Latest Articles Include:
- The lure of reviews
- Nat Immunol 12(10):915 (2011)
Nature Immunology | Editorial The lure of reviews Journal name:Nature ImmunologyVolume: 12,Page:915Year published:(2011)DOI:doi:10.1038/ni.2122Published online20 September 2011 The powers and perils of review articles for the design and publication of research. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg "Can you recommend a good review?" is a question every scientist will ask many times in his or her career. For trainees making their debut in research, for postdoctoral fellows starting new projects or for seasoned scientists following intriguing results into a new field, a review is the first step into the unknown. Review articles are also the ultimate resource for staying updated. In a world in which huge amounts of information are constantly being generated, it is difficult to monitor every primary research article published, especially in fields that are not of immediate interest. Most scientists depend on syntheses from their peers to keep track of progress in such areas and, of course, read reviews published in their own field. Beyond delivering a synthesis of the most recent advances, a review is an attempt to organize new and old data sets into conceptual frameworks that make sense biologically and evolutionarily. Written by experts in the field, reviews reflect ! the personal views and interpretation of the authors on the present state of things. Everyone appreciates a good synthesis and—why not—a peek into the brains of their mentors and colleagues. When commissioning review articles, the editors of Nature Immunology advise authors to provide fresh scientific insight and a novel synthesis of the data so it is obvious to the reader which directions will most probably bear fruit in the near future. New ideas and hypotheses for research are born from seeing the big picture and from the data as an ensemble, not from disparate, singular results. A good review opens new avenues of research by formulating questions and suggesting directions for future studies. It is no surprise, then, that review articles have become very popular. Journals such as Cell, Immunity, The Journal of Immunology, The European Journal of Immunology, Mucosal Immunology and Nature Immunology publish at least one review article per issue. These are usually commissioned directly by the journal from leaders in the field and are intended to cover topics considered of high interest for the journal's audience. Because they are highly cited (on average, a review article is cited almost twice as often as a research paper), they help boost the impact factor of the journal. Thus, both the scientific community and journals seem to benefit from the publication of reviews. Occasionally, there are complaints about a perceived inflation of reviews, especially in trendy areas in which the progress made in the field does not justify the number of published reviews (for example, does it seem that regulatory T cells and TH17 cells have been over-reviewed?). However, a well-written, coherent piece with the right mixture of data synthesis, data integration and informed guesses can constitute a highly read, highly cited and highly influential article. Some reviews are so influential, in fact, that sometimes they become a source of information as important as primary research articles. Occasionally, Nature Immunology receives submissions that experimentally dismiss predictions of models proposed in highly cited review articles. The dismissal of such predictions is considered sufficiently important to be of interest to a large audience. Although such studies do not challenge but instead reproduce the original data that generated the model, they do offer a different interpretation of those data. Depending on the case, the editors may choose to reject such manuscripts. Models, no matter how fitting they may seem at a certain point or how influential they may become, are simply interpretations; they are influenced by the state of knowledge in the field and, to a certain degree, by the authors' ability to analyze and interpret data. By trying to integrate the data into working models, reviews can oversimplify, omit contradictor! y data sets or suggest implications that go beyond the more cautious interpretations of the original primary research articles. In contrast, if the assays are done correctly, the raw data are infallible and can fit into radically different models. The difference is made by the additional data sets that build context. From this perspective, when assessing novelty, the editors of Nature Immunology seek original data that will help build a new context in which old and new data can be successfully integrated. This does not mean that results that disprove various predictions or corollaries of current models should not be published. Disproving theories is an essential part of the entire process of research and discovery. However, from case to case, it may not be considered a sufficiently large step forward in the understanding of a certain process. The baseline for assessing novelty should always be the primary data (that is, has this been shown before?) rather than a certain interpretation made of the data (that is, has this been suggested before?). Reviews can be great guides, tools or engaging intellectual exercises. However, because models are born and refitted constantly, primary data should always be the main point of reference. In the end, no one reads an old review. Additional data - Baruj Benacerraf 1920–2011
- Nat Immunol 12(10):917 (2011)
Article preview View full access options Nature Immunology | Obituary Baruj Benacerraf 1920–2011 * Kenneth L Rock1Journal name:Nature ImmunologyVolume: 12,Page:917Year published:(2011)DOI:doi:10.1038/ni.2110Published online20 September 2011 The scientific community mourns the loss of Baruj Benacerraf, who died recently at the age of 90. He had a long and storied career, to which we owe much as a field. Baruj Benacerraf was born in Venezuela to Spanish Moroccan and Algerian parents. When he was 5 years of age, his family moved to Paris, France, where he received most of his primary education. The original plan was that Baruj would go into law and assist the family business in Venezuela, but then Hitler intervened. His family heard the distant drumbeat of war, and as they were Jews in Paris, his father had the foresight to move the family back to Venezuela in advance of Fall Rot, the Nazi invasion of France. As his schooling had been unexpectedly interrupted, Baruj was sent to the United States to complete his education. It was after his transfer to Columbia University that his interest in science was kindled and he decided to apply to medical school, rather than pursuing law. However, he had not appreciated how difficult a goal this would be for someone of his ethnic background at that point in history. Rejected from all the medical schools to which he had applied, he gaine! d last-minute admission to the Medical College of Virginia only through the intervention of a friend's father. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Affiliations * Kenneth L. Rock is at the University of Massachusetts Medical School, Worcester, Massachusetts, USA. Author Details * Kenneth L Rock Search for this author in: * NPG journals * PubMed * Google Scholar - ChIP-Seq: technical considerations for obtaining high-quality data
- Nat Immunol 12(10):918-922 (2011)
Nature Immunology | Commentary ChIP-Seq: technical considerations for obtaining high-quality data * Benjamin L Kidder1 * Gangqing Hu1 * Keji Zhao1 * Affiliations * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:918–922Year published:(2011)DOI:doi:10.1038/ni.2117Published online20 September 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Chromatin immunoprecipitation followed by next-generation sequencing analysis (ChIP-Seq) is a powerful method with which to investigate the genome-wide distribution of chromatin-binding proteins and histone modifications in any genome with a known sequence. The application of this technique to a variety of developmental and differentiation systems has provided global views of the cis-regulatory elements, transcription factor function and epigenetic processes involved in the control of gene transcription. Here we describe several technical aspects of the ChIP-Seq assay that diminish bias and background noise and allow the consistent generation of high-quality data. View full text Figures at a glance * Figure 1: ChIP-Seq experimental design. ChIP-Seq is a powerful tool with which to investigate protein-DNA interactions on a global scale. It is important that the appropriate controls for antibody specificity be determined before ChIP-Seq is begun. After isolation of the ideal number of cells, chromatin is sheared into an ideal size range by sonication or enzymatic means (micrococcal nuclease (MNase)). Next, high-quality antibodies are used for ChIP to enrich for factor-occupied DNA sequences. After purification of ChIP-enriched DNA, a library is constructed to allow sequencing on next-generation sequencing (NGS) platforms. Library construction typically includes end-repair, the addition of single adenosine residues, adaptor ligation and PCR with primers compatible with the sequencing platform. After cluster generation, single- or paired-end sequencing is performed on next-generation sequencing platforms. RNAi, RNA-mediated interference; bp, base pairs. * Figure 2: Common procedures for ChIP-seq data analysis. After base-calling, short-read sequences are aligned to a reference genome. Data quality is assessed by a combination of various strategies, such as visual inspection with a genome browser, motif identification and confirmation by ChIP and quantitative PCR. The initial inspection or prior knowledge provides information about whether the peaks are broad or sharp or both. Various algorithms (bottom right) have been developed for the identification of peaks of these three groups. Author information * Abstract * Author information Affiliations * Benjamin L. Kidder, Gangqing Hu and Keji Zhao are in the Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Benjamin L Kidder or * Keji Zhao Author Details * Benjamin L Kidder Contact Benjamin L Kidder Search for this author in: * NPG journals * PubMed * Google Scholar * Gangqing Hu Search for this author in: * NPG journals * PubMed * Google Scholar * Keji Zhao Contact Keji Zhao Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - ASCertaining cytoskeletal rearrangements in antigen presentation and migration
- Nat Immunol 12(10):923-925 (2011)
Article preview View full access options Nature Immunology | News and Views ASCertaining cytoskeletal rearrangements in antigen presentation and migration * Ioannis Karakasiliotis1 * Dimitris L Kontoyiannis1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:923–925Year published:(2011)DOI:doi:10.1038/ni.2114Published online20 September 2011 ASC has emerged as an adaptor for inflammasome sensors in cells of the innate immune response. New inflammasome-independent roles have been identified for ASC in the control of adaptive immunity; these include the post-transcriptional regulation of cytoskeletal rearrangements. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Ioannis Karakasiliotis and Dimitris L. Kontoyiannis are with the Institute of Immunology, Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dimitris L Kontoyiannis Author Details * Ioannis Karakasiliotis Search for this author in: * NPG journals * PubMed * Google Scholar * Dimitris L Kontoyiannis Contact Dimitris L Kontoyiannis Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Damage control: how HIV survives the editor APOBEC3G
- Nat Immunol 12(10):925-927 (2011)
Article preview View full access options Nature Immunology | News and Views Damage control: how HIV survives the editor APOBEC3G * J Ludovic Croxford1 * Stephan Gasser1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:925–927Year published:(2011)DOI:doi:10.1038/ni.2115Published online20 September 2011 The antiviral factor APOBEC3G upregulates the expression of ligands for the activating receptor NKG2D via DNA damage induced by the viral protein Vpr in cells infected with human immunodeficiency virus. The virus overcomes greater susceptibility to natural killer cell–mediated lysis by targeting APOBEC3G for degradation. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * J. Ludovic Croxford and Stephan Gasser are with the Immunology Programme, Department of Microbiology, National University of Singapore, Singapore. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephan Gasser Author Details * J Ludovic Croxford Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Gasser Contact Stephan Gasser Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Peli1 (rel)ieves autoimmunity
- Nat Immunol 12(10):927-929 (2011)
Article preview View full access options Nature Immunology | News and Views Peli1 (rel)ieves autoimmunity * Paul N Moynagh1Journal name:Nature ImmunologyVolume: 12,Pages:927–929Year published:(2011)DOI:doi:10.1038/ni.2108Published online20 September 2011 T cell tolerance is essential to the prevention of autoimmunity. The ubiquitin E3 ligase Peli1 acts as a negative regulator of T cell activation and contributes to the maintenance of self-tolerance. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Paul N. Moynagh is with the Institute of Immunology, National University of Ireland Maynooth, Maynooth, Ireland. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Paul N Moynagh Author Details * Paul N Moynagh Contact Paul N Moynagh Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - STING-dependent signaling
- Nat Immunol 12(10):929-930 (2011)
Article preview View full access options Nature Immunology | News and Views STING-dependent signaling * Glen N Barber1Journal name:Nature ImmunologyVolume: 12,Pages:929–930Year published:(2011)DOI:doi:10.1038/ni.2118Published online20 September 2011 The sensing of pathogen-associated DNA in the cytoplasm is an important trigger of host-defense responses that include the production of type I interferon. A new study suggests that the DExDc helicase DDX41 may function in dendritic cells as a DNA sensor to activate STING-dependent innate immune responses. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Glen N. Barber is in the Department of Cell Biology, University of Miami School of Medicine, Miami, Florida, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Glen N Barber Author Details * Glen N Barber Contact Glen N Barber Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Malignant pirates of the immune system
- Nat Immunol 12(10):933-940 (2011)
Nature Immunology | Review Malignant pirates of the immune system * Lixin Rui1 * Roland Schmitz1 * Michele Ceribelli1 * Louis M Staudt1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:933–940Year published:(2011)DOI:doi:10.1038/ni.2094Published online20 September 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg At great human cost, cancer is the largest genetic experiment ever conducted. This review highlights how lymphoid malignancies have genetically perverted normal immune signaling and regulatory mechanisms for their selfish oncogenic goals of unlimited proliferation, perpetual survival and evasion of the immune response. View full text Figures at a glance * Figure 1: Origin of human lymphoid malignancies. The later stages of B cell differentiation give rise to a variety of cancer subtypes. Bcl-6, SPIB, IRF4 and Blimp-1 are key transcription factors with differences in expression during normal B cell differentiation. Plasmacytic differentiation is initiated when IRF4 is activated, which causes upregulation of Blimp-1 and downregulation of Bcl-6. Bcl-6 forms a double negative regulatory loop with Blimp-1 such that an initial drop in Bcl-6 expression will swing the cell toward Blimp-1 expression, thereby reinforcing plasmacytic differentiation. Each cancer subtype 'inherits' the expression of key transcription factors from its normal counterpart and dies when these transcription factors are inactivated, an example of non-oncogene addiction. ABC DLBCL is initiated by various genetic lesions that activate NF-κB, thereby inducing IRF4 expression and plasmacytic differentiation. Also required are genetic lesions that inactivate Blimp-1, which results in the arrest of differentiatio! n at the plasmablast stage. Recurrent genetic alterations and oncogenic pathways characteristic of each subtype of lymphoid malignancy are also presented. Amp, amplification; Chr, chromosome, * Figure 2: Model of the genesis of ABC DLBCL from an autoreactive B cell. * Figure 3: Oncogenic MYD88 mutations in human lymphomas and leukemias activate multiple downstream signaling pathways. 'P' in a red circle indicates phosphorylation. IL-6R, receptor for IL-6; gp130, signal transducer; IL-10Rα and IL-10Rβ, subunits of the receptor for IL-10; p38, mitogen-activated protein kinase (MAPK); IFN-β, interferon-β; IFNAR1 and IFNAR2, receptors for interferon; Tyk2, tyrosine kinase. * Figure 4: Model for the pathogenesis of PMBL and Hodgkin lymphoma. Autocrine IL-13 signaling activates Jak2, which translocates to the nucleus to modify chromatin and enhance gene expression. In PMBL and Hodgkin lymphoma, amplification of an interval on chromosome band 9p24 upregulates the expression of the genes encoding Jak2, JMJD2C, PD-L1 and PD-L2. Jak2 and JMJD2C act together to diminish heterochromatin and increase gene expression by preventing the recruitment of HP1α. Some of the targets of Jak2 epigenetic regulation that participate in positive feedback loops that amplify the response are included here. γc, common γ-chain; IL-13RA1 and IL-4R, interleukin receptors. Author information * Abstract * Author information Affiliations * Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. * Lixin Rui, * Roland Schmitz, * Michele Ceribelli & * Louis M Staudt Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Louis M Staudt Author Details * Lixin Rui Search for this author in: * NPG journals * PubMed * Google Scholar * Roland Schmitz Search for this author in: * NPG journals * PubMed * Google Scholar * Michele Ceribelli Search for this author in: * NPG journals * PubMed * Google Scholar * Louis M Staudt Contact Louis M Staudt Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - IL-22 bridges the lymphotoxin pathway with the maintenance of colonic lymphoid structures during infection with Citrobacter rodentium
- Nat Immunol 12(10):941-948 (2011)
Nature Immunology | Article IL-22 bridges the lymphotoxin pathway with the maintenance of colonic lymphoid structures during infection with Citrobacter rodentium * Naruhisa Ota1, 4 * Kit Wong1, 4 * Patricia A Valdez1, 3, 4 * Yan Zheng1, 3 * Natasha K Crellin1, 3 * Lauri Diehl2 * Wenjun Ouyang1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:941–948Year published:(2011)DOI:doi:10.1038/ni.2089Received01 June 2011Accepted15 July 2011Published online28 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Colonic patches (CLPs) and isolated lymphoid follicles (ILFs) are two main lymphoid structures in the colon. Lymphoid tissue–inducer cells (LTi cells) are indispensable for the development of ILFs. LTi cells also produce interleukin 17 (IL-17) and IL-22, signature cytokines secreted by IL-17-producing helper T cells. Here we report that IL-22 acted downstream of the lymphotoxin pathway and regulated the organization and maintenance of mature CLPs and ILFs in the colon during infection with Citrobacter rodentium. Lymphotoxin (LTα1β2) regulated the production of IL-22 during infection with C. rodentium, but the lymphotoxin-like protein LIGHT did not. IL-22 signaling was sufficient to restore the organization of CLPs and ILFs and host defense against infection with C. rodentium in mice lacking lymphotoxin signals, which suggests that IL-22 connects the lymphotoxin pathway to mucosal epithelial defense mechanisms. View full text Figures at a glance * Figure 1: Blockade of lymphotoxin signaling affects CLP and ILF structure in colons from mice infected with C. rodentium. () Portion of whole-mount colon stained by immunofluorescence for CD4 (green) and B220 (red), imaged at 10 d after infection with C. rodentium (top). Arrowheads indicate CLPs; arrows indicate ILFs. Below, enlargement of outlined area above, showing an ILF (by IHC) and a CLP. Scale bars, 1 mm (top row and bottom right) or 100 μm (bottom left). () Size of CLPs 0–16 d after infection with C. rodentium (n = 5 mice). () Follicles in colons from mice treated with control protein (Ctrl) or LTβR-Fc, imaged on day 10 after infection with C. rodentium. () Images of whole colons from . Arrowheads indicate CLPs. Original magnification, ×5 () or ×1 (). () Quantification of normal and aberrant follicles with a dissecting microscope (n = 4 mice). () Immunofluorescence images of portions of whole-mount colon tissues from mice treated with control protein or LTβR-Fc (left) assessed at 10 d after infection, stained for CD4 (green) and B220 (red). Right, enlargement of outlined area at ! left. Arrowheads indicate CLPs; arrow indicates ILF. Scale bars, 1 mm. () Immunofluorescence images of CLPs in colon sections from mice treated as in , stained for CD11c (green), CD4 (red) and B220 (blue). Scale bar, 100 μm. () Quantification of normal CLPs from the mice in by whole-mount immunofluorescence staining (n = 5 mice). () Quantification of normal CLPs from the mice in by IHC (n = 4 mice). () Quantification of normal ILFs per colon, as in . *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of at least two independent experiments (error bars (,,–), s.e.m.). * Figure 2: Blockade of the lymphotoxin pathway inhibits IL-22 expression in colons of mice infected with C. rodentium. (,) Survival () and weight loss () of wild-type mice (n = 5 per group) infected with C. rodentium and treated with control protein, LTβR-Fc or anti-IL-22. () Il22 mRNA expression in colons of mice left uninfected (Uninf) or infected (Inf) as in , (n = 15 per group), assessed on day 4 after infection; results are presented relative to those of uninfected mice. () Enzyme-linked immunosorbent assay of IL-22 in supernatants of colons of mice treated as in (n = 15 per group), collected on day 4 after infection and cultured for 48 h. () Il22 mRNA expression in the colons of the mice in , (n = 2 per group) at 0–8 d after infection; results are presented relative to those of uninfected mice. () Expression of Il22 mRNA in colons from wild-type mice (WT), LTβ-deficient mice (Ltb−/−) and LIGHT-deficient mice (Tnfsf14−/−; called 'Lgt−/−' here) on day 4 after infection with C. rodentium (n = 6 mice per group); results are presented relative to those of wild-type mice. *P ! < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of at least two independent experiments (error bars, s.e.m.). * Figure 3: Exogenous IL-23 restores IL-22 induction and host defense during infection with C. rodentium when the lymphotoxin pathway is blocked. (–) Leukocytes (), ILCs (), NKp46+ cells (), LTi cells () and DCs () in the colon lamina propria of mice (n = 5 per group) left uninfected or infected with C. rodentium and treated with control protein or LTβR-Fc, assessed on day 4 after infection. () Enzyme-linked immunosorbent assay of IL-22 in supernatants of colons collected from mice (n = 5 per group) on day 4 after treatment as in – and cultured for 48 h in the presence (+) or absence (−) of IL-23 (10 ng/ml). (,) Weight loss () and survival () of mice (n = 5 per group) given hydrodynamic injection of IL-23-expressing or control plasmid into the tail vein, then infected with C. rodentium the next day and treated with control protein or LTβR-Fc. *P < 0.05 (Student's t-test). Data are representative of at least two independent experiments (error bars, s.e.m.). * Figure 4: Normal lymphoid structure development in IL-22-deficient mice. () Ileal cryptopatches from naive wild-type and Il22−/− mice, stained for CD11c (blue), the IL-7 receptor α-chain (IL-7Rα; green), the cell surface marker c-Kit (orange) and B220 (magenta). Scale bar, 10 μm. () Quantification of cryptopatches (CPs) per area of ileum in naive wild-type and Il22−/− mice (n = 3 per group). () Immunofluorescence images of CLPs in whole-mount colon samples from uninfected wild-type and Il22−/− mice, stained for CD4 (green) and B220 (red). Scale bar, 1 mm. () Quantification of CLPs in (n = 3 per group). () Immunofluorescence images of ILFs in IHC colon sections as in . Scale bar, 100 μm. () Quantification of ILFs in (n = 3 per group). Data are representative of at least two independent experiments (error bars (,,), s.e.m.). * Figure 5: IL-22 blockade disrupts the normal organization of CLPs and ILFs during infection with C. rodentium. () Whole colons from mice infected with C. rodentium and treated with control protein or anti-IL-22, imaged on day 10 after infection. Arrowheads indicate CLPs. () Quantification of CLPs in mice treated as in (n = 4 per group), assessed with a dissecting microscope. () Quantification of normal CLPs and ILFs in mice treated as in (n = 5 per group), by whole-mount immunostaining. () Quantification of normal CLPs in mice treated as in (n = 4 per group), by IHC. () Brightfield image of the entire colon of mice treated as in (left), and immunofluorescence image (top right) of the area outlined in red at left, stained for CD4 (green) and B220 (red): arrowheads indicate CLPs, and arrows indicate ILFs. Bottom right, enlargement of CLP outlined in white above. Scale bars, 1 mm. () Immunofluorescence images of CLPs from colon sections of mice treated as in , stained for CD11c (green), CD4 (red) and B220 (blue). Scale bar, 100 μm. () Quantification of DC, T cell and B cell populations! isolated from colon lamina propria of mice treated as in (n = 5 per group), assessed on day 10 after infection. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of at least two independent experiments (error bars (,,,), s.e.m.). * Figure 6: IL-22–Fc rescues mice treated with LTβR-Fc during infection with C. rodentium. (,) Survival () and weight loss () of mice (n = 12 per group) infected with C. rodentium and treated with control protein, LTβR-Fc alone or LTβR-Fc and IL-22–Fc. (,) Image () and length () of whole colons from mice treated as in , (n = 5 per group). (,) Quantification of bacterial burden in the spleen () and liver () of mice treated as in , (n = 5 per group). CFU, colony-forming units. () Colon histology of mice treated as in ,, showing epithelial proliferation, enterocyte shedding into the gut lumen (arrows), bacterial colonies on gut surface and in colon crypts (arrowheads). Scale bar, 100 μm. () Clinical scores of mice treated as in , (n = 4 per group), determined by histological analysis. *P < 0.01 and **P < 0.001 (Student's t-test). Data are representative of at least two independent experiments (error bars (,–,), s.e.m.). * Figure 7: IL-22–Fc restores the structure of CLPs and ILFs in colons of mice treated LTβR-Fc during infection with C. rodentium. () Brightfield image of the entire colon of mice infected with C. rodentium and treated with control protein, LTβR-Fc alone or LTβR-Fc and IL-22–Fc, assessed on day 10 after infection (left), and immunofluorescence image (top right) of the area outlined in red at left, stained for CD4 (green) and B220 (red); arrowheads indicate CLPs, and arrows indicate ILFs. Bottom right, enlargement of area outlined in white above. Scale bars, 1 mm. () Quantification of normal CLPs of mice treated as in (n = 5 per group), by whole-mount immunofluorescence staining. () CLPs in colon sections from mice treated as in , stained for CD11c (green), CD4 (red) and B220 (blue). Scale bar, 100 μm. () Quantification of ILFs as in . (,) Weight loss () and survival () of Rag2−/−Il2rg−/− mice (n = 10 per group) either left uninfected or infected with C. rodentium and treated with control protein or IL-22–Fc. *P < 0.05 (Student's t-test). Data are representative of at least two independent! experiments (error bars (,,), s.e.m.). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Naruhisa Ota, * Kit Wong & * Patricia A Valdez Affiliations * Department of Immunology, Genentech, South San Francisco, California, USA. * Naruhisa Ota, * Kit Wong, * Patricia A Valdez, * Yan Zheng, * Natasha K Crellin & * Wenjun Ouyang * Department of Pathology, Genentech, South San Francisco, California, USA. * Lauri Diehl * Present addresses: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA (P.A.V.), Target Discovery & Validation, Novo Nordisk Inflammation Research Center, Seattle, Washington, USA (Y.Z.) and Centers for Therapeutic Innovation–San Francisco, Pfizer, San Francisco, California, USA (N.K.C.). * Patricia A Valdez, * Yan Zheng & * Natasha K Crellin Contributions N.O., K.W. and P.A.V. did most of experiments and analyzed the data; Y.Z. contributed to Figure 2; L.D. analyzed the histological results in Figure 6g,h; N.K.C. contributed to Supplementary Figure 3; W.O. devised and planned the project; and W.O., N.O., K.W. and P.A.V. wrote the manuscript. Competing financial interests All authors are employees of Genentech. Corresponding author Correspondence to: * Wenjun Ouyang Author Details * Naruhisa Ota Search for this author in: * NPG journals * PubMed * Google Scholar * Kit Wong Search for this author in: * NPG journals * PubMed * Google Scholar * Patricia A Valdez Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Zheng Search for this author in: * NPG journals * PubMed * Google Scholar * Natasha K Crellin Search for this author in: * NPG journals * PubMed * Google Scholar * Lauri Diehl Search for this author in: * NPG journals * PubMed * Google Scholar * Wenjun Ouyang Contact Wenjun Ouyang Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (872K) Supplementary Figures 1–10 and Table 1 Additional data - Notch signaling is necessary for adult, but not fetal, development of RORγt+ innate lymphoid cells
- Nat Immunol 12(10):949-958 (2011)
Nature Immunology | Article Notch signaling is necessary for adult, but not fetal, development of RORγt+ innate lymphoid cells * Cécilie Possot1, 2, 3 * Sandrine Schmutz1, 3 * Sylvestre Chea1, 2, 3 * Laurent Boucontet1, 3 * Anne Louise4 * Ana Cumano1, 3 * Rachel Golub1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:949–958Year published:(2011)DOI:doi:10.1038/ni.2105Received03 May 2011Accepted15 August 2011Published online11 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The transcription factor RORγt is required for the development of several innate lymphoid populations, such as lymphoid tissue–inducer cells (LTi cells) and cells that secrete interleukin 17 (IL-17) or IL-22. The progenitor cells as well as the developmental stages that lead to the emergence of RORγt+ innate lymphoid cells (ILCs) remain undefined. Here we identify the chemokine receptor CXCR6 as an additional marker of the development of ILCs and show that common lymphoid progenitors lost B cell and T cell potential as they successively acquired expression of the integrin α4β7 and CXCR6. Whereas fetal RORγt+ cells matured in the fetal liver environment, adult bone marrow–derived RORγt+ ILCs matured outside the bone marrow, in a Notch2-dependent manner. Therefore, fetal and adult environments influence the differentiation of RORγt+ cells differently. View full text Figures at a glance * Figure 1: Fetal liver RORγt+ cells differentiate from CLPs. () Fractionation of fetal liver CLPs from Cxcr6GFP/+ embryos at embryonic day 15.5 (E15.5) according expression of α4β7, Flt3, CXCR6 and RORγt (left), and quantitative RT-PCR analysis of Rorc expression (far right) in RORγt− and RORγt+ CLPs (top) or CXCR6− and CXCR6+ CLPs (bottom), presented in arbitrary units (AU) relative to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase). ND, not detectable (<0.01 relative expression). Numbers adjacent to outlined areas indicate percent cells in each. () Differentiation potential of α4β7−Flt3− and α4β7−Flt3+ fetal liver CLPs cultured for 12 d on OP9 or OP9-DL4 stroma (50 cells per well) with IL-7, IL-2 and the ligands for c-Kit and Flt3; below, absolute cell number. Numbers in quadrants indicate percent cells in each throughout. () Differentiation potential of single α4β7−Flt3− and α4β7−Flt3+ fetal liver CLPs cultured on OP9 stroma (310 wells) or single CXCR6− fetal liver C! LPs cultured on OP9-DL4 stroma (190 wells), presented as the frequency of wells containing each population (left), and combination of lineage potential obtained from clonal assays, showing the presence (gray boxes) or absence (white boxes) of each lineage potential (right) and the frequency of wells with each combination of lineage potential (far right margin). () Expression of NKp46, CD19, CD3 and RORγt on the progeny of 1 × 104 Ly5.2+α4β7−Flt3− or α4β7−Flt3+ CLPs (CD45.2+) injected into sublethally irradiated Ly5.1+Rag2−/−Il2rg−/− host mice, assessed in bone marrow (BM), spleen and lamina propria (LP) 2 weeks after injection. Data are representative of three (,) or five () experiments or three experiments with two mice each (; mean ± s.d.). * Figure 2: Expression of α4β7 and CXCR6 defines two steps during differentiation into RORγt+ cells. () Differentiation potential of α4β7−CXCR6−, α4β7+CXCR6− and CXCR6+ fetal liver CLPs from Cxcr6GFP/+ embryos at E15.5, plated on OP9 or OP9-DL4 stroma (20 cells per well) and cultured for 12 d as in Figure 1b. () Quantitative RT-PCR analysis of the expression of various transcripts in α4β7−CXCR6−, α4β7+CXCR6− and CXCR6+ fetal liver CLPs, presented relative to Hprt1 expression. *P < 0.05 and **P < 0.01 (unilateral Student's t-test). () Analysis of genomic DJH rearrangements (bottom) in Flt3− (1) and Flt3+ (2) LSK cells, α4β7−CXCR6− (3), α4β7+CXCR6− (4) and CXCR6+ (5) CLPs (Lin−Sca1loc-KitintIL-7Rα+), and Lin+ cells (6) from Cxcr6GFP/+ fetal liver at E15.5. kb, kilobases. Data are representative of three experiments with at least 12 wells each (), three experiments (; mean ± s.d.) or two experiments (). * Figure 3: Both progenitors and mature cells migrate from the fetal liver to the periphery. () Differentiation potential of α4β7+Flt3−IL-7Rαhi fetal liver CLPs from C57BL/6 embryos at E15.5, plated on OP9 or OP9-DL4 stroma (10 cells per well) and cultured for 12 d as in Figure 1b. () Quantitative RT-PCR analysis of the expression of various transcripts in CXCR6− and CXCR6+IL-7Rαhi fetal liver CLPs and Lin−CXCR6+IL-7Rα+ fetal spleen cells from Cxcr6GFP/+ embryos at E15.5, left unstimulated (Lta and Ltb, which encode lymphotoxin-α and lymphotoxin-β, respectively) or stimulated with the phorbol ester PMA and ionomycin (Il17a, Il17f and Il22); results are presented relative to the expression of Gapdh (encoding glyceraldehyde phosphate dehydrogenase). *P < 0.05 (unilateral Student's t-test). () Fractionation of Lin−IL-7Rα+c-KitintSca-1lo cells according the expression of α4β7, CXCR6 and RORγt in spleen, mesenteric lymph nodes (mLN) and blood from E15.5 Cxcr6GFP/+ embryos. Numbers adjacent to outlined areas (left) indicate percent cells in each; number! s in plots (far right) indicate percent RORγt+ cells. () Expression of various receptors in C57BL/6 RORγt− and RORγt+ fetal liver CLPs at E15.5. () Expression of IL-7Rα, NKp46, CD19 and RORγt on the progeny of 2 × 103 Ly5.2+α4β7−Flt3− or α4β7−Flt3+ (CD45.2+) CLPs seeded on irradiated Ly5.1+ fetal liver explants and cultured for 6 d. Number in outlined area (top) indicates percent cells. Data are representative of three experiments with at least 24 wells each (), three experiments (– mean ± s.d.) or three experiments with two FLOCs each (). * Figure 4: Bone marrow CLPs are the progenitors of adult RORγt+ cells. () Expression of RORγt (far right) in bone marrow LSK cells and CLPs (Lin−IL-7Rα+c-KitloSca-1int) from 10-week-old Rag2−/− mice (left, sorting of cells). Numbers adjacent to outlined areas (left) indicate percent cells in each. () Fractionation of bone marrow CLPs from 10-week-old Cxcr6GFP/+ mice according cell surface expression of α4β7 and CXCR6 (far right). Numbers adjacent to outlined areas indicate percent cells in each. () Quantitative RT-PCR analysis of Rorc expression in CXCR6− and CXCR6+ bone marrow CLPs and CXCR6+ fetal liver CLPs at E15.5, presented relative to Hprt1 expression. () Differentiation potential of Rag2−/− bone marrow CLPs plated on OP9 or OP9-DL4 stroma (50 cells per well) and cultured for 12 d as in Figure 1b (left) and absolute cell number for each population (right). () Expression of NKp46, CD19, CD3 and RORγt on the progeny of 1 × 104 Ly5.2+Rag2−/− (CD45.2+) bone marrow CLPs injected into sublethally irradiated Ly5.1+Rag2−/! −Il2rg−/− host mice, assessed in bone marrow, spleen and lamina propria 2 weeks after injection. Numbers adjacent to outlined areas (left) indicate percent cells in each. Data are representative of five experiments (), three experiments (), three experiments (; mean ± s.d.), five experiments with at least 24 wells each (; mean ± s.d.) or three experiments with two mice each (). * Figure 5: The expression of α4β7 and CXCR6 defines bone marrow CLP subsets. () Differentiation potential of α4β7−CXCR6−, α4β7+CXCR6− and CXCR6+ bone marrow CLPs from 10-week-old Cxcr6GFP/+ mice plated on OP9 or OP9-DL4 stroma (20 cells per well) and cultured for 12 d as in Figure 1b (above) and absolute cell number for each population (below). () Quantitative RT-PCR analysis of various transcripts in α4β7−CXCR6−, α4β7+CXCR6− and CXCR6+ bone marrow CLPs, presented relative to Hprt1 expression. *P < 0.05 and **P < 0.01 (unilateral Student's t-test). Data are representative of three experiments with at least 12 wells each () or three experiments (; mean ± s.d.). * Figure 6: Peripheral CLP-like populations are the source of adult RORγt+ cells. () Fractionation of Lin−Sca-1loc-KitintIL-7Rα+ cells according their expression of CXCR6 and RORγt (right) in the spleen and lamina propria of 10-week-old Cxcr6GFP/+ mice. Numbers adjacent to outlined areas (left) indicate percent cells in each. () Quantitative RT-PCR analysis of Rorc expression in CXCR6− and CXCR6+ CLP-like (Lin−Sca-1loc-KitintIL-7Rα+) cells from the spleen and lamina propria, presented relative to Hprt1 expression. () Fractionation of Lin−Sca-1loc-KitintIL-7Rα+ cells from the blood of 10-week-old Cxcr6GFP/+ mice according cell surface expression of α4β7 and CXCR6 (far right). Numbers adjacent to outlined areas (left) indicate percent cells in each. () Differentiation potential of Lin−IL-7Rα+c-KitintSca1lo cells from the spleen and the lamina propria of Cxcr6GFP/+ mice, plated on OP9 or OP9-DL4 stroma (20 cells per well) and cultured for 12 d as in Figure 1b. Data are representative of five experiments (,), three experiments (; mean ± s.d.! ) or three experiments with at least 24 wells each (). * Figure 7: The Notch pathway is required for the differentiation of adult RORγt+ cells. () Differentiation potential of Rag2−/− Lin−IL-7Rα+c-KitintSca-1lo bone marrow cells plated on OP9 or OP9-DL4 stroma (50 cells per well) and cultured for 12 d as in Figure 1b with (+) or without (−) DAPT. () Differentiation potential of Cxcr6GFP/+ Lin−Sca-1loc-KitintIL-7Rα+ bone marrow cells plated on OP9 or OP9-DL4 stroma (50 cells per well) and cultured as in . () Quantitative RT-PCR analysis of the expression of Hes1 and Notch2 in CXCR6− and CXCR6+ Lin−Sca-1loc-KitintIL-7Rα+ cells from the spleen and the lamina propria and in α4β7−CXCR6−, α4β7+CXCR6− and CXCR6+ fetal liver (FL) and bone marrow CLPs; results are presented relative to Hprt1 expression. *P < 0.05 (unilateral Student's t-test). () Expression of NKp46, CD19 and RORγt on the progeny of 2 × 103 Ly5.2+Rag2−/− bone marrow CLPs or 2 × 103 Ly5.2+ C57BL/6 (CD45.2+) fetal liver CLPs seeded on irradiated Ly5.1+ fetal liver explants and cultured for 6 d with or without DAPT. Data are re! presentative of three experiments with at least 24 wells each (,), three experiments (; mean ± s.d.) or three experiments with two FLOCs each (). Author information * Abstract * Author information * Supplementary information Affiliations * Institut Pasteur, Unité de Lymphopoièse, Paris, France. * Cécilie Possot, * Sandrine Schmutz, * Sylvestre Chea, * Laurent Boucontet, * Ana Cumano & * Rachel Golub * L'Université Paris Diderot, Sorbonne Paris Cité (Cellule Pasteur), Paris, France. * Cécilie Possot, * Sylvestre Chea & * Rachel Golub * Institut National de la Santé et de la Recherche Médicale U668, Paris, France. * Cécilie Possot, * Sandrine Schmutz, * Sylvestre Chea, * Laurent Boucontet, * Ana Cumano & * Rachel Golub * Institut Pasteur, Plate Forme de Cytometrie, Paris, France. * Anne Louise Contributions C.P. and R.G. designed the experiments; C.P. and S.S. did the experiments with assistance from L.B. for quantitative RT-PCR, S.C. for supplementary figures, and A.L. for cell sorting; and C.P. and R.G. analyzed the data and wrote the manuscript with contributions from A.C. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rachel Golub Author Details * Cécilie Possot Search for this author in: * NPG journals * PubMed * Google Scholar * Sandrine Schmutz Search for this author in: * NPG journals * PubMed * Google Scholar * Sylvestre Chea Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Boucontet Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Louise Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Cumano Search for this author in: * NPG journals * PubMed * Google Scholar * Rachel Golub Contact Rachel Golub Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–13 Additional data - The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells
- Nat Immunol 12(10):959-965 (2011)
Nature Immunology | Article The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells * Zhiqiang Zhang1 * Bin Yuan1 * Musheng Bao1 * Ning Lu1 * Taeil Kim1 * Yong-Jun Liu1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:959–965Year published:(2011)DOI:doi:10.1038/ni.2091Received15 June 2011Accepted19 July 2011Published online04 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The recognition of pathogenic DNA is important to the initiation of antiviral responses. Here we report the identification of DDX41, a member of the DEXDc family of helicases, as an intracellular DNA sensor in myeloid dendritic cells (mDCs). Knockdown of DDX41 expression by short hairpin RNA blocked the ability of mDCs to mount type I interferon and cytokine responses to DNA and DNA viruses. Overexpression of both DDX41 and the membrane-associated adaptor STING together had a synergistic effect in promoting Ifnb promoter activity. DDX41 bound both DNA and STING and localized together with STING in the cytosol. Knockdown of DDX41 expression blocked activation of the mitogen-activated protein kinase TBK1 and the transcription factors NF-κB and IRF3 by B-form DNA. Our results suggest that DDX41 is an additional DNA sensor that depends on STING to sense pathogenic DNA. View full text Figures at a glance * Figure 1: DDX41 senses cytosolic DNA in mDCs. () Immunoblot analysis of the knockdown efficiency of nontargeting scrambled shRNA (Control) or shRNA targeting mRNA encoding DDX41 (three shRNAs: X41-a, X41-b and X41-c), STING, RIG-I or IPS1 (above lanes) in D2SC mDCs; β-actin (bottom) serves as a loading control throughout. (–) ELISA of IFN-α and IFN-β in D2SC cells either treated with scrambled shRNA and left unstimulated (N-STM) or treated with scrambled shRNA (Control) or shRNA targeting mRNA encoding DDX41, STING, RIG-I or IPS1 (as in ; horizontal axis), then stimulated for 16 h with B-form DNA (1 μg/ml; ), Z-form DNA (1 μg/ml; ), poly(I:C) (2.5 μg/ml; ), HSV-1 (multiplicity of infection (MOI), 10; ) or influenza A virus (MOI, 10; ). Each symbol represents the result of one experiment; small horizontal lines indicate the average. () *P < 1.5 × 10−6 and **P < 3.5 × 10−5; () *P < 8.3 × 10−6 and **P < 2.8 × 10−5; () *P < 5.2 × 10−5 and **P < 9.5 × 10−5; () *P < 8.10 × 10−7 and **P < 6.0 × 10! −6; and () *P < 3.2 × 10−5 and **P < 2.0 × 10−4 (Student's t-test). Data are from at least three independent experiments. * Figure 2: Knockdown of DDX41 or STING in BMDCs abolishes their cytokine responses to DNA alone and DNA viruses. () Immunoblot analysis of the knockdown efficiency of shRNA (as in Fig. 1a) in BMDCs. (–) ELISA (as in Fig. 1a) of IFN-α and IFN-β in DCs either treated with scrambled shRNA and left unstimulated or treated with scrambled shRNA or shRNA (as in ), then stimulated for 16 h with B-form DNA (1 μg/ml; ), Z-form DNA (1 μg/ml; ), L. monocytogenes (20 colony-forming units; ), adenovirus (MOI, 10; ) or HSV-1 (MOI, 10; ). () *P < 8.3 × 10−7 and **P < 4.0 × 10−6; () *P < 6.0 × 10−6 and **P < 1.8 × 10−5; () *P < 5.7 × 10−5 and **P < 2.5 × 10−4; () *P < 4.4 × 10−4 and **P < 6.4 × 10−4; and () *P < 3.4 × 10−5 and **P < 1.9 × 10−4 (Student's t-test). Data are from at least three independent experiments. * Figure 3: Knockdown of DDX41 or STING in THP-1 cells abolishes their cytokine responses to DNA or HSV-1. () Immunoblot analysis of the knockdown efficiency of scrambled shRNA or shRNA targeting mRNA encoding DDX41 (two shRNAs: X41-a and X41-b), IFI16 (two shRNAs: IFI16-a and IFI16-b) or STING (above lanes ) in THP-1 cells. (–) ELISA (as in Fig. 1a) of IFN-β and IL-6 in THP-1 cells either treated with nontargeting scrambled shRNA and left unstimulated or treated with shRNA (as in ), then stimulated for 16 h with vaccinia virus (VACV) DNA (1 μg/ml; ), Z-form DNA (1 μg/ml; ), HSV DNA (1 μg/ml; ) or HSV-1 (MOI, 10; ). () *P < 4.0 × 10−6 and **P < 2.0 × 10−5; () *P < 3.6 × 10−5 and **P < 2.2 × 10−5; () *P < 9.3 × 10−5 and **P < 5.10 × 10−5; () *P < 7.0 × 10−5 and **P < 7.8 × 10−5 (Student's t-test). () ELISA of IFN-β (as in Fig. 1a; top) and immunoblot analysis of the expression of DDX41 and IFI16 (below) in THP-1 cells treated with nontargeting shRNA or shRNA targeting DDX41 or IFI16 and stimulated for 0–16 h with B-form DNA (1 μg/ml). GAPDH (glyce! raldehyde phosphate dehydrogenase) serves as a loading control (bottom). Data are from at least three independent experiments. * Figure 4: DDX41 interacts with DNA but not with RNA. () Immunoblot analysis of immunoprecipitation (IP) assays of purified HA-tagged DDX41, STING or DHX9 incubated with biotinylated B-form or Z-form DNA or poly(I:C), probed with anti-HA. () Immunoblot analysis of immunoprecipitation assays of purified HA-tagged full-length DDX41 (A) and serial truncations of DDX41 (B–E) incubated individually with biotinylated DNA, probed with anti-HA. Top, full-length and serial truncations of DDX41. DEADc, Asp-Glu-Ala-Asp motif; HELICc, helicase C-terminal domain; numbers indicate positions of amino acids. () Immunoblot analysis of nucleic acid–immunoprecipitation competition assays of increasing concentrations of DNA or poly(U) (0.5, 5 or 50 μg/ml; wedges) or no nucleic acid (−) added to a mixture of HA-tagged DDX41 plus biotinylated B-form DNA, probed with anti-HA. () Immunoblot analysis of immunoprecipitation assays of purified HA-tagged DDX41 incubated with avidin beads alone (No DNA) or with various biotinylated RNA or DNA substr! ates plus avidin beads, probed with anti-HA. Input, 10% of the purified HA-tagged DDX41; GC-25, GC-50 or GC-100, Z-form DNA 25, 50 or 100 nucleotides in length; GC, full-length Z-form DNA; VACV, vaccinia virus. () ELISA of IFN-α in D2SC cells treated with control shRNA (sh-control) or shRNA targeting DDX41 (sh-DDX41) or STING (sh-STING) and left unstimulated (N-STM) or stimulated with poly(dG:dC) of various lengths (as in ). () Immunoblot analysis of endogenous DDX41, p204 and RIG-I in D2SC cells incubated for 0 or 60 min with biotinylated B-form DNA. Input, 10% of the D2SC lysate. Data are representative of three independent experiments (mean and s.d. in ). * Figure 5: Recombinant DDX41 can 'rescue' the defect in DNA-activated production of interferon caused by knockdown of DDX41 via siRNA. () Immunoblot analysis of endogenous DDX41 in D2SC mDCs (Control) or of recombinant HA-tagged full-length or truncated DDX41 in D2SC mDCs with selective knockdown of endogenous DDX41 alone (siDDX41) or along with expression of full-length DDX41 (si+X41-a) or DDX41 with deletion of the DNA-binding domain (si+X41-c). () ELISA (as in Fig. 1a) of IFN-β in D2SC mDCs either left untreated and unstimulated (N-STM) or treated as in and stimulated for 16 h with B-form DNA (1 μg/ml), Z-form DNA (1 μg/ml) or poly(I:C) (2.5 μg/ml). Data are from at least three independent experiments. * Figure 6: Interaction of DDX41 with STING. () Immunoblot analysis of proteins (left margin) precipitated with anti-DDX41 or immunoglobulin G (IgG; control) from whole-cell lysates of D2SC cells left unstimulated (−) or stimulated with B-form DNA (+). Input, 10% of the D2SC cells lysate. () Immunoblot analysis of immunoprecipitation assays of purified HA-tagged STING incubated with Myc-tagged DDX41 or STING, probed with anti-Myc. () Immunoblot analysis of immunoprecipitation assays of purified HA-tagged full-length or truncated DDX41 (as in Fig. 4b) incubated with Myc-tagged STING, probed with anti-HA. () Immunoblot analysis of purified HA-tagged full-length STING (A) or truncated STING (B–F), probed with anti-HA (middle), and immunoblot analysis of immunoprecipitation assays of HA-tagged STING (as above) incubated with Myc-tagged DDX41, probed with anti-HA (bottom). Top, full-length STING (A) and serial truncations of STING (B–F); numbers indicate positions of amino acids. () Activation of the Ifnb promoter in ! mouse L929 cells transfected with an IFN-β luciferase reporter (IFN-β–Luc; 100 ng) plus increasing concentrations (20, 100 or 200 ng; wedges) of expression vectors for DDX41, IPS1 or STING individually; or expression vector for DDX41 (20 ng; solid bar) together with increasing concentrations (20, 100 or 200 ng; wedges) of expression vectors for IPS1 or STING. Results are presented relative to those of cells transfected with empty vector alone (Vector). Data are representative of three independent experiments (mean and s.d. in ). * Figure 7: The Walker motifs in DDX41 are essential for sensing DNA. () Full-length DDX41 (X41) and serial deletion mutants of DDX41 lacking the HELICc domain (X41-e), both the Walker A motif and the HELICc domain (X41-dA) or both the Walker B motif and the HELICc domain (X41-dM). () Immunoblot analysis of immunoprecipitation assays of purified HA-tagged DDX41 (as in ) incubated with biotinylated Z-form DNA, followed by addition of avidin beads (top), or with Myc-tagged STING, followed by the addition of anti-Myc beads (bottom), probed with anti-HA. Input, 10% of the purified HA-tagged DDX41. () Activation of the Ifnb promoter in L929 cells transfected with the IFN-β luciferase reporter (100 ng) plus increasing concentrations (20, 100 or 200 ng; wedges) of expression vectors for DDX41 (as in ); a renilla luciferase reporter (2 ng) was transfected simultaneously as an internal control. Results are presented relative to those of cells transfected with empty vector alone (Vector). Data are representative of three independent experiments (mean a! nd s.d. in ). * Figure 8: STING is the key adaptor for DDX41 signaling. () Confocal microscopy of HEK293T cells transfected with expression plasmid for HA-tagged STING, Myc-tagged DDX41 (X41) and/or Myc-tagged DDX41 with truncation of the C terminus (X41-c), then left unstimulated (top and third rows) or stimulated for 4 h with B-form DNA (second and bottom rows). Nuclei are stained with the DNA-intercalating dye DAPI; staining of calreticulin serves as a marker of the endoplasmic reticulum (ER). Original magnification, ×100. Data are representative of three independent experiments. () Immunoblot analysis of the fractionation of unstimulated D2SC cells (Mock) or D2SC cells stimulated with B-form DNA (1 μg/ml) or HSV-1 DNA (1 μg/ml), probed with anti-STING, anti-DDX41, anti-calreticulin (to detect the endoplasmic reticulum), anti-Sigma1R (to detect the mitochondria-associated ER membrane (MAM)) or anti-COXIV (to detect mitochondria (Mit)). Total, 15% of the D2SC lysate; Mic, microsome. Data are representative of three experiments. () Immunoblo! t analysis of phosphorylated (p-) and total Erk1/2, p38, Jnk, TBK1, IRF3 and p65 in lysates of BMDCs treated with shRNA (as in Fig. 4e) and stimulated for 0–120 min with B-form DNA (1 μg/ml). Data are representative of three experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Immunology, Center for Cancer Immunology Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. * Zhiqiang Zhang, * Bin Yuan, * Musheng Bao, * Ning Lu, * Taeil Kim & * Yong-Jun Liu Contributions Z.Z. designed and did most of experiments; B.Y., M.B., N.L. and T.K. helped with experiments; and Y.-J.L. designed the research and supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Yong-Jun Liu Author Details * Zhiqiang Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Bin Yuan Search for this author in: * NPG journals * PubMed * Google Scholar * Musheng Bao Search for this author in: * NPG journals * PubMed * Google Scholar * Ning Lu Search for this author in: * NPG journals * PubMed * Google Scholar * Taeil Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Yong-Jun Liu Contact Yong-Jun Liu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (556K) Supplementary Figures 1–7, Table 1 and Methods Additional data - Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria
- Nat Immunol 12(10):966-974 (2011)
Nature Immunology | Article Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria * Yuki Kinjo1, 2, 13 * Petr Illarionov3, 13 * José Luis Vela1, 13 * Bo Pei1 * Enrico Girardi4 * Xiangming Li5 * Yali Li4 * Masakazu Imamura6 * Yukihiro Kaneko2 * Akiko Okawara2 * Yoshitsugu Miyazaki2 * Anaximandro Gómez-Velasco7 * Paul Rogers8 * Samira Dahesh9 * Satoshi Uchiyama9 * Archana Khurana1 * Kazuyoshi Kawahara10 * Hasan Yesilkaya11 * Peter W Andrew11 * Chi-Huey Wong6 * Kazuyoshi Kawakami12 * Victor Nizet9 * Gurdyal S Besra3 * Moriya Tsuji5 * Dirk M Zajonc4 * Mitchell Kronenberg1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:966–974Year published:(2011)DOI:doi:10.1038/ni.2096Received21 December 2010Accepted27 July 2011Published online04 September 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria. View full text Figures at a glance * Figure 1: CD1d-dependent cytokine production by Vα14i NKT cells. () Expression of intracellular IFN-γ and IL-17 by CD19− lung mononuclear cells positive for α-GalCer-loaded CD1d tetramer, measured in uninfected mice (Control) or 13 h after intratracheal infection with S. pneumoniae (SPN; cells combined from at least five mice per condition). Isotype, isotype-matched control antibody. Numbers in quadrants indicate percent cells in each throughout. () Expression of intracellular IFN-γ by tetramer-positive CD19− spleen cells from an uninfected mouse (Control), a mouse 6 h after intravenous infection with S. pneumoniae (SPN; n = 3) or a mouse injected with α-GalCer 1.5 h before tissue collection (α-GC). (,) Expression of intracellular IFN-γ by tetramer-positive CD19− spleen cells from an uninfected mouse or a mouse injected with α-GalCer as in , and from mice treated with anti-CD1d (SPN anti-CD1d) or isotype-matched control antibody (SPN isotype) and infected intravenously with S. pneumoniae, assessed 6 h later (n = 3 per group).! MFI, mean fluorescence intensity. Numbers adjacent to outlined areas (,) indicate percent IFN-γ+TCRβ+ cells. () Enzyme-linked immunosorbent assay of IL-2 produced by Vα14i NKT cell hybridoma clones 1.2 and DN32 cultured with CD11c+ cells from spleens of mice injected with buffer containing Tween 20 vehicle (Veh), infected 16 h earlier with S. pneumoniae (SPN), injected 16 h earlier with synthetic glycosphingolipid antigen from Sphingomonas bacteria (GSL) or injected with α-GalCer (α-GC). () Bacterial burden in lungs of mice treated with anti-CD1d or isotype-matched control antibody (immunoglobulin G), assessed 3 d after infection with S. pneumoniae. Each symbol represents an individual mouse. CFU, colony-forming units. *P < 0.05 (Mann-Whitney test). Data are representative of at least two experiments with similar results (–; mean and s.d. in ) or two independent experiments (; mean and s.d. of triplicate wells) or are from two independent experiments (). * Figure 2: Structure of S. pneumoniae glycolipids. () Enzyme-linked immunosorbent assay of IL-2 in supernatants of Vα14i NKT cell hybridoma 1.2 assessed in an APC-free assay of PBS alone, E. coli (E-coli), S. typhimurium (Salm), group A Streptococcus (GAS), group B Streptococcus (GBS), S. pneumoniae (SPN) or α-GalCer (α-GC; 5 ng/well). The sonicate volumes 0.01, 0.1 and 1 (key) are equivalent to 1 × 106, 1 × 107 and 1 × 108 bacteria per well, respectively. () Structure of S. pneumoniae glycolipids SPN-Glc-DAG (left) and SPN-Gal-Glc-DAG (right). () Electrospray-ionization mass spectrometry analysis of SPN-Glc-DAG (left) and SPN-Gal-Glc-DAG (right); fatty acid composition is in parentheses. m/z, mass/charge. Data are representative of two experiments (; mean and s.e.m. of triplicate wells) or three experiments (). * Figure 3: Microbial glycolipids stimulate Vα14i NKT cells in vitro. () Release of IL-2 from cells of the Vα14i NKT cell hybridoma 1.2 cultured with A20 cells (A20) or A20 cells transfected to express mouse CD1d (A20-CD1d), pulsed for 20 h with buffer containing Tween 20 (vehicle), Sphingomonas GalA-GSL, SPN-Glc-DAG or SPN-Gal-Glc-DAG at various concentrations (horizontal axis; in μg/ml). () Release of IL-2 from cells of Vα14i NKT cell hybridoma 1.2 cultured for 20 h in mouse CD1d–coated wells with SPN-Glc-DAG, SPN-Gal-Glc-DAG or BbGL-IIc at various concentrations (key). () Release of IL-2 from cells of the non-Vα14-expressing but CD1d-reactive hybridoma 19 in wells coated with CD1d, cultured with Tween 20 (vehicle) alone, SPN-Glc-DAG, SPN-Gal-Glc-DAG or BbGL-IIc (2,000 ng/well) or self antigen presented by A20 cells transfected to express mouse CD1d. ND, not detected. () Release of IL-2 from cells of the Vα14i NKT hybridoma 1.2 cultured with A20 cells, transfected to express mouse CD1d and pulsed with buffer containing Tween 20 (vehic! le), Sphingomonas GalA-GSL, SPN-Glc-DAG (Glc), SPN-Gal-Glc-DAG (Gal-Glc), GBS-Glc-DAG (Glc) or GBS-Glc-Glc-DAG (Glc-Glc) at various concentrations (key). Data are representative of at least three (,,) or two () experiments (mean and s.e.m. of triplicate wells). * Figure 4: In vivo stimulation of Vα14i NKT cells by purified glycolipids. (,) Expression of CD25 () and intracellular IFN-γ and IL-4 () by tetramer-positive Vα14i NKT cells (liver mononuclear cells) obtained from mice 14 h after transfer of DCs pulsed with α-GalCer (0.1 μg/ml), Sphingomonas GalA-GSL (10 μg/ml), SPN-Glc-DAG (20 μg/ml), SPN-Gal-Glc-DAG (20 μg/ml) or vehicle alone. () Expression of intracellular IFN-γ and IL-4 by tetramer-positive liver mononuclear cells obtained from mice 14 h after transfer of DCs pulsed with vehicle, SPN-Glc-DAG, GBS-Glc-DAG or GBS-Glc-Glc-DAG (20 μg/ml). () Intracellular expression of IFN-γ and IL-4 by liver mononuclear cells (positive for CD1d tetramer loaded with α-GalCer) obtained from wild-type or Myd88−/− mice 14 h after transfer of Myd88−/−TrifLps2/Lps2 DCs pulsed with vehicle, SPN-Gal-Glc-DAG (20 μg/ml), SPN-Glc-DAG (20 μg/ml) or α-GalCer (0.1 μg/ml). () Expression of intracellular IFN-γ and IL-4 by tetramer-positive liver mononuclear cells obtained from IL-12p35-deficient mice 4 h ! after transfer of wild-type or IL-12p35-deficient (IL-12-KO) DCs pulsed with vehicle, SPN-Glc-DAG (20 μg/ml) or α-GalCer (0.1 μg/ml). NT, not tested. Data are representative of at least two independent experiments with similar results (n = 3 mice per group, except n = 2 mice for α-GalCer in ). * Figure 5: Stringent requirement for vaccenic acid in the stimulation of iNKT cells. (,) Expression of CD25 () and intracellular IFN-γ and IL-4 () by tetramer-positive liver mononuclear cells obtained from mice (n = 3 per group) 14 h after transfer of DCs pulsed with α-GalCer (0.1 μg/ml), synthetic variants of Glc-DAG (Glc-DAG-s1 through Glc-DAG-s5 (fatty acid composition, Table 1); 20 μg/ml) or vehicle. () Secretion of IFN-γ by human Vα24i NKT cell lines (n = 5) cultured for 24 h with human Hela cells transfected to express CD1d, in the presence of vehicle alone, purified glycolipids (Glc-DAG and Gal-Glc-DAG), synthetic glycolipids (Glc-DAG-s1 through Glc-DAG-s5) or α-GalCer. Data are representative of two independent experiments with similar results (,) or two experiments (; mean and s.d. of triplicate wells). * Figure 6: Crystal structure of the mouse CD1d–Glc-DAG-s2 complex. () Conformation of Glc-DAG-s2 in the binding groove. Side view with the α2 helix removed for clarity and the 2Fo – Fc electron density for the ligand (1σ) presented as a blue mesh. Green indicates the unsaturation of vaccenic acid. () Top view of CD1d with the Glc-DAG-s2 ligand in yellow (sugar removed for clarity) and the corresponding 2Fo – Fc electron density in blue; additional, unmodeled electron density (Fo – Fc map at 3σ in green) is visible at the bottom of the A′ pocket. (,) Hydrogen-bond network between CD1d and the ligands Glc-DAG-s2 (yellow) and α-GalCer (green; Protein Data Bank accession code, 1Z5L; ) or Glc-DAG-s2 (yellow) and BbGL-IIc (cyan; Protein Data Bank accession code, 3ILQ; ). Dashed lines indicate potential hydrogen bonds: blue, Glc-DAG-s2; green, α-GalCer; cyan, BbGL-IIc. (,) Top view onto the molecular surface of the CD1d binding pocket, including its electrostatic potentials: yellow, Glc-DAG-s2 ligand; green, α-GalCer (); cyan, BbGL-I! Ic (). () Binding response of mouse CD1d–Glc-DAG-s2 to immobilized Vα14Vβ8.2 TCR, measured by surface plasmon resonance and presented as response units (RU). Inset, binding of increasing concentrations (0.3125–20 μM) of the CD1d–DAG antigen complex. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 3T1F * 1Z5L * 3ILQ * 3T1F * 1Z5L * 3ILQ Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yuki Kinjo, * Petr Illarionov & * José Luis Vela Affiliations * Division of Developmental Immunology, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA. * Yuki Kinjo, * José Luis Vela, * Bo Pei, * Archana Khurana & * Mitchell Kronenberg * Department of Chemotherapy and Mycoses, National Institute of Infectious Diseases, Tokyo, Japan. * Yuki Kinjo, * Yukihiro Kaneko, * Akiko Okawara & * Yoshitsugu Miyazaki * School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK. * Petr Illarionov & * Gurdyal S Besra * Division of Cell Biology, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA. * Enrico Girardi, * Yali Li & * Dirk M Zajonc * HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, Affiliate of the Rockefeller University, New York, New York, USA. * Xiangming Li & * Moriya Tsuji * Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA. * Masakazu Imamura & * Chi-Huey Wong * Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, Canada. * Anaximandro Gómez-Velasco * Kyowa Hakko Kirin California Inc, La Jolla, California, USA. * Paul Rogers * Department of Pediatrics and Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA. * Samira Dahesh, * Satoshi Uchiyama & * Victor Nizet * Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, Yokohama, Japan. * Kazuyoshi Kawahara * Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK. * Hasan Yesilkaya & * Peter W Andrew * Department of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan. * Kazuyoshi Kawakami Contributions Y. Kinjo and M.K. designed most the study, except D.M.Z. designed the crystal structure study and the Biacore assay; Y. Kinjo, P.I., J.L.V., E.G., V.N., D.M.Z. and M.K. prepared the manuscript; Y. Kinjo, J.L.V. and B.P. did most of the immunology experiments; P.I., K. Kawahara and A.G.-V. analyzed bacterial glycolipids; P.I., M.I. and C.-H.W. synthesized glycolipids; G.S.B. provided informational support; E.G., Y.L. and D.M.Z. determined the crystal structure of the CD1d-Glc-DAG-s2 complex and did the Biacore assay; X.L., P.R. and M.T. did the human NKT cell experiments; Y. Kinjo, J.L.V., Y. Kaneko, A.O., Y.M. and K. Kawakami did S. pneumoniae infection experiments; S.D., S.U. and V.N. prepared bacterial sonicates and provided advice on bacterial culture and infection; A.K. made the mouse CD1d protein; H.Y. and P.W.A. prepared bacteria for glycolipid analysis; and M.K. provided overall supervision. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mitchell Kronenberg Author Details * Yuki Kinjo Search for this author in: * NPG journals * PubMed * Google Scholar * Petr Illarionov Search for this author in: * NPG journals * PubMed * Google Scholar * José Luis Vela Search for this author in: * NPG journals * PubMed * Google Scholar * Bo Pei Search for this author in: * NPG journals * PubMed * Google Scholar * Enrico Girardi Search for this author in: * NPG journals * PubMed * Google Scholar * Xiangming Li Search for this author in: * NPG journals * PubMed * Google Scholar * Yali Li Search for this author in: * NPG journals * PubMed * Google Scholar * Masakazu Imamura Search for this author in: * NPG journals * PubMed * Google Scholar * Yukihiro Kaneko Search for this author in: * NPG journals * PubMed * Google Scholar * Akiko Okawara Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshitsugu Miyazaki Search for this author in: * NPG journals * PubMed * Google Scholar * Anaximandro Gómez-Velasco Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Rogers Search for this author in: * NPG journals * PubMed * Google Scholar * Samira Dahesh Search for this author in: * NPG journals * PubMed * Google Scholar * Satoshi Uchiyama Search for this author in: * NPG journals * PubMed * Google Scholar * Archana Khurana Search for this author in: * NPG journals * PubMed * Google Scholar * Kazuyoshi Kawahara Search for this author in: * NPG journals * PubMed * Google Scholar * Hasan Yesilkaya Search for this author in: * NPG journals * PubMed * Google Scholar * Peter W Andrew Search for this author in: * NPG journals * PubMed * Google Scholar * Chi-Huey Wong Search for this author in: * NPG journals * PubMed * Google Scholar * Kazuyoshi Kawakami Search for this author in: * NPG journals * PubMed * Google Scholar * Victor Nizet Search for this author in: * NPG journals * PubMed * Google Scholar * Gurdyal S Besra Search for this author in: * NPG journals * PubMed * Google Scholar * Moriya Tsuji Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk M Zajonc Search for this author in: * NPG journals * PubMed * Google Scholar * Mitchell Kronenberg Contact Mitchell Kronenberg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (9M) Supplementary Figures 1–18, Table 1, Results, Methods and References Additional data - The antiviral factor APOBEC3G enhances the recognition of HIV-infected primary T cells by natural killer cells
- Nat Immunol 12(10):975-983 (2011)
Nature Immunology | Article The antiviral factor APOBEC3G enhances the recognition of HIV-infected primary T cells by natural killer cells * Jason M Norman1 * Michael Mashiba2 * Lucy A McNamara1, 3 * Adewunmi Onafuwa-Nuga4 * Estelle Chiari-Fort4 * Wenwen Shen4 * Kathleen L Collins1, 2, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:975–983Year published:(2011)DOI:doi:10.1038/ni.2087Received19 April 2011Accepted12 July 2011Published online28 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif), which promotes degradation of A3G. We report that viral protein R (Vpr), which interacts with a uracil glycosylase, also counteracted A3G by diminishing the incorporation of uridine. However, this process resulted in activation of the DNA-damage–response pathway and the expression of natural killer (NK) cell–activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells. View full text Figures at a glance * Figure 1: Expression of NKG2D ligands in HIV-infected cells. () Genome of the HIV vector NL-PI (derived from the NL4-3 HIV molecular clone12, 40). Downward arrows indicate locations of mutations in open reading frames. tat and rev, regulatory genes; gag, group-associated antigen gene; pol, polymerase gene; env, envelope gene; plap-IRES, placental alkaline phosphatase gene–internal ribosomal entry site. () Flow cytometry analysis of the expression of NKG2D ligands by CD3+ T cells infected with wild-type NL-PI (solid black lines) or mutant NL-PI (dashed black lines) lacking the accessory proteins Vif (NL-PIvif−), Vpr (NL-PIvpr−), Vpu (NL-PIvpu−) or Nef (NL-PInef−) or both Vif and Nef (NL-PIvif−nef−), and in mock-treated control cells (shaded gray curves). () Expression of NKG2D ligand on the infected T cells in relative to that on mock-treated controls. Each symbol represents a different donor (n = 9); small horizontal lines indicate the mean. () Flow cytometry analysis of the expression of NKG2D ligands by CD3+ T cells in! fected with NL-PIvpr− (solid black lines) or NL-PI mutants (dashed black lines) lacking both Vif and Vpr (NL-PIvif−vpr−), Nef (NL-PInef−) or both Vpr and Nef (NL-PIvpr−nef−), and in mock-treated control cells (shaded gray curves). () Expression of NKG2D ligand on cells in , presented as in . () Expression of NKG2D ligand on primary T cells infected with NL-PI or NL-PIvif− and also transduced with control lentivirus (Target: vif−) or Vif-expressing lentivirus (Target: vif+; right) or infected with NL-PIvif− HIV prepared from control 293T cells (Producer: vif−) or Vif-expressing 293T cells (Producer: vif+; right); results presented as in . () Immunoblot analysis of Vif and tubulin (loading control) in whole-cell lysates of the primary T cell targets (left) and 293T producer cells (right) in ; – (far left), mock-infected primary T cells. Lenti, lentivirus. *P < 0.05 and **P < 0.01 (paired t-test). Data are representative of six experiments (,), four exper! iments (,), two experiments with two independent donors assaye! d in triplicate () or one experiment with one donor (). * Figure 2: Upregulation of A3G correlates with upregulation of NKG2D ligands. () Immunoblot analysis of A3G and tubulin in T cells treated with medium alone (−) or 16 h before (−16) or 2 h after (+2) the addition of conditioned supernatants (Sup) from peripheral blood mononuclear cell cultures depleted of CD8+ cells (as in ). Numbers below lanes indicate densitometry of A3G relative (rel) to that of tubulin and normalized to that of cells treated with medium alone. () Experimental timeline. () Flow cytometry of CD3+ T cells treated with control medium (Control med) or conditioned supernatant (+ Sup) and mock-infected (Mock) or infected with NL-PIvif− and stained for NKG2D ligands and HIV-1 Gag (left and middle) or left unnstained (right). Numbers in quadrants indicate mean fluorescence intensity of NKG2D ligands for the total Gag− population (top left) or Gag+ population (top right). () A3G expression (below lanes in ) versus the expression of NKG2D ligand in NL-PIvif− Gag+ cells (relative to that in mock-infected cells treated with medium);! dashed line indicates the linear best-fit line (R2 = 0.83). (,) Immunoblot analysis of mock-infected T cells (Mock) or T cells treated with the supernatant of 293T cells (293T sup) or infected with various NL-PI mutants (above lanes). Numbers below lanes, as in . NL-PI Vif (Y44A), HIV expressing mutant Vif defective in binding and degrading A3G. (,) Immunoblot analysis () and flow cytometry analysis () of A3G expression by cells infected with wild-type or mutant NL-PI (horizontal axis; ,) and also (in ) made permeable and stained for A3G and Gag (Gag+, HIV infected; Gag−, uninfected). Results are presented relative to those of control cells treated with medium. Each symbol represents a different donor (independent donors in and (n = 5)); small horizontal lines indicate the mean. *P < 0.05 (paired t-test). () A3G expression (as in ) versus the expression of NKG2D ligand by mock-infected cells or cells infected with NL-PIvif− (as in ); each symbol represents a different ! donor. R2 = 0.66 (mock-infected) or 0.84 (NL-PIvif−); P < 0.! 0001 (one-tailed t-test). () Expression of NKG2D ligand by cells infected with wild-type or mutant NL-PI, presented as in . *P < 0.02 (paired t-test). Data are representative of three experiments with three independent donors (,–), five experiments with five independent donors (), three experiments with three independent donors () or eight experiments with eight independent donors (). * Figure 3: Activation of the DNA-damage response in HIV-infected primary T cells. () Immunoblot analysis of phosphorylated (p-) Chk1 and Chk2 and total A3G, Gag and tubulin (loading control) in primary T cells mock infected or infected with wild-type or mutant NL-PI (above lanes). (,) Phosphorylation of Chk2 at Thr68 () and of Chk1 at Ser345 () in cells infected as in , presented relative to results obtained with mock-infected cells. *P < 0.05 (paired t-test). (,) Expression of A3G (relative that in mock-infected cells) versus phosphorylation of Chk2 at Thr68 () or of Chk1 Ser345 (; each relative that of mock-infected cells) in cells mock infected or infected with NL-PIvif− plus conditioned supernatant (as in Fig. 2a). () R2 = 0.75 (mock) or 0.92 (NL-PIvif−); P < 0.02 (one-tailed t-test). () R2 = 0.02. () Immunoblot analysis of the phosphorylation of ATM at Ser1981, and of Gag and tubulin, in primary T cells mock infected or infected with wild-type NL-PI or NL-PIvif−. Below lanes, band intensity adjusted for background, normalized to the intensity o! f tubulin and presented relative to mock-infected cells. () Expression of NKG2D ligand on primary T cells infected with wild-type NL-PI or NL-PIvif− and treated with the solvent dimethyl sulfoxide (DMSO; control) or the ATM inhibitor KU55933 (10 μM); results are presented relative to those obtained with mock-infected cells. *P < 0.05 (paired t-test). () Immunoblot analysis of phosphorylated Chk2 and total tubulin in primary T cells mock infected or infected with NL-PIvif− and treated with KU55933 (10 μM), dimethyl sulfoxide (DMSO) or etoposide (10 μM). Below lanes, band intensity adjusted for background, normalized to the intensity of tubulin and presented relative to mock-infected cells treated with dimethyl sulfoxide. Each symbol represents a different donor; small horizontal lines indicate the mean (–,). Data are representative of three experiments (–), one experiment (), five experiments with five independent donors () or two experiments (). * Figure 4: HIV-infected T cells are resistant to recognition by NK cells unless an NKG2D ligand is overexpressed. () Genomes of the HIV-1 reporter virus NL4-3-ΔE–EGFP (NL-G; which expresses a fusion protein of truncated Env (Δenv) and enhanced green fluorescent protein (gfp)) and the HIV plasmid NL-GIulbp1+ (which overexpresses ULBP1 via an internal ribosomal entry site and sequence encoding ULBP (IRES-ulbp1) added to NL-G just after gfp. Downward arrow indicates mutation in nef (as in ). () Flow cytometry of peripheral blood mononuclear cell populations depleted of CD8+ cells, infected with viruses in and incubated alone (PBMC) or with interleukin 2–stimulated autologous NK cells at an effector/target ratio of 2:1 (PBMC + NK cells) in a 4-hour cytotoxicity assay, showing the expression of MHC class I (MHCI) and GFP by 7-AAD−CD56−CD16− live target cells. Arrows at top indicate infected cells (Inf); numbers in plots indicate total GFP+ cells normalized to counting beads. () Quantification of an NK cell assay as in , showing the survival of mock-infected cells or cells infecte! d with the viruses in , or of K562 cells, incubated at various effector/target ratios (horizontal axis). () Flow cytometry analysis of ULBP1 expression in GFP+ phytohemagglutinin-activated primary T cells transduced with NL-GIulbp1+ (dotted lines) or NL-G (solid lines), with (nef−) or without (nef+) the nef mutation in . Solid gray shading, isotype-matched control antibody. Numbers in plots indicate ULBP1 expression relative to that in cells infected with control virus (NL-G). () Flow cytometry of peripheral blood mononuclear cells prepared as in and infected with the viruses in , with the nef mutation, and incubated alone or with autologous NK cells (as in ). () Quantification of an NK cell assay as in , presented as in . Data are representative of three experiments with three independent donors (–) or two experiments with two independent donors (,). * Figure 5: The effect of Vif on lysis by NK cells. () NK cell killing assay (as in Fig. 4c) of T cells infected with wild-type NL-PI or NL-PIvif− and incubated with autologous NK cells at a ratio of 5:1 or 2:1 (bottom). *P < 0.05 and **P < 0.01 (paired t-test). () Immunoblot analysis of whole-cell lysates of 293T cells (far left) or 293T cells expressing hemagglutinin-tagged A3G (HA-A3G; right), left untransfected (−) or transfected with negative control shRNA (NC) or shRNA targeting A3G (above lanes). Numbers below lanes indicate band intensity of A3G, normalized to tubulin expression and presented relative to that of untransfected control cells. () Flow cytometry of peripheral blood mononuclear cells (prepared as in Fig. 4b), infected with NL-PIvif− plus GFP-expressing lentivirus encoding negative control shRNA (shNC) or shRNA targeting A3G (shA3G) and incubated alone or with interleukin 2–stimulated autologous NK cells at an effector/target ratio of 1:1 in a 4-hour cytotoxicity assay, showing the expression of MHC! class I and placental alkaline phosphatase (PLAP) by GFP+7-AAD−CD56−CD16− live target cells. Numbers in bottom right corners indicate percent infected cells with low expression of MHC class I remaining after incubation with NK cells; numbers in parentheses indicate percent survival after incubation with NK cells relative to survivial after incubation without NK cells. () NK cell assay showing the survival of target cells treated as in , incubated with effector cells at ratio of 1:1. Numbers in plots indicate mean survival. Each symbol represents a different donor. In one of three experiments, conditioned supernatant (as in Fig. 2b) was required for detection of the effect of A3G knockdown. *P < 0.05 (paired t-test). Data are representative of five experiments with five independent donors (), three experiments () or three experiments with three independent donors (,). * Figure 6: Vif and Vpr limit the incorporation of uridine into primary T cells. () Agarose gel electrophoresis of the products of PCR with NL-PI DNA containing dTTP or dUTP treated for 1 h with recombinant UDG (+) or heat-inactivated UDG (–) as the template. () Quantitative PCR analysis of HIV-1 or β-actin DNA among total genomic DNA from primary T cells infected with wild-type or mutant NL-PI; results are presented as amplification in cells treated with UDG (as in ; UDG+) relative to amplification in cells treated with heat-inactivated UDG (as in ; UDG−). *P < 0.05 and **P < 0.002, compared with the theoretical mean of 1.0 (no effect of treatment with UDG; one-sample t-test). () Quantitative PCR results from versus the infection rate of primary T cells infected with NL-PIvif−vpr−. R2 = 0.86 and *P = 0.07 (HIV-1 DNA; one-tailed t-test); R2 = 0.96 and **P = 0.02 (β-actin DNA; one-tailed t-test). (,) Quantitative PCR analysis of total HIV DNA () and β-actin DNA () isolated from primary T cells infected with viruses as in , presented relative to! results obtained with cells infected with wild-type virus. In , the mean was normalized for infection rate and β-actin DNA content. *P < 0.002 () or 0.05 (; one-sample t-test). () Quantitative PCR analysis of serial dilutions (wedge) of NL-PI DNA templates containing dUTP or dTTP. Data are representative of two experiments (), four experiments with four independent donors (–; error bars, s.e.m.) or one experiment (). * Figure 7: Binding of UNG2 by Vpr induces the expression of NKG2D ligands. () Expression of NKG2D ligand on activated primary T cells infected with NL-PI mutants (horizontal axis), presented relative to its expression in mock-infected cells. NL-PInef− Vpr(W54R), Nef-deficient NL-PI containing vpr with a mutation in sequence encoding the UNG2-binding domain. Each symbol represents a different donor (n = 8); small horizontal lines indicate the mean. *P < 0.01 (paired t-test). () Immunoblot analysis of UNG2 and tubulin in CEM-SS T cells transduced with lentivirus expressing negative control shRNA or shRNA targeting UNG2 (shUNG2). () Expression of NKG2D ligand on primary T cells infected with wild-type NL-PI or NL-PIvpr− and transduced with lentivirus expressing negative control shRNA (shNC) or shRNA targeting UNG2 (shUNG2), presented relative to expression in mock-infected cells. Each symbol represents a different donor (n = 4); small horizontal lines indicate the mean. *P = 0.03 (paired t-test). Data are representative of nine experiments (), thr! ee experiments () or four experiments (). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA. * Jason M Norman, * Lucy A McNamara & * Kathleen L Collins * Graduate Program in Immunology, University of Michigan, Ann Arbor, Michigan, USA. * Michael Mashiba & * Kathleen L Collins * Department of Epidemiology, University of Michigan, Ann Arbor, Michigan, USA. * Lucy A McNamara * Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA. * Adewunmi Onafuwa-Nuga, * Estelle Chiari-Fort, * Wenwen Shen & * Kathleen L Collins Contributions J.M.N. and K.L.C. designed the experiments and prepared the manuscript; J.M.N., M.M., L.A.M., A.O.-N., W.S. and E.C.-F. did experiments; and all authors read and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kathleen L Collins Author Details * Jason M Norman Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Mashiba Search for this author in: * NPG journals * PubMed * Google Scholar * Lucy A McNamara Search for this author in: * NPG journals * PubMed * Google Scholar * Adewunmi Onafuwa-Nuga Search for this author in: * NPG journals * PubMed * Google Scholar * Estelle Chiari-Fort Search for this author in: * NPG journals * PubMed * Google Scholar * Wenwen Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen L Collins Contact Kathleen L Collins Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (541K) Supplementary Figures 1–2 and Tables 1–3 Additional data - Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell responses by targeting the aminopeptidase ERAP1
- Nat Immunol 12(10):984-991 (2011)
Nature Immunology | Article Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell responses by targeting the aminopeptidase ERAP1 * Sungchul Kim1 * Sanghyun Lee1 * Jinwook Shin2 * Youngkyun Kim1 * Irini Evnouchidou3 * Donghyun Kim1 * Young-Kook Kim4 * Young-Eui Kim5 * Jin-Hyun Ahn5 * Stanley R Riddell6 * Efstratios Stratikos3 * V Narry Kim4 * Kwangseog Ahn1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:984–991Year published:(2011)DOI:doi:10.1038/ni.2097Received20 June 2011Accepted27 July 2011Published online04 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Major histocompatibility complex (MHC) class I molecules present peptides on the cell surface to CD8+ T cells, which is critical for the killing of virus-infected or transformed cells. Precursors of MHC class I–presented peptides are trimmed to mature epitopes by the aminopeptidase ERAP1. The US2–US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and encodes three microRNAs (miRNAs). We show here that HCMV miR-US4-1 specifically downregulated ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides was inhibited, which led to less susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings identify a previously unknown viral miRNA–based CTL-evasion mechanism that targets a key step in the MHC class I antigen-processing pathway. View full text Figures at a glance * Figure 1: Expression of HCMV miR-US4-1 results in less ERAP1b mRNA and protein but not less ERAP1a mRNA or protein. (–) Change in cellular mRNA by viral miR-US4-1 (), miR-US5-1 () and miR-US5-2 () versus mRNA expression in response to control miRNA (siGFP) in U373MG cells; red dot, ERAP1 expression. (,) Quantitative real-time PCR analysis of ERAP1a mRNA () and ERAP1b mRNA () in HEK293T cells treated with miR-US4-1, siERAP1, siERAP1a or siERAP1b, presented relative to the expression of GAPDH mRNA (encoding glyceraldehyde 3-phosphate dehydrogenase). *P < 0.05, versus control miRNA (two-tailed Student's t-test). () Immunoblot analysis (IB) of ERAP1 and GAPDH (loading control), RNase-protection assay (RPA) of miR-US4-1 expression, and RNA blot analysis (RNA) of hsa-miR-16 expression (loading control) in HEK293T cells transfected with control vector or vector expressing miR-US4-1, siERAP1 or miR-US4-1(M), followed by selection for 1 week with 2 μg puromycin. () Immunoblot analysis (IB) of ERAP1 and GAPDH, and RNA blot analysis of the expression of miR-US4-1 and hsa-miR-16, in HEK293T cells ! transfected with control miRNA (C) or increasing concentrations of vector encoding miR-US4-1 (2 μg, 4 μg and 10 μg; wedge). Data are representative of three independent experiments (mean ± s.d. in ,). * Figure 2: HCMV miR-US4-1 targets the 3′ UTR of ERAP1b and physically binds to ERAP1b mRNA in the RISC. (,) Predicted binding site for miR-US4-1 in the 3′ UTR of ERAP1a mRNA () and ERAP1b mRNA (). Bold indicates the expected seed region interaction site; red, nucleotide sequence replaced with the sequence indicated by the arrow in mutant 3′ UTRs; blue, HCMV miR-US4-1. Bottom right, minimum free energy of hybridization. () Dual-luciferase assay of HEK293T cells transfected with 5 μg vector expressing miRNA (key), 10 ng luciferase reporter vector for wild-type (WT) or mutated (Mut) 3′ UTR of ERAP1a or ERAP1b (horizontal axis) and 5 ng renilla vector (control); results are presented relative to the luminescence of renilla luciferase. () Dual-luciferase assay as in except for the use of 1 μg, 2 μg or 5 μg (wedge) of vector expressing miR-US4-1 and 5 μg vector expressing control miRNA (C). () RNase-protection assay to detect miR-US4-1 in HEK293T cells transfected with vector for miR-US4-1 or miR-US4-1(M) and empty vector (Mock) or vector for human AGO1–AGO4 tagged with! Flag at the N terminus (AGO), followed by RISC immunoprecipitation 48 h after transfection and extraction of total RNA. NT-NR, no target RNA or RNase; NT, no target RNA; *, undigested probe; **, HCMV miR-US4-1. () Immunoblot analysis of aliquots of the RISC immunoprecipitates and cell lysates in , probed with antibody to Flag (α-Flag). Ab HC, heavy-chain immunoglobulin. (,) Quantitative real-time PCR analysis of ERAP1a mRNA () and ERAP1b mRNA () among RNA extracted from RISC immunoprecipitates or total samples of the cells in , presented relative to GAPDH mRNA. *P < 0.05 and **P < 0.01 (,,), versus control miRNA (two-tailed Student's t-test). Data are representative of three independent experiments (mean ± s.d. in ,,,). * Figure 3: Downregulation of ERAP1 in HCMV-infected HFF cells. () RNase-protection assay (as in Fig. 2e) to measure miR-US4-1 in HFF cells left uninfected (U) or infected for 1 h with wild-type HCMV AD169 (WT), HCMVΔUS4 (ΔUS4 Mut) or revertant HCMV (Rev) at a multiplicity of infection of 5, followed by extraction of total RNA 0 h, 24 h, 48 h or 72 h after infection. *, undigested probe; **, HCMV miR-US4-1. () Immunoblot analysis of ERAP1, IE1-IE2 (IE1/2) and GAPDH (loading control) in aliquots of the cells in . Data are representative of three independent experiments. * Figure 4: HCMV miR-US4 inhibits the trimming of OVA8 peptide from OVA precursor peptide by ERAP1. () Quantitative real-time PCR analysis of ERAP1a and ERAP1b mRNA in H-2Kb-expressing HeLa cells transfected with control miRNA, miR-US4-1, siERAP1 or miR-US4-1(M), followed by 1 week of selection with puromycin; results are presented relative to GAPDH. (,) Activity of β-galactosidase (lacZ) in H-2Kb-expressing HeLa cells (target cells) treated as in , then transfected for 48 h with vector for N5OVA8 () or OVA8 () and cultured (1.0 × 104 HeLa cells) for 16 h with B3Z cells (effector cells) at an effector/target ratio of 1 (1.0 × 104 B3Z cells), 3 (3.0 × 104 B3Z cells) or 9 (9.0 × 104 B3Z cells); β-galactosidase production assessed with the lacZ substrate CPRG is presented relative to that obtained with control miRNA. *P < 0.001, versus control miRNA (two-tailed Student's t-test). Data are representative of three independent experiments (mean ± s.e.m.). * Figure 5: HCMV miR-US4-1 inhibits the generation of HCMV-derived antigenic peptides and CD8+ CTL responses. () Chromatograms of epitope production in vitro by ERAP1: top, precursor alone; middle, precursor plus a moderate amount of enzyme; and bottom, precursor plus more enzyme. *, peak corresponding to the mature epitope; right margin, reaction conditions; below, precursor sequence, with N-terminal extension of two amino acids underlined. () Chromium-release assay of the lysis of autologous fibroblasts transfected twice with control miRNA, miR-US4-1 or siERAP1 and then, 24 h later, infected for 1 h with HCMV RV798 (multiplicity of infection, 2) and, at 48 h after infection, collected and incubated for 2 h in a 37 °C in a CO2 incubator with 51Cr, then used as target cells (1.0 × 104) incubated with CTLs (clone identification at top) as effector cells at an effector/target ratio of 10 (1.0 × 105 CTLs), 5 (5.0 × 104 CTLs), 2.5 (2.5 × 104 CTLs) or 1.25 (1.25 × 104 CTLs). *P < 0.001, versus control miRNA (two-tailed Student's t-test). Data are representative of three independent! experiments (mean ± s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * National Creative Research Initiative Center for Antigen Presentation, Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea. * Sungchul Kim, * Sanghyun Lee, * Youngkyun Kim, * Donghyun Kim & * Kwangseog Ahn * Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA. * Jinwook Shin * Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research Demokritos, Athens, Greece. * Irini Evnouchidou & * Efstratios Stratikos * Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea. * Young-Kook Kim & * V Narry Kim * Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Seoul, Republic of Korea. * Young-Eui Kim & * Jin-Hyun Ahn * Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. * Stanley R Riddell Contributions S.K., D.K., Y.-K.K. and V.N.K. designed and did biochemical and cell biological experiments; J.S. and Y.K. did microarray experiments; S.L., Y.-E.K. and J.-H.A. generated HCMV mutants; I.E. and E.S. did in vitro ERAP1 trimming assays; S.R.R. cloned the HCMV-specific CTLs; and S.K. and K.A. designed the overall study and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kwangseog Ahn Author Details * Sungchul Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Sanghyun Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Jinwook Shin Search for this author in: * NPG journals * PubMed * Google Scholar * Youngkyun Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Irini Evnouchidou Search for this author in: * NPG journals * PubMed * Google Scholar * Donghyun Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Young-Kook Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Young-Eui Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Jin-Hyun Ahn Search for this author in: * NPG journals * PubMed * Google Scholar * Stanley R Riddell Search for this author in: * NPG journals * PubMed * Google Scholar * Efstratios Stratikos Search for this author in: * NPG journals * PubMed * Google Scholar * V Narry Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Kwangseog Ahn Contact Kwangseog Ahn Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–11 and Table 1 Additional data - The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells
- Nat Immunol 12(10):992-1001 (2011)
Nature Immunology | Article The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells * Masaki Miyazaki1 * Richard R Rivera1 * Kazuko Miyazaki1 * Yin C Lin1 * Yasutoshi Agata2 * Cornelis Murre1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:992–1001Year published:(2011)DOI:doi:10.1038/ni.2086Received04 April 2011Accepted08 July 2011Published online21 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell–B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells. View full text Figures at a glance * Figure 1: Analysis of Id3 expression in thymocytes and peripheral T cells from an Id3-GFP reporter mouse strain. () Wild-type (WT) Id3 locus (top) and Id3 locus with insertion of sequence encoding GFP into the ATG initiation codon (Id3gfp; bottom). Dotted lines link BamH1 sites (B) in the wild-type and mutated loci; the sequence encoding neomycin resistance (neo) was deleted in the Id3gfp locus. kb, kilobases. () Expression of GFP and CD27 in Id3−/− mouse thymocytes gated on the DN3 compartment (left), and GFP expression in Id3+/+ and Id3gfp/+ thymocytes in the DN3a compartment (middle) and DN3b compartment (right). Numbers adjacent to outlined areas (left) indicate percent GFP−CD27int cells (bottom left) or GFP+CD27+ cells (top right); numbers in plots (middle and right) indicate mean fluorescence intensity. () Intracellular (ic) staining of TCRβ and TCRγδ in sorted DN3 CD27lo GFP− cells (left) and DN3 CD27hiGFP+ cells (middle), and analysis of cell size (right), gated on DN3a GFP− and DN3b GFP+ cells derived from an Id3gfp/+ mouse. Numbers in quadrants indicate percent c! ells in each. FSC, forward scatter. () GFP expression in CD5−CD69−, CD5+CD69− and CD5+CD69+ DP cells. Numbers in plots indicate mean fluorescence intensity. () GFP expression at various stages of thymocyte development, presented as mean fluorescence intensity (MFI). ETP, early T lineage precursor. () GFP expression in CD62L+CD44lo, CD62L+CD44hi and CD62L−CD44hi cells gated on CD4+ (CD8+) CD3+TCRβ+TCRγδ− cells isolated from Id3+/+ and Id3gfp/+ spleens. () Flow cytometry of the expression of GFP and CD127 or CD122 by cells gated on the CD25−CD4+ compartment (left), and GFP expression in CD44lo, CD44hiCD122− or CD44hiCD122+ cells isolated from Id3+/+ and Id3gfp/+ spleens (right). Numbers in quadrants indicate percent cells in each. Data are representative of two (–) or three (,) experiments. * Figure 2: Global occupancy by E2A decreases during β-selection. Genome-wide occupancy by E2A and H3K4me1 patterns in thymocytes isolated from untreated Rag2−/− mice (DN3) or Rag2−/− mice injected with antibody to CD3ε (DN4). () Distribution of E2A-binding sites (numbers in plots) in DN3 cells versus DN4 cells. () Cis-regulatory sequences associated with occupancy by E2A in DN3 cells and DN4 cells, identified by comparison of enriched peaks with randomly selected genomic DNA sequences (letter size indicates nucleotide frequency). P values indicate enrichment for a motif relative to its presence in randomly selected regions. () Occupancy by E2A and H3K4me1 patterns across the Hes1, Ptcra, Notch1, Notch3, Rag2-Rag1, Cd3e, Rorc and Gfi1 loci in the DN3 compartment (E2A, gray; H3K4me1, blue) and DN4 compartment (E2A, black; H3K4me1, green). Numbers in plots indicate total tags observed; red arrows indicate transcription start sites; double-headed arrows indicate position of PCR amplicons used for ChIP (Supplementary Fig. 3). () Occup! ancy by E2A and H3K4me1 patterns across the Zap70 locus (as in ). () Expression of Zap70 in wild-type (Id3+/+) and Id3-deficient (Id3−/−) thymocytes in the ETP and DN2–DN4 compartments. () Expression of CD27 and Zap70 in fetal wild-type (E2a+/+) and E2A-deficient (E2a−/−) thymocytes in the DN3 compartment at day 16.5 after conception (top), and Zap70 expression in E2a+/+ and E2a−/− DN4 and ISP thymocytes (bottom). Numbers adjacent to outlined areas (top) indicate percent CD27+Zap70+ cells; lines above peaks (bottom) indicate Zap70+ cells. Data are representative of one experiment (–) or two independent experiments () with littermates. * Figure 3: Id3 acts to maintain the naive fate of T cells. () Expression of CD8 and CD4 by wild-type and Id3−/− thymocytes, gated on the γδ− population (top left); proportion of γδ T cells in the entire thymocyte population (bottom left); lineage-negative compartments (top right); and c-Kit expression in wild-type and Id3−/− thymocytes in the DN1 compartment (bottom right). Numbers in outlined areas (top left) or quadrants (top right) indicate percent cells in each; numbers adjacent to outlined areas (bottom left) indicate percent TCDγδ+CD3+ cells; and numbers above bracketed lines indicate percent c-Kit+ cells. () Flow cytometry analysis of CD44 expression in wild-type and Id3−/− ISP cells, DP cells, CD4SP (CD4+CD8−TCRβhiCD3εhi) cells and CD8SP (CD4−CD8+TCRβhiCD3εhi) cells (top); bottom, CD44 expression in DP and CD4SP thymocytes, presented as mean fluorescence intensity. () Intracellular staining of IFN-γ in gated CD4SP (CD3ε+TCRγδ−TCRβ+CD4+CD8−) thymocytes (top); numbers above bracketed lines ! indicate percent IFN-γ+ cells. Bottom, frequency of IFN-γ+ cells. () Flow cytometry analysis of the expression of Eomes and PLZF (left) in DP (top) and CD4SP (bottom) thymocytes from 4- to 6-week-old Id3+/+ and Id3−/− mice, gated on the TCRγδ− population; numbers in quadrants indicate percent cells in each. Right, frequency of PLZF+ or Eomes+ cells. *P < 0.05 (Student's t-test). Data are representative of four experiments (), four experiments with four mice each (), three independent experiments with three mice each () or three experiments with four mice each (; mean ± s.d. in –). * Figure 4: Aberrant development of TFH-like cells in the Id3−/− thymus. () Expression of CXCR5 and CD44 in wild-type and Id3−/− CD4SP thymocytes, gated on the CD3εhiTCRγδ−TCRβhiCD4+CD8− compartment (left top), and of PD-1 and ICOS in those cells, gated on the CD44hiCXCR5+CD4SP compartment (left bottom). Numbers adjacent to or in outlined areas indicate percent cells in each. Right, frequency (top) and absolute number (bottom) of CD44hiCXCR5+PD-1hiICOShi CD4SP thymocytes. () Ly108 (Slamf6) expression in the CXCR5− and CXCR5+CD4SP compartments of wild-type and Id3−/− thymocytes. () Flow cytometry analysis of the expression of CXCR5 and CD44 in wild-type and Id3−/− thymocytes, gated on the lineage-negative CD25−c-Kit−(DN4) compartments (left); numbers in outlined areas indicate percent CXCR5+CD44hi cells. Right, frequency of CXCR5+CD44hi cells. () Flow cytometry analysis of the expression of CXCR5 and B220 on the total thymocyte population (left); numbers in outlined areas indicate percent B220+CXCR5+ cells. Right, frequen! cy (left) and absolute number (right) of B220+CXCR5+ cells among total thymocytes. () Immunostaining of B220 in thymi from 4-week-old (top) and 4-month-old (bottom) wild-type and Id3−/− mice. Original magnification, ×50. () Flow cytometry analysis of the expression of B220 and forward scatter of gated CD44hiCXCR5+CD4SP, CD44hiCXCR5+CD8SP and DP Id3+/+ or Id3−/− thymocytes. Numbers adjacent to outlined areas indicate percent B220+ cells. () Cxcr5, Slamf1 and Bcl6 transcripts in Id3+/+ or Id3−/− CD4SP thymocytes (top) and in sorted CXCR5+ cells (bottom), presented relative to the abundance of Hprt1 transcript (encoding hypoxanthine guanine phosphoribosyl transferase). Bottom (key): naive CD4+CD62LhiCD44lo cells isolated from unimmunized wild-type mouse spleen (Id3+/+ naive); CD4+CD44hiCXCR5+ TFH cells derived from wild-type mice 10 d after immunization (Id3+/+ TFH); and CD4+CD8−TCRβhiCD44hiCXCR5+ cells isolated from Id3−/− thymi from unimmunized 4-week-old! mice (4 weeks Id3−/− CXCR5+) or 4-month-old mice (4 month! s Id3−/− CXCR5+). *P < 0.05 (Student's t-test). Data are representative of four experiments with four mice each (), one experiment (,), three experiments with four mice each (), three experiments with five mice each (), three experiments with ten 4-week-old mice or twelve 4-month-old mice () or two experiments (; mean ± s.d. in ,,,). * Figure 5: Id3 acts to enforce the naive fate of T cells by suppressing intrinsic E2A activity. () Absolute number of thymocytes in 4- to 6-week-old Id3+/+, Id3−/− and E2af/fCd4-CreId3−/− mice. () Expression of IFN-γ on gated CD4SP (CD3ε+TCRγδ−TCRβ+CD4+CD8−) thymocytes, assessed by intracellular staining (left); numbers above bracketed lines indicate percent IFN-γ+ cells. Right, frequency of IFN-γ+ cells. () Flow cytometry analysis of Eomes expression in CD4SP thymocytes derived from 4-week-old Id3+/+, Id3−/− and E2af/fCd4-CreId3−/− mice (top), and expression of Eomes and PLZF in Id3−/− and E2af/fCd4-CreId3−/− CD4SP cells (bottom). Numbers in quadrants indicate percent Eomes+PLZF− cells (top left) or Eomes−PLZF+ cells (bottom right). () Flow cytometry analysis of the expression of CXCR5 and CD44 (left) or CD44 and CD150 (right) in CD4SP thymocytes. Numbers in outlined areas (top) indicate percent CXCR5+CD44+ cells (top right), CXCR5−CD44− cells (bottom left) or CXCR5−CD44+ cells (bottom right). () Flow cytometry analysis of ! the expression of CXCR5 and CD44 on Id3+/+, Id3−/− and E2af/fCd4-CreId3−/− DN4 thymocytes. Numbers in outlined areas indicate percent CXCR5+CD44+ cells. () Flow cytometry analysis of the expression of CXCR5 and B220 on total thymocytes from Id3+/+, Id3−/− and E2af/fCd4-CreId3−/− mice. Numbers above outlined areas indicate percent CXCR5+B220+ cells. *P < 0.05 (Student's t-test). Data are representative of six experiments with six mice each (; mean ± s.d.), three independent experiments (; mean ± s.d.), two independent experiments (), two experiments with four mice () or two experiments (). * Figure 6: Effector-memory–like CD4+ T cells in the Id3−/− thymus. () Expression of CD127 and CD44 by wild-type and Id3−/− CD4SP thymocytes (top); numbers above outlined areas indicate percent CD127+CD44+ cells. Bottom, frequency of CD127+CD44+ cells. () Expression of CD127 and CD44 by wild-type and Id3−/−CD4SP thymocytes (top left), and CD127 expression, gated on CD44hiCD122+ CD4SP cells (bottom left). Numbers above outlined areas (top) indicate percent CD127+CD44+ cells, and numbers above bracketed lines (bottom) indicate percent CD127+ cells. Right, frequency (top) and absolute number (bottom) of CD44hiCD122+CD127+ cells in the CD4SP compartment. () Occupancy by E2A and H3K4me1 islands across the Cxcr5 and Il2rb loci in DN3 cells (E2A, gray; H3K4me1, blue) and the E2a−/− E47-reconstituted T cell line A12 (E2A, black; H3K4me1, green) Numbers in plots indicate total tags observed; red arrows indicate transcription start sites; double-headed arrows indicate position of PCR amplicons used in . () ChIP analysis of occupancy by E2A! at the Cxcr5 and Il2rb loci in Id3+/+ and Id3−/− DP and CD4SP thymocytes, followed by real-time PCR (primers in ). IgG, ChIP with nonspecific rabbit immunoglobulin G (negative control). *P < 0.05 (Student's t-test). Data are representative of one experiment with three mice (,; mean ± s.d.) or one experiment (,). * Figure 7: Abnormal effector-memory and TFH-like compartments in Id3−/− spleen. () Flow cytometry analysis of the expression of CD62L and CD44 in splenocytes from 6-week-old Id3+/+ and Id3−/− mice, gated on CD3ε+TCRγδ−TCRβ+CD4+ cells. Numbers in quadrants indicate percent cells in each. () Expression of IL-4 and IFN-γ in CD3ε+TCRγδ−TCRβ+CD4+ T cells isolated from spleens of 4-week-old Id3+/+ or Id3−/− mice (left); numbers in quadrants indicate percent cells in each. Below, frequency of cells positive for IFN-γ, IL-4 or IL-17. () Flow cytometry analysis (right) of the expression of CXCR5 and CD44 by Id3+/+ and Id3−/− splenocytes, gated on CD3ε+TCRγδ−TCRβ+CD4+ cells (top), and of ICOS and PD-1, gated on CXCR5+CD44hi CD4+ T cells (bottom); numbers in outlined areas indicate percent CXCR5+CD44+ cells (top) or ICOS+PD-1+ cells (bottom). Right, frequency of CD44hiCXCR5+PD-1hiICOShi cells. () Immunostaining of spleens with antibody to CD3ε. Original magnification, ×200. () Quantitative real-time RT-PCR analysis of Cxcr5, Slamf! 1 and Bcl6 in CD4+ T cells (top) and CXCR5+ cells sorted from Id3+/+ or Id3−/− spleen (bottom; cells as in Fig. 4g). () Flow cytometry analysis (left) of the expression of CD127 and CD44 (top) or of CD122 and CD44 (middle) on splenocytes from 4-week-old Id3+/+ or Id3−/− mice, gated on TCRγδ−TCRβ+CD3ε+CD4+ cells; or of CXCR5 and CD127, gated on CD44hiCD122+ CD4+ T cells (bottom). Right, frequency of cells at top. *P < 0.05 (Student's t-test). Data are representative of three experiments (,), three experiments with three mice (), three experiments with five mice (), one experiment () or one experiment with three mice (; mean ± s.d. in ,,,). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Biology, University of California, San Diego, La Jolla, California, USA. * Masaki Miyazaki, * Richard R Rivera, * Kazuko Miyazaki, * Yin C Lin & * Cornelis Murre * Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Kyoto, Japan. * Yasutoshi Agata Contributions M.M. designed and did experiments, analyzed data and wrote portions of the manuscript; R.R.R. generated Id3-GFP mice; K.M. did ChIP analyses; Y.C.L. contributed to the analysis of data from ChIP followed by deep sequencing; Y.A. provided advice on ChIP experiments with small number of cells; and C.M. designed experiments, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Cornelis Murre Author Details * Masaki Miyazaki Search for this author in: * NPG journals * PubMed * Google Scholar * Richard R Rivera Search for this author in: * NPG journals * PubMed * Google Scholar * Kazuko Miyazaki Search for this author in: * NPG journals * PubMed * Google Scholar * Yin C Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Yasutoshi Agata Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelis Murre Contact Cornelis Murre Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–10 Additional data - The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity
- Nat Immunol 12(10):1002-1009 (2011)
Nature Immunology | Article The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity * Mikyoung Chang1, 6 * Wei Jin1, 5, 6 * Jae-Hoon Chang1 * Yichuan Xiao1 * George C Brittain1 * Jiayi Yu1 * Xiaofei Zhou1 * Yi-Hong Wang1 * Xuhong Cheng1 * Pingwei Li2 * Brian A Rabinovich3 * Patrick Hwu4 * Shao-Cong Sun1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1002–1009Year published:(2011)DOI:doi:10.1038/ni.2090Received21 June 2011Accepted19 July 2011Published online28 August 2011Corrected online20 September 2011 Abstract * Abstract * 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 T cell activation is subject to tight regulation to avoid inappropriate responses to self antigens. Here we show that genetic deficiency in the ubiquitin ligase Peli1 caused hyperactivation of T cells and rendered T cells refractory to suppression by regulatory T cells and transforming growth factor-β (TGF-β). As a result, Peli1-deficient mice spontaneously developed autoimmunity characterized by multiorgan inflammation and autoantibody production. Peli1 deficiency resulted in the nuclear accumulation of c-Rel, a member of the NF-κB family of transcription factors with pivotal roles in T cell activation. Peli1 negatively regulated c-Rel by mediating its Lys48 (K48) ubiquitination. Our results identify Peli1 as a critical factor in the maintenance of peripheral T cell tolerance and demonstrate a previously unknown mechanism of c-Rel regulation. View full text Figures at a glance * Figure 1: Peli1-deficient T cells are hyper-responsive to TCR and CD28 signals. Enzyme-linked immunosorbent assay (ELISA) of IL-2 and interferon-γ (IFN-γ) in CD4+ T cells (), CD8+ T cells (,) or naive CD4+ T cells () purified from splenocytes of 7-week-old age- and sex-matched wild-type (WT) and Peli1−/− (KO) mice and stimulated for 48 h with various doses (horizontal axes) of plate-bound anti-CD3 (α-CD3) plus anti-CD28 (α-CD28) or PMA plus ionomycin (Iono). Data are representative of four independent experiments with at least three mice per group (mean ± s.e.m.). * Figure 2: A cell-autologous role for Peli1 in regulating T cell homeostasis in vivo. () Frequency of naive (CD44loCD62Lhi) and memory (CD44hiCD62Llo) CD4+ or CD8+ T cells among total splenocytes from age- and sex-matched wild-type and Peli1−/− mice (10 weeks or 6 months old), gated on CD3+CD25− cells. Numbers in quadrants indicate percent cells in each throughout. () Naive and memory CD4+ or CD8+ T cells (assessed as in ) from age-matched 6- to 8-month-old wild-type and Peli1−/− mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). *P < 0.001 (two-tailed unpaired t-test). () Flow cytometry of peripheral lymph node cells from recipients of bone marrow cells (2 × 106) derived from Peli1−/− (CD45.1−) mice (backcrossed to the C57BL/6 background) and wild-type (CD45.1+) B6.SJL mice, mixed at a ratio of 1:1 and adoptively transferred into γ-irradiated mice deficient in recombination-activating gene 1, assessed 10 weeks later as the frequency of Peli1−/− and wild-type CD4+ or CD8+ T cells based! on CD45.1 expression (left). Numbers adjacent to outlined areas indicate percent Peli1−/− (left) or wild-type (right) CD4+ T cells (top row) and CD8+ T cells (bottom row). Right, frequency of memory and naive T cells among the Peli1−/− and wild-type CD4+ or CD8+ T cell populations (determined as in ). Data are representative of five independent experiments with at least three mice per group (), five experiments with three to seven mice per group () or three experiments with four recipients per group (). * Figure 3: Peli1 deficiency renders naive T cells refractory to suppression by Treg cells and TGF-β. (,) Flow cytometry analysis of the frequency of Treg cells (among CD3+CD4+ cells) in the thymus, spleen and mesenteric lymph nodes (mLN) of 10-week-old mice () or 6-month-old mice (). () Proliferation of naive CD4+ T cells (effector T cells (Teff)) from 7-week-old wild-type and Peli1−/− mice activated in vitro by incubation with wild-type Treg cells at various ratios (left margin), assessed as CFSE dilution. Numbers above bracketed lines indicate undivided cells. () Proliferation of naive CD4+ or CD8+ T cells cultured for 48 h without inducers (NT) or induced with various combinations of TGF-β and anti-CD3 and anti-CD28 (horizontal axis), assessed as incorporation of [3H]thymidine. () Proliferation of splenic CD4+ T cells obtained from wild-type and Peli1−/− OT-II mice given 20 mg OVA or PBS orally daily for 4 d, then stimulated in vitro with OVA peptide–pulsed antigen-presenting cells (assessed as in ). Data are representative of three (,,,) or two () independent! experiments (mean ± s.e.m. in ). * Figure 4: Spontaneous development of signs of autoimmune disease by Peli1−/− mice. () Lymph nodes isolated from age- and sex-matched 10-week-old wild-type and Peli1−/− mice. () Total cells in the spleen, peripheral lymph nodes (pLN) and mesenteric lymph nodes of 6-month-old mice (presented as in Fig. 2b). *P < 0.0002, **P < 0.0001 and ***P = 0.3457 (two-tailed unpaired t-test). () Hematoxylin-and-eosin staining of kidney, liver and lung sections from 6-month-old wild-type and Peli1−/− mice, showing the infiltration of cells of the immune response. Original magnification, ×100 (kidney) or ×200 (liver and lung). () Kidney tissue sections from a Peli1−/− mouse, stained with hematoxylin and eosin (H&E) or analyzed by immunohistochemistry with anti-CD4 or anti-CD8. Original magnification, ×200. () ELISA of antinuclear antibodies (ANA) in serum from age-matched 10- to 24-month-old wild-type and Peli1−/− mice (n = 8 per genotype; presented as in Fig. 2b). *P < 0.001 (two-tailed unpaired t-test). () Immunofluorescence staining of kidney tissue s! ections of wild-type and Peli1−/− mice to detect deposits of immune complexes (red); counterstaining with the DNA-intercalating dye DAPI (blue) indentifies glomeruli. Original magnification, ×40. Data are representative of three experiments (,), five experiments (), three experiments with at least three mice per genotype (,) or two experiments with four mice per genotype (). * Figure 5: Peli1 deficiency causes hyperactivation of late-phase NF-κB. () Electrophoretic mobility-shift assay (EMSA) of nuclear extracts of total T cells isolated from 7-week-old wild-type and Peli1−/− mice and left untreated (−) or stimulated (+) with various combinations of plate-bound anti-CD3 and anti-CD28 (1 μg/ml), assessed with 32P-radiolabeled probes for NF-κB or the constitutively active nuclear transcription factor NF-Y. (,) EMSA of nuclear extracts of total T cells () or CD8+ T cells () left untreated or stimulated for 8 or 16 h (above lanes) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) or with PMA (5 ng/ml) and ionomycin (100 ng/ml), assessed with 32P-radiolabeled probes for various transcription factors (left margins). () EMSA of naive T cells left untreated or stimulated for 2 or 16 h (above lanes) with anti-CD3 and anti-CD28, assessed as in . () Protein kinase assay (KA) or immunoblot analysis (IB) of IKK activity (top blot) and Erk phosphorylation (p-; third blot) and the expression of various proteins (other blots! ; left margin) in total lysates of T cells stimulated with PMA plus ionomycin. GST, glutathione S-transferase; hsp60, 60-kilodalton heat-shock protein (loading control). Data are representative of four independent experiments with at least three mice per genotype. * Figure 6: Peli1 negatively regulates c-Rel. () EMSA of nuclear extracts of Peli1−/− T cells stimulated for 16 h with anti-CD3 and anti-CD28 in the presence of control immunoglobulin (Ig) or anti-c-Rel. () Immunoblot analysis of nuclear extracts (NE) and cytoplasmic extracts (CE) of total T cells obtained from 7-week-old wild-type and Peli1−/− mice and incubated for 16 h in the presence (+) or absence (−) of plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml). kDa, kilodaltons. Lamin B serves as a loading control throughout. () Immunoblot analysis of nuclear extracts of purified CD8+ T cells obtained from 7-week-old wild-type and Peli1−/− mice and left untreated (0) or stimulated for 8 or 16 h with anti-CD3 and anti-CD28 or with PMA (5 ng/ml) and ionomycin (100 ng/ml). () Immunoblot analysis of nuclear extracts of naive CD4+ T cells left untreated or stimulated for 2 or 16 h with anti-CD3 and anti-CD28. HDAC1, histone deacetylase 1 (loading control). () Immunoblot analysis of cytoplasmic and nuclear ! extracts of EL4 T cells infected with lentivirus containing empty vector (EV) or vector encoding short hairpin RNA targeting Peli1 (shPeli1), then left untreated or stimulated for 8 h with PMA (5 ng/ml) and ionomycin (100 ng/ml). () Immunoblot analysis of cells infected as in , then left untreated or stimulated for 8 or 16 h with PMA and ionomycin. () Immunoblot analysis of nuclear proteins (left margin) in EL4 cells infected with empty retroviral vector (EV) or retroviral vector encoding wild-type Peli1 or Peli1ΔC, then left untreated or stimulated for 16 h with PMA and ionomycin. Data are representative of three experiments (,,,,), five experiments () or four experiments (). * Figure 7: Peli1 induces ubiquitination of c-Rel. () Immunoassay of wild-type and Peli1−/− T cells incubated for 16 h in the presence (+) or absence (−) of anti-CD3 plus anti-CD28, then further incubated for 6 h with medium or MG132, followed by immunoprecipitation (IP) of proteins from lysates with anti-c-Rel and immunoblot analysis with anti-ubiquitin (α-Ub) to detect ubiquitin-conjugated c-Rel (c-Rel–Ub) or with anti-c-Rel. () Immunoassay of wild-type T cells left untreated (−) or stimulated (+) for 16 h with anti-CD3 plus anti-CD28, followed by immunoblot analysis of cell lysates with (top two blots) or without (Lysates; below) immunoprecipitation with preimmune serum (Pre; control) or anti-Peli1. () Immunoassay of HEK293T cells transfected with expression vector for c-Rel alone (−) or along with vector for hemagglutinin-tagged (HA-Peli1) full-length Peli1 (FL) or Peli1ΔC (ΔC), followed by immunoblot analysis of total cell lysates with (top two blots) or without (Lysates; below) immunoprecipitation with a! nti-c-Rel. () Immunoblot analysis of c-Rel immunoprecipitated from HEK293T cells transfected with c-Rel plus hemagglutinin-tagged ubiquitin (HA-Ub) alone or with Peli1 constructs as in . (,) Immunoassay of HEK293T cells transfected with c-Rel and E-tag–Peli1 (E-Peli1), along with hemagglutinin-tagged wild-type or mutant (K48R or K63R) ubiquitin, followed by immunoprecipitation with anti-c-Rel and immunoblot analysis () or immunoblot analysis without immunoprecipitation (). Data are representative of three independent experiments with at least three mice per genotype (,) or three independent experiments (–). Change history * Abstract * Change history * Author information * Supplementary informationCorrected online 20 September 2011In the version of this article initially published, in the Online Methods subsection "Analysis of TCR-proximal signaling," the catalog number for goat antibody to hamster immunoglobulin was incorrect. The correct number is 127-005-160. The error has been corrected in the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Mikyoung Chang & * Wei Jin Affiliations * Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. * Mikyoung Chang, * Wei Jin, * Jae-Hoon Chang, * Yichuan Xiao, * George C Brittain, * Jiayi Yu, * Xiaofei Zhou, * Yi-Hong Wang, * Xuhong Cheng & * Shao-Cong Sun * Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA. * Pingwei Li * Immunology Laboratory of Physician Scientists, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. * Brian A Rabinovich * Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. * Patrick Hwu * Present address: Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Wei Jin Contributions M.C. and W.J. designed and did the research and prepared the figures; J.-H.C. did the in vitro Treg cell assays and OVA tolerance assays; Y.X., J.Y. and X.Z. did the EAE experiment; G.C.B. did the experiments with knockdown and overexpression of Peli1 in EL4 cells; Y.-H.W. did the histology and immunohistochemistry; X.C. constructed Peli1 expression vectors; P.L., B.A.R. and P.H. contributed reagents; and S.-C.S. designed the research and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shao-Cong Sun Author Details * Mikyoung Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Jin Search for this author in: * NPG journals * PubMed * Google Scholar * Jae-Hoon Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Yichuan Xiao Search for this author in: * NPG journals * PubMed * Google Scholar * George C Brittain Search for this author in: * NPG journals * PubMed * Google Scholar * Jiayi Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaofei Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Yi-Hong Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Xuhong Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Pingwei Li Search for this author in: * NPG journals * PubMed * Google Scholar * Brian A Rabinovich Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Hwu Search for this author in: * NPG journals * PubMed * Google Scholar * Shao-Cong Sun Contact Shao-Cong Sun Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–12 and Methods Additional data - The inflammasome adaptor ASC regulates the function of adaptive immune cells by controlling Dock2-mediated Rac activation and actin polymerization
- Nat Immunol 12(10):1010-1016 (2011)
Nature Immunology | Article The inflammasome adaptor ASC regulates the function of adaptive immune cells by controlling Dock2-mediated Rac activation and actin polymerization * Sirish K Ippagunta1, 7 * R K Subbarao Malireddi1, 7 * Patrick J Shaw1, 7 * Geoffrey A Neale2 * Lieselotte Vande Walle3, 4 * Douglas R Green1 * Yoshinori Fukui5, 6 * Mohamed Lamkanfi3, 4 * Thirumala-Devi Kanneganti1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:1010–1016Year published:(2011)DOI:doi:10.1038/ni.2095Received13 June 2011Accepted27 July 2011Published online04 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide–exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by mod! ulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes. View full text Figures at a glance * Figure 1: ASC controls antigen uptake and presentation independently of inflammasomes. (–) Dose-dependent antigen-specific proliferation of lymphocytes among DCs obtained from naive wild-type (WT) and Asc−/− mice (n = 4–6 per group) and cultured for 72 h with wild-type CD4+ T cells in the presence of 0–100 μg BSA, assessed as uptake of [3H]thymidine () and as the concentration of interferon-γ (IFN-γ; ), IL-6 () and IL-17 (). *P < 0.01 (two-tailed Student's t-test). () Flow cytometry analysis of the phagocytosis of fluorescein-labeled zymosan A or polystyrene beads by wild-type, Nlrp3−/−, Asc−/− and Casp1−/− BMDCs after incubation together for 3 h at 37 °C. MFI, mean fluorescence intensity. *P < 0.0005 (two-tailed Student's t-test). () Flow cytometry analysis of the macropinocytosis of fluorescein-labeled luciferase yellow (LY), OVA or dextran by wild-type, Nlrp3−/−, Asc−/− and Casp1−/− BMDCs after incubation as in . *P < 0.005 (two-tailed Student's t-test). Data are representative of three independent experiments (–; mea! n and s.d.) or at least three independent experiments (,; mean and s.e.m. of triplicates). * Figure 2: ASC is required for lymphocyte migration in vitro and in vivo. () Total number of various cell populations (horizontal axis) among wild-type and Asc−/− spleen cells (left) and lymph node cells (LN; right) stained for CD4, CD8, CD19, CD11b and CD11c. () Migration of congenically marked wild-type and Asc−/− CD4+ T cells and B cells into the spleen and axillary lymph nodes of naive wild-type mice 48 h after injection of an equal ratio of congenically marked wild-type and Asc−/− CD4+ T cells or B cells, presented as a percentage of the cells injected. () Frequency of congenically marked CD4+ T cells and B cells in the spleen, blood and axillary lymph nodes (LN) of lethally irradiated mice 6 weeks after injection of an equal ratio of congenically marked wild-type and Asc−/− bone marrow cells, presented as a percentage of total bone marrow–derived lymphocytes. (,) Transwell chemotaxis assay of the migration of wild-type and Asc−/− splenocytes in vitro (n = 3–4 mice per group) toward SDF-1, SLC or BLC, presented as freq! uency among total migratory CD4+ T lymphocytes () or B cells (). *P < 0.005 (two-tailed Student's t-test). Data are representative of three independent experiments (mean and s.d. of triplicates). * Figure 3: ASC is essential for Rac activation and actin polymerization, induced by antigens or chemokines in DCs or lymphocytes, respectively. (,) Flow cytometry analysis of Rac GTPase activity () and F-actin polymerization () in wild-type and Asc−/− BMDCs treated for 0–60 s () or 0–30 s () with OVA, presented as relative light units (RLU; ) or mean fluorescence intensity () relative to baseline, set as 100. () Small G protein–activation assay of Rac activation in lysates of CD4+ T cells and B cells isolated from spleens of wild-type and Asc−/− mice and treated for 0–60 s in vitro with SDF-1 (500 ng/ml). () Flow cytometry analysis of F-actin polymerization in CD4+ T cells and B cells isolated from wild-type and Asc−/− mice (n = 1–3 per group) and treated for 0–60 s in vitro with SDF-1 (500 ng/ml). *P <0.05 (two-tailed Student's t-test). Data are representative of at least three independent experiments (mean and s.d. of triplicates). * Figure 4: ASC regulates Dock2 expression independently of inflammasomes and TLRs. () Microarray analysis gene expression among RNA from naive wild-type and Asc−/− BMDCs; right margin, genes with a transcript difference of threefold or more in Asc−/− cells relative to the expression in wild-type cells. () Quantitative PCR analysis of Dock2 mRNA expression in naive wild-type, Nlrp3−/−, Asc−/− and Casp1−/− BMDCs, presented relative to the expression of Gapdh (encoding glyceraldehyde phosphate dehydrogenase). () Quantitative PCR analysis of Dock2 mRNA expression in purified wild-type and Asc−/− CD4+ T cells and B cells, presented as in . () Immunoblot analysis of Dock2, FSTL1, FABP4 and galanin in naive wild-type, Nlrp3−/−, Asc−/− and Casp1−/− BMDCs. kDa, kilodaltons. () Immunoblot analysis of Dock2 and ASC in purified wild-type, Nlrp3−/−, Asc−/− and Casp1−/− CD4+ T cells and B cells. () Immunoblot analysis of Dock2 in lysates of naive wild-type, Tlr2−/−, Tlr4−/−, Myd88−/− and Trif−/− BMDCs. Acti! n serves as a loading control throughout. *P < 0.005 (two-tailed Student's t-test). Data are representative of at least three independent experiments (mean and s.d. in ,). * Figure 5: ASC localizes to the nucleus and controls the stability of Dock2 mRNA. () Immunoblot analysis of ASC and caspase-1 (Casp1) in the cytosolic (Cyto) and nuclear (Nuc) compartments of wild-type and Asc−/− BMDCs left untreated (UT) or primed for 4 h with LPS (1 μg/ml), in the presence of ATP (5 mM) for the final 15 min (LPS + ATP). GAPDH and lamin-B serve as compartment-specific markers for the cytosolic and nuclear compartments, respectively. () Microscopy of wild-type and Asc−/− BMDCs left untreated or primed and treated as in , then washed in PBS, fixed, made permeable and stained for ASC and caspase-1. The DNA-intercalating dye DAPI stains nuclei. Original magnification, ×63. () Luciferase activity of wild-type and Asc−/− BMDCs transfected by nucleofection with empty reporter vector (EV) or luciferase vector containing the Dock2 promoter (Dock2P); results are presented relative to renilla luciferase activity. () Quantitative RT-PCR analysis of Dock2 and Actb mRNA among total RNA from wild-type and Asc−/− BMDCs treated with DRB! (50 μM) or actinomycin D (ActD; 5 μg/ml), normalized to the expression of Gapdh mRNA and presented relative to baseline expression, set as 100%; the half-life of mRNA (upward arrows) was calculated as the time required for decay to 50% of baseline. *P < 0.005 (Student's t-test). Data are representative of at least three independent experiments (mean and s.d. in ,). * Figure 6: Dock2 is critical for antigen uptake by DCs and restores immune-cell functions in the absence of ASC. (–) Macropinocytosis of FITC-labeled dextran (), OVA () or lucifer yellow () by wild-type and Dock2−/− BMDCs after incubation together for 3 h at 37 °C. (–) Phagocytosis of fluorescein-labeled zymosan A () or polystyrene beads () by wild-type and Dock2−/− BMDCs after incubation as in . () Macropinocytosis of fluorescein-labeled OVA by wild-type and Asc−/− BMDCs transfected by nucleofection with plasmid expressing GFP or GFP-Dock2, assessed by flow cytometry 24 h after transfection. () Transwell chemotaxis assay of the in vitro migration of wild-type and Asc−/− CD4+ T lymphocytes expressing either GFP or GFP-Dock2 toward SLC; results are presented as the frequency among the total migrating T cell population. NS, not significant; *P < 0.05 (two-tailed Student's t-test). Data are representative of at least three independent experiments (–; mean and s.e.m. of triplicates). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sirish K Ippagunta, * R K Subbarao Malireddi & * Patrick J Shaw Affiliations * Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Sirish K Ippagunta, * R K Subbarao Malireddi, * Patrick J Shaw, * Douglas R Green & * Thirumala-Devi Kanneganti * Department of Hartwell Center for Bioinformatics & Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Geoffrey A Neale * Department of Biochemistry, Ghent University, Ghent, Belgium. * Lieselotte Vande Walle & * Mohamed Lamkanfi * Department of Medical Protein Research, VIB, Ghent, Belgium. * Lieselotte Vande Walle & * Mohamed Lamkanfi * Division of Immunogenetics, Kyushu University, Kyushu, Japan. * Yoshinori Fukui * Department of Immunobiology and Neuroscience Medical Institute of Bioregulation, Kyushu University, Kyushu, Japan. * Yoshinori Fukui Contributions T.-D.K., M.L., S.K.I., P.J.S. and R.K.S.M. designed research; S.K.I., P.J.S., R.K.S.M., did research; G.A.N. did bioinformatic analyses; L.V.W. confirmed ASC-dependent Dock2 expression in an independently generated line of ASC-deficient mice; D.R.G. contributed to the writing of the manuscript and conceptual insights; Y.F. provided reagents; T.-D.K., M.L., S.K.I., P.J.S., R.K.S.M., G.A.N. and Y.F. analyzed data; P.J.S., M.L. and T.-D.K. wrote the paper; and T.-D.K. conceived of the study, designed the experiments and provided overall direction. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Mohamed Lamkanfi or * Thirumala-Devi Kanneganti Author Details * Sirish K Ippagunta Search for this author in: * NPG journals * PubMed * Google Scholar * R K Subbarao Malireddi Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick J Shaw Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey A Neale Search for this author in: * NPG journals * PubMed * Google Scholar * Lieselotte Vande Walle Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas R Green Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshinori Fukui Search for this author in: * NPG journals * PubMed * Google Scholar * Mohamed Lamkanfi Contact Mohamed Lamkanfi Search for this author in: * NPG journals * PubMed * Google Scholar * Thirumala-Devi Kanneganti Contact Thirumala-Devi Kanneganti Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (856K) Supplementary Figures 1–11 Additional data
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