
Nature Immunology | Article T-bet represses TH17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt * Vanja Lazarevic1 Contact Vanja Lazarevic Search for this author in: * NPG journals * PubMed * Google Scholar * Xi Chen1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jae-Hyuck Shim1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Eun-Sook Hwang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Eunjung Jang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandra N Bolm1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mohamed Oukka3 Search for this author in: * NPG journals * PubMed * Google Scholar * Vijay K Kuchroo4 Search for this author in: * NPG journals * PubMed * Google Scholar * Laurie H Glimcher1, 5 Contact Laurie H Glimcher Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:96–104Year published:(2011)DOI:doi:10.1038/ni.1969Received01 June 2010Accepted12 November 2010Published online12 December 2010 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 Overactive responses by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) are tightly linked to the development of autoimmunity, yet the factors that negatively regulate the differentiation of this lineage remain unknown. Here we report that the transcription factor T-bet suppressed development of the TH17 cell lineage by inhibiting transcription of Rorc (which encodes the transcription factor RORγt). T-bet interacted with the transcription factor Runx1, and this interaction blocked Runx1-mediated transactivation of Rorc. T-bet Tyr304 was required for formation of the T-bet–Runx1 complex, for blockade of Runx1 activity and for inhibition of the TH17 differentiation program. Our data reinforce the idea of master regulators that shape immune responses by simultaneously activating one genetic program while silencing the activity of competing regulators in a common progenitor cell. View full text Figures at a glance * Figure 1: T-bet deficiency promotes IL-17A production in vitro independently of IFN-γ. () Flow cytometry of Tbx21−/−, Ifng−/− and wild-type (WT) CD4+ T cells cultured for 5 d in the presence of IL-2 (TH0), IL-2, IL-12 and antibody to IL-4 (anti-IL-4; TH1), or TGF-β, IL-6, anti-IL-4 and anti-IFN-γ with (TH17 + IL-23) or without (TH17) subsequent IL-23 treatment, then stimulated for 4 h with PMA and ionomycin, followed by intracellular cytokine staining with anti-IL-17A and anti-IFN-γ. Numbers in plots indicate percent IL-17A+IFN-γ− cells (top left) or IL-17A−IFN-γ+ cells (bottom right). () Enzyme-linked immunosorbent assay (ELISA) of IL-17A in supernatants of cells differentiated and stimulated as in . () RT-PCR analysis of Rorc mRNA expression in Tbx21−/− and wild-type cells differentiated and stimulated as in ; results are presented in relative units (RU) relative to the expression of the housekeeping gene Hprt1. Data are representative of four independent experiments (,) or are from two independent experiments (; mean and s.e.m. in ,). * Figure 2: T-bet deficiency promotes TH17 responses in the CNS during EAE. () Production of IL-17A and IFN-γ by CNS-infiltrating CD4+ lymphocytes on day 17 after immunization of Tbx21−/− and wild-type mice with MOG(35–55) plus CFA. Numbers in plots indicate percent positive cells in the CD4+ gate. () Quantification of IL-17A- and IFN-γ-producing CD4+ cells in the CNS of Tbx21−/− and wild-type mice on day 17 after immunization as in . () ELISA of IL-17A and IFN-γ in purified CNS-infiltrating CD4+ T cells, normalized to 1 × 106 cells per ml. () Flow cytometry of cells from the lymph nodes (LN), spleen (Spl) and CNS of Tbx21−/− and wild-type IL-23R.GFP mice on day 17 after EAE induction as in . Numbers in plots (above) indicate percent CD4+IL-23R.GFP+ cells (left group) or CD4+CCR6+ cells (right group) in the CD4+ gate (mean and s.e.m.); each symbol (below) represents an individual mouse and small horizontal lines indicate the mean. () Expression of genes encoding TH17 signature cytokines in CD4+ T cells purified from the CNS of Tbx2! 1−/− and wild-type mice on day 17 after immunization as in , then stimulated for 4 h with PMA and ionomycin before RNA extraction (cells pooled from four mice per group); results are presented relative to Hprt1 expression. *P < 0.05, **P < 0.01 and ***P < 0.0001 (Student's t-test). Data are representative of three independent experiments () or are from three independent experiments (–; mean and s.e.m.) with three to four mice per group in each. * Figure 3: T-bet expression in naive helper T cell precursors and fully differentiated TH17 cells inhibits the TH17 response. () Flow cytometry of the expression of IL-17A and IFN-γ by naive CD4+ T cells transduced with empty retrovirus expressing GFP alone (EV-RV) or retrovirus expressing T-bet and GFP (T-bet–RV) under TH17-polarizing conditions, then stimulated for 4 h with PMA and ionomycin, followed by intracellular cytokine staining of sorted GFP+ cells. () Real-time PCR analysis of Rorc, Il23r, Il17a, Il17f, Il21 and Il22 mRNA in naive CD4+ T cells transduced with empty or T-bet-expressing retrovirus under TH17-polarizing conditions. () Expression of Tbx21 and Rorc mRNA in cells obtained from Tbx21−/− mice transgenic for T-bet expression induced in response to doxycycline treatment, left untreated (–) or treated (+) with doxycycline (Dox), then activated under TH17-polarizing conditions in the presence (+) or absence (–) of anti-IFN-γ (α-IFN-γ). () Immunoblot analysis (IB) of T-bet and RORγt in the cells in , probed with anti-RORγt (α-RORγt) or anti-T-bet (α-T-bet). () Flow! cytometry analysis of the expression of IL-17A and IFN-γ by fully differentiated TH17 cells transduced with empty or T-bet-expressing retrovirus under TH17-polarizing conditions, then stimulated for 4 h with PMA and ionomycin, followed by intracellular cytokine staining. () Real-time PCR analysis of genes encoding TH17 signature cytokines in fully differentiated TH17 cells transduced with empty or T-bet-expressing retrovirus in the presence of TH17-polarizing cytokines. Numbers in plots (,) indicate percent positive cells in each quadrant; gene or mRNA results (,,) are presented relative to Hprt1 expression (,) or expression of the housekeeping gene Actb (). Data are representative of three independent experiments (mean and s.e.m. in ,,). * Figure 4: T-bet blocks Runx1-mediated transactivation of the Rorc promoter. (,) ChIP analysis of the binding of T-bet to the Rorc promoter (numbers along horizontal axis correspond to Rorc at bottom left) in wild-type (WT (anti-T-bet)) and Tbx21−/− (anti-T-bet) TH0 cells () and differentiated TH1 cells () after 6 h of stimulation with PMA and ionomycin, as well as in preimmune serum (WT (mock)); the Ifng and Il4 promoters (right) serve as positive and negative controls, respectively. Results are presented relative to input DNA. () Luciferase activity in HEK293 cells transfected with empty or Rorc firefly luciferase reporter (Rorc-luc; constructed from a 2-kb fragment of the mouse Rorc promoter), plus renilla luciferase reporter, along with increasing concentrations of Runx1 (wedges) in the presence or absence of T-bet; activity was normalized to that of renilla luciferase for transfection efficiency and is presented relative to that of cells transfected with empty vector, set as 1. () Luciferase activity in HEK293 cells transfected with the Rorc! luciferase reporter and Runx1 in the presence or absence of increasing concentrations of T-bet (wedge), presented as in . Data are representative of three experiments (,; mean ± s.e.m.) or three () and two () independent experiments (mean and s.e.m. of duplicate samples). * Figure 5: Interaction of T-bet with Runx1. () Immunoprecipitation (IP; with anti-T-bet) of lysates of HEK293 cells transfected with various combinations of empty vector or expression vector for T-bet or Myc-tagged Runx1, Runx2 or Runx3 (above blot), followed by immunoblot analysis (IB) with anti-Myc or anti-T-bet. () Expression of Runx1 and T-bet protein in Tbx21−/− and wild-type TH0, TH1 and TH17 cells after 6 h of stimulation with PMA and ionomycin. () Immunoprecipitation (with control immunoglobulin G (IgG), anti-Runx1 or anti-T-bet) of lysates of Tbx21−/− and wild-type TH0 cells, followed by immunoblot analysis immunoprecipitates (top) or input protein (bottom) with anti-T-bet or anti-Runx1. IgH, additional loading control. () DNA-precipitation assay (DNA precip) and immunoblot analysis of HEK293 cells transfected with oligonucleotide containing wild-type Runx1- and T-bet-binding sites (WT oligo) or a mutated Runx1-binding site (R-mt oligo) or T-bet-binding site (T-mt oligo) at a site 2 kb upstream of Ror! c exon 1, in the presence of Myc-tagged Runx1 or T-bet. Below, oligonucleotide sequence; underlining indicates binding sites. () DNA-precipitation assay and immunoblot analysis (top two blots) of HEK293 cells transfected with oligonucleotide with a wild-type or mutated Runx1-binding site (as in ), plus Myc-tagged Runx1, in the presence or absence of increasing doses of T-bet (wedge; 0.1, 0.5 and 1 μg). Bottom two blots, immunoblot analysis of input DNA (samples without precipitation). Data are representative of one to two independent experiments. * Figure 6: Runx1 overexpression restores IL-17A production in TH17 cells expressing T-bet. () Flow cytometry of CD4+ T cells transduced with various combinations of retrovirus expressing GFP, Thy-1.1, Runx1-GFP or T-bet–Thy-1.1 (above plots) within 24 h of activation, then cultured for 5 d under TH17-polarizing conditions and stimulated for 4 h with PMA and ionomycin before intracellular cytokine staining for IL-17A. Numbers adjacent to outlined areas indicate percent IL-17A-producing cells in the GFP+Thy-1.1+ gate. FSC, forward scatter. () Production of IL-17A and expression of Il17a and Rorc by CD4+ T cells transduced with various combinations of retroviruses as in , then differentiated for 5 d under TH17-polarizing conditions, followed by sorting of GFP+Thy-1.1+ cells and stimulation for 4 h with PMA and ionomycin. () Flow cytometry of activated CD4+ T cells transduced with various combinations of retrovirus expressing GFP, Thy-1.1, RORγt-GFP or T-bet–Thy-1.1 (above plots), then cultured for 5 d under TH17 conditions and stimulated for 4 h with PMA and ion! omycin before intracellular cytokine staining for IL-17A. () ELISA of IL-17A production and RT-PCR analysis of Il17a and Rorc mRNA transcripts of cells transduced under TH17 conditions as described in . Numbers adjacent to outlined areas (,) indicate percent IL-17A-producing cells in the GFP+Thy-1.1+ gate; gene or mRNA expression (,) is presented relative to Hprt1 expression. Data are representative of two independent experiments (error bars (,), s.e.m.). * Figure 7: T-bet Tyr304 is essential for the interaction of T-bet with Runx1 and for inhibition of the TH17 differentiation program. () Interaction of Myc-tagged Runx1 with wild-type T-bet or T-bet point mutants (above lanes) in coimmunoprecipitation experiments in HEK293 cells. () Luciferase activity in HEK293 cells transfected with the Rorc luciferase reporter construct (as in Fig. 4c) in the presence or absence of Runx1, wild-type T-bet or T-bet(Y304F) or T-bet(Y525F) (below graph), assessed as in Figure 4c. () Flow cytometry analysis of the expression of IL-17A and IFN-γ by naive CD4+ T cells transduced with control (empty) retrovirus (EV-RV) or retrovirus encoding wild-type T-bet (T-bet–RV), T-bet(Y304F) (T-bet(Y304F)–RV) or T-bet(Y525F) (T-bet(Y525F)–RV) under TH17-polarizing conditions. Numbers in plots indicate percent IL-17A+IFN-γ− cells (top left) or IL-17A−IFN-γ+ cells (bottom right) in the CD4+ gate. () RT-PCR analysis of genes encoding TH17 signature cytokines by CD4+ T cells transduced with as in ; results are presented relative to Hprt1 expression. () DNA-precipitation assay an! d immunoblot analysis of HEK293 cells transfected with wild-type T-bet or T-bet(Y304F) in the presence of oligonucleotide containing a wild-type or mutated T-bet-binding site (as in Fig. 5d). () DNA-precipitation assay and immunoblot analysis of HEK293 cells transfected with Myc-tagged Runx1, wild-type T-bet or T-bet(Y304F) in the presence of oligonucleotide containing wild-type or mutated Runx1- or T-bet-binding sites (as in Fig. 5d), probed with anti-Myc and anti-T-bet. Data are representative of three independent experiments (–; mean and s.e.m. in , or one experiment (,). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Xi Chen & * Jae-Hyuck Shim Affiliations * Harvard School of Public Health, Department of Immunology and Infectious Diseases, Boston, Massachusetts, USA. * Vanja Lazarevic, * Xi Chen, * Jae-Hyuck Shim, * Alexandra N Bolm & * Laurie H Glimcher * College of Pharmacy, Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, South Korea. * Eun-Sook Hwang & * Eunjung Jang * Department of Pediatrics, Seattle Children's Research Institute, Seattle, Washington, USA. * Mohamed Oukka * Center for Neurologic Diseases, Brigham & Women's Hospital, Boston, Massachusetts, USA. * Vijay K Kuchroo * Department of Medicine, Harvard Medical School, and the Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts, USA. * Laurie H Glimcher Contributions V.L. designed and did experiments and prepared the manuscript; X.C. did ChIP assays; J.-H.S. did DNA-precipitation and coimmunoprecipitation assays; E.-S.H. created T-bet mutant retroviral constructs; E.J. did doxycycline transgenic T cell experiments; M.O. generated IL-23R.GFP mice; V.K.K. contributed to discussions and manuscript preparation; A.N.B. provided technical assistance; and L.H.G. supervised the research, designed experiments and participated in preparing the manuscript. Competing financial interests L.H.G. is on the Board of Directors and holds equity in Bristol Myers Squibb. Corresponding authors Correspondence to: * Vanja Lazarevic or * Laurie H Glimcher Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–4 and Supplementary Tables 1–2 Additional data
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