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- Nat Immunol 11(11):971 (2010)
The European Union has passed new laws for the protection of laboratory animals according to the 'three Rs' concept. - IL-1 family nomenclature
- Nat Immunol 11(11):973 (2010)
Newly cloned interleukin 1 (IL-1) family members1, 2, 3 were originally given an IL-1 family (IL-1F) designation4, but as functions have now been elucidated for several of these5, 6, we propose that each now be assigned an individual interleukin designation. IL-1F6, IL-1F8 and IL-1F9 are encoded by distinct genes but use the same receptor complex (IL-1Rrp2 and AcP), are proinflammatory and deliver nearly identical signals7, 8, 9, 10, 11, 12. - A model for harmonizing flow cytometry in clinical trials
- Nat Immunol 11(11):975-978 (2010)
Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues. - Host DNase TREX1 hides HIV from DNA sensors
- Nat Immunol 11(11):979-980 (2010)
Human immunodeficiency virus type 1 (HIV-1) seems to avoid detection by nucleic acid sensors. This is probably due to the host exonuclease TREX1, which degrades HIV DNA generated during HIV-1 infection. - A novel axis of innate immunity in cancer
- Nat Immunol 11(11):981-982 (2010)
Neutrophils can function as chief effector cells in inflammation but can also regulate excessive inflammatory responses by secreting anti-inflammatory cytokines. The acute-phase reactant SAA-1 seems to be pivotal in the control of such plasticity. - Outfoxing Foxo1 with miR-182
- Nat Immunol 11(11):983-984 (2010)
Induction of the microRNA miR-182 by interleukin 2 in helper T lymphocytes targets the transcription factor Foxo1 and promotes clonal expansion. Targeting this process opens new possibilities for adjuvancy, immunosuppression and anti-inflammatory therapeutics. - PYHIN proteins: center stage in DNA sensing
- Nat Immunol 11(11):984-986 (2010)
Innate immune responses to pathogens are often triggered by nucleic acids, including DNA delivered to the cytoplasm of cells. IFI16 is a newly identified cytoplasmic DNA sensor that induces the transcription of genes involved in the innate response. - Research Highlights
- Nat Immunol 11(11):987 (2010)
- B cell follicles and antigen encounters of the third kind
- Nat Immunol 11(11):989-996 (2010)
Nature Immunology | Review B cell follicles and antigen encounters of the third kind * Jason G Cyster1jason.cyster@ucsf.edu Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 11 ,Pages:989–996Year published:(2010)DOI:doi:10.1038/ni.1946Published online19 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Defining where and in what form lymphocytes encounter antigen is fundamental to understanding how immune responses occur. Although knowledge of the recognition of antigen by CD4+ and CD8+ T cells has advanced greatly, understanding of the dynamics of B cell–antigen encounters has lagged. With the application of advanced imaging approaches, encounters of this third kind are now being brought into focus. Multiple processes facilitate these encounters, from the filtering functions of lymphoid tissues and migration paths of B cells to the antigen-presenting properties of macrophages and follicular dendritic cells. This Review will discuss how these factors work together in the lymph node to ensure efficient and persistent exposure of B cells to diverse forms of antigen and thus effective triggering of the humoral response. View full text Figures at a glance * Figure 1: Multiple cues direct B cell migration through the lymph node follicle. A B cell enters from an HEV, migrates into a follicle in a CXCR5-CXCL13–dependent manner and then is attracted to the outer follicle in response to both CXCL13 and EBI2 ligand (EBI2L). After migrating back through the FDC network, the cell travels through the T cell zone (T zone)–proximal follicle using CCR7 to respond to CCL21 (and CCL19; not presented here) and CXCR5 before exiting into a LYVE1+ cortical sinus in an sphingosine 1-phosphate receptor type 1 (S1P1)-dependent manner. CCL21 is produced by fibroblastic reticular cells (FRC) and HEVs; CXCL13 is produced by MRCs and FDCs; and the source of EBI2L is not known, but it is suggested to be more abundant in the interfollicular regions and outer follicle than in the follicle center. The morphology of MRCs are not well defined and there is probably additional heterogeneity in the follicular stroma not presented here. Some of the stromal cells are associated with collagen fibers (not presented here) and the cells form ! an interconnected network. MΦ, macrophage. * Figure 2: SCS macrophages overlying a lymph node follicle. Confocal microscopy of a peripheral lymph node frozen section stained for various markers (bottom left corners); CD169+CD11b+ macrophages project through the LYVE1+ SCS-lining cells. Original magnification, ×63. Images by Elizabeth E. Gray. * Figure 3: Multiple modes for the encounter of follicular B cells with antigen. The modes by which particulate antigens (such as immune complexes and viral particles; ), soluble antigens of small hydrodynamic radius (in some cases released from large antigens by proteolysis; ), or antigens too large to enter passively into lymphatic vessels () encounter B cells. () Antigen is captured and displayed by SCS macrophages and then is encountered directly by cognate B cells or is transported by noncognate B cells to FDCs for later encounter with cognate B cells. () Antigen accesses the follicle via gaps in the SCS lining or via conduits and is then encountered by cognate B cells. () Antigen is carried into the follicle-proximal cortex inside DCs, is recycled to their cell surface and is encountered by cognate B cells. * Figure 4: Models for the translocation of opsonized particulate antigen from the sinus to the follicular surface of the SCS macrophage. Particles in the lymph percolate through the SCS and some are captured by unknown receptors on the SCS macrophage, whereas excess material reaches medullary sinuses and is phagocytosed and degraded by medullary macrophages. In model 1 (top right), particulate antigen is retained on the SCS macrophage surface, undergoes fragmentation and moves unidirectionally on the cell surface by unknown mechanisms from the SCS to the follicle, where it is displayed to migrating B cells. In model 2 (bottom right), the particulate antigen is endocytosed into vesicles, transported directionally within the cell and then returned to the cell surface for display. Over a period of hours, antigen that is not removed by migrating B cells is probably degraded by the SCS macrophage. Author information * Abstract * Author information Affiliations * Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, USA. * Jason G Cyster Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Jason G Cyster (jason.cyster@ucsf.edu) Additional data - IFI16 is an innate immune sensor for intracellular DNA
- Nat Immunol 11(11):997-1004 (2010)
Nature Immunology | Article IFI16 is an innate immune sensor for intracellular DNA * Leonie Unterholzner1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Sinead E Keating1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Marcin Baran1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kristy A Horan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Søren B Jensen2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Shruti Sharma3 Search for this author in: * NPG journals * PubMed * Google Scholar * Cherilyn M Sirois3 Search for this author in: * NPG journals * PubMed * Google Scholar * Tengchuan Jin4 Search for this author in: * NPG journals * PubMed * Google Scholar * Eicke Latz3, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * T Sam Xiao4 Search for this author in: * NPG journals * PubMed * Google Scholar * Katherine A Fitzgerald3 Search for this author in: * NPG journals * PubMed * Google Scholar * Søren R Paludan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew G Bowie1agbowie@tcd.ie Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:997–1004Year published:(2010)DOI:doi:10.1038/ni.1932Received06 July 2010Accepted09 August 2010Published online03 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly associated with IFN-β-inducing viral DNA motifs. STING, a critical mediator of IFN-β responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-β induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs). View full text Figures at a glance * Figure 1: Induction of IFN-β by a VACV DNA motif. () Reporter gene assay of IFNB promoter activity in HEK293T cells mock transfected (MT) or transfected for 16 h with nucleic acids (0.5 or 5 μg/ml; wedges). s-VACV, sonicated VACV DNA. () IFN-β mRNA in PMA-treated THP-1 cells mock transfected or transfected for 6 h with 1 μg/ml of poly(dA:dT) or with 200 ng/ml of DNA isolated from VACV, calf thymus or Listeria monocytogenes. (,) IFN-β mRNA in THP-1 cells () or immortalized MEFs () mock transfected or transfected for 6 h with nucleic acids at a concentration of 1 μg/ml () or 5 μg/ml (). ss-FVACV 70mer, forward strand only of dsVACV 70mer; ss-RVACV 70mer, reverse strand only of dsVACV 70mer. () IFNB promoter activity in HEK293T cells mock transfected or transfected for 16 h with nucleic acids at a concentration of 5 μg/ml. (,) IFN-β mRNA in THP-1 cells () or RAW264.7 cells () mock transfected or transfected for 6 h with DNA oligonucleotides or sequences of various length (horizontal axis; in base pairs) derived from VA! CV 70mer (sequences, Supplementary Methods), GC-rich VACV 70mer (70 (GC)) or ISD, each at a concentration of 1 μg/ml. Results for mRNA and promoter activity are presented relative to those of mock-transfected cells. Data are from one experiment representative of two (error bars, s.d.). * Figure 2: Induction of IFN-β by a VACV DNA motif is independent of known DNA-sensing pathways. (–) IFN-β mRNA in immortalized BMDMs derived from mice containing (+/+) or lacking (−/−) the signaling components MyD88 (Myd88; ,), TRIF (Ticam1; ,) or DAI (Zbp1; ,) and transfected for 6 h with 5 μg/ml of dsVACV 70mer (,,) or poly(dA:dT) () or stimulated for 6 h with LPS () or poly(I:C) (); results are presented relative to those of cells from wild-type mice (WT). Data are from three independent experiments in triplicate (mean and s.e.m). () IFN-β mRNA (left) in immortalized BMDMs mock transfected or transfected for 6 h with nucleic acids, and IFNB promoter activity (right) in HEK293T cells transfected for 16 h with 100 ng of the RNA extracted from BMDMs (results presented as in Fig. 1). Data are from one experiment representative of three (error bars, s.d.). (–) IFN-β mRNA in immortalized BMDMs or MEFs derived from mice lacking the signaling component IRF3 (Irf3; ) or TBK1 (Tbk1; ,) and transfected for 6 h with dsVACV 70mer (5 μg/ml); results are presented as ! in –. Data are from three independent experiments in triplicate (mean and s.e.m). * Figure 3: IFI16 binds to immunostimulatory viral DNA. () Human PYHIN proteins (left), including the pyrin, HINa and HINb domains (scale bar (right), size in amino acids (aa)). () Immunoblot analysis of IFI16 among proteins precipitated from cytoplasmic extracts of PMA-treated THP-1 cells incubated with biotinylated ssVACV 70mer or dsVACV 70mer immobilized on streptavidin beads. () IFN-β mRNA (left) in THP-1 cells transfected for 6 h with 1 μg/ml of dsVACV 70mer or poly(I:C) alone (control (Ctrl)) or in the presence of 1 μg/ml of the reverse strand only of dsVACV 70mer (ss-RVACV 70mer), and IFNB promoter activity (right) in HEK293T cells transfected for 16 h with 50 ng/ml of poly(dA:dT) alone (Ctrl) or together with 50 ng/ml of the reverse strand only of dsVACV 70mer (ss-RVACV 70mer) or dsVACV 70mer (results presented relative to control). () Microscopy of PMA- and IFN-α-treated THP-1 cells grown on coverslips and mock transfected or transfected for 1 h with 2.5 μg/ml of dsVACV 70mer or poly(I:C), then fixed and stained wit! h antibody to IFI16 (red) and with the DNA-intercalating dye DAPI (blue), for visualization of DNA and poly(I:C). () Microscopy of PMA-treated THP-1 cells transfected for 3 h with fluorescein isothiocyanate–labeled HSV 60mer (FITC-60mer; green), then fixed and stained for IFI16 (red) or with DAPI (blue; DNA). Inset (,), magnification of area outlined in main image. Scale bar (,), 10 μm. () AlphaScreen assessment of the binding of dsVACV 70mer to the IFI16 domains HINa, HINb or HINab (both HINa and HINb) or a GB1 expression tag: top, 30 nM DNA with increasing concentrations of HIN or GB1; bottom, 30 nM HIN or GB1 with increasing concentrations of biotin-labeled dsVACV 70mer. Data are from one experiment representative of three (mean and s.d. in ). * Figure 4: Role for STING in IFI16-mediated IFN-β induction. () Immunoblot analysis of TBK1 and DDX3 among proteins precipitated from cytoplasmic extracts of PMA-treated THP-1 cells incubated with biotinylated ssVACV 70mer or dsVACV 70mer immobilized on streptavidin beads. () Immunoassay of THP-1 cells pretreated with PMA and IFN-α and mock transfected or transfected for 4 h with 1 μg/ml of dsVACV 70mer (+); lysates were immunoprecipitated (IP) with antibody to STING and then analyzed by immunoblot (IB) for IFI16 and STING. () Immunoassay of the association of IFI16, from lysates of DNA-treated THP-1 cells, with Myc-tagged human (hu) or mouse (mu) STING overexpressed in HEK293 cells and immobilized on sepharose beads; coimmunoprecipitated IFI16 was detected by immunoblot analysis. pCMV, vector; Ab, antibody heavy chain. (,) Release of IFN-β protein from BMDMs containing (Tmem173+/+) or lacking (Tmem173−/−) STING, mock transfected or transfected for 18 h with either dsVACV 70mer or HSV 60mer or mock transfected or infected for 1! 8 h with HSV-1 or Sendai virus (SeV). () Secretion of IFN-β protein from immortalized Asc−/− BMDMs mock transfected or transfected for 18 h with DNA. Data are from one experiment representative of two (mean and s.d. in –). * Figure 5: IFI16 is required for DNA-mediated gene induction. () IFI16 mRNA (left) in THP-1 cells, PMA-treated THP-1 cells and HEK293 cells, and IFN-β mRNA (right) in untreated or PMA-treated THP-1 cells transfected for 6 h with dsVACV 70mer (results presented as in Fig. 1). (,) Immunoblot analysis of IFI16 protein () and real-time PCR analysis of IFN-β mRNA () in THP-1 cells treated with control (Ctrl) or IFI16-specific siRNA (key) and mock transfected or transfected for 6 h with 1 μg/ml of dsVACV 70mer. () Domain organization (left) of human IFI16 (top) and members of the mouse PYHIN family (below), drawn to scale (right). Boxes indicate conserved domains. *, mouse PYHIN protein most similar to human IFI16 (p204). (,) Immunoblot analysis of p204 () and real-time PCR analysis of p204, IFN-β, CCL5 and TNF mRNA () in RAW264.7 cells treated with control (Ctrl) or p204-specific siRNA (key) and mock transfected or transfected for 6 h with 1 μg/ml of dsVACV 70mer. () Analysis of p204, IFN-β, CCL5 and TNF mRNA in siRNA-treated MEFs moc! k transfected or transfected for 6 h with 1 μg/ml of dsVACV 70mer. () IFN-β mRNA in siRNA-treated RAW264.7 cells mock transfected or transfected for 6 h with HSV 60mer DNA. () Release of IFN-β protein from BMDMs electroporated with siRNA and then mock transfected or transfected with oligomers for 18 h; results are presented relative to those of mock-transfected cells not treated with siRNA. *P < 0.05, compared with control siRNA (Student's t-test). Data are from one experiment representative of two or three (mean and s.d. in ,,–). * Figure 6: IFI16 is required for VACV 70mer DNA-stimulated transcription factor activation. () Confocal microscopy of siRNA-treated RAW264.7 cells grown on glass coverslips and mock transfected or transfected for 6 h with 2.5 μg/ml of dsVACV 70mer or poly(I:C), then fixed and stained for NF-κB p65 (red) and IRF3 (green); nuclei were visualized by DAPI (blue). Original magnification, ×60. () Staining of p65 (top) or IRF3 (bottom) in the nucleus of cells treated as in a; cells with nuclear staining are presented as a percentage of total cells (n = 200 cells per sample). Data are representative of four experiments. * Figure 7: IFI16 is required for the innate immune response to HSV-1. () IFN-β mRNA in wild-type (WT) or Myd88−/−Ticam1−/− BMDMs mock infected or infected for 6 h with HSV-1 (multiplicity of infection, 10). () IFN-β mRNA in RAW264.7 cells given no pretreatment (Ctrl) or pretreated for 2 h with ML60812, then mock infected or infected for 6 h with HSV-1, or mock transfected or transfected for 6 h with poly(dA:dT). (,) IFN-β mRNA (,), CXCL10 mRNA (), interleukin 6 (IL-6) mRNA () and TNF mRNA () in siRNA-treated RAW264.7 cells mock infected or infected for 6 h with HSV-1 () or Sendai virus (). () IFN-β protein expression in siRNA-treated RAW264.7 cells mock infected or infected for 20 h with HSV-1 or Sendai virus. () Confocal microscopy of siRNA-treated RAW264.7 cells grown on glass coverslips and mock infected or infected for 6 h with HSV-1 or Sendai virus, then fixed and stained for NF-κB p65 (red) and IRF3 (green); nuclei were visualized by DAPI staining (blue). Original magnification, ×60. () Staining of p65 (left) or IRF3 (right! ) in the nucleus of cells treated as in ; cells with nuclear staining are presented as a percentage of total cells (n = 200 cells per sample). () Plaque assay of virus in culture supernatants of RAW264.7 cells mock transfected or transfected with HSV 60mer (2 μg/ml) and infected with HSV-1 (multiplicity of infection, 1). PFU, plaque-forming units. Results for mRNA (–) are presented relative to those of mock-transfected cells. *P < 0.001, compared with control siRNA (Student's t-test). Data are from one experiment representative of three (mean and s.d. in –,). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Leonie Unterholzner & * Sinead E Keating Affiliations * School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland. * Leonie Unterholzner, * Sinead E Keating, * Marcin Baran & * Andrew G Bowie * Department of Medical Microbiology and Immunology, Aarhus University, Aarhus, Denmark. * Kristy A Horan, * Søren B Jensen & * Søren R Paludan * Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Søren B Jensen, * Shruti Sharma, * Cherilyn M Sirois, * Eicke Latz & * Katherine A Fitzgerald * Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Tengchuan Jin & * T Sam Xiao * Institute of Clinical Chemistry and Pharmacology, University of Bonn, Bonn, Germany. * Eicke Latz Contributions L.U. and S.E.K. did experiments, analyzed data and cowrote the manuscript; M.B., K.A.H., S.B.J., S.S., C.M.S. and T.J. did experiments and analyzed data; E.L. provided expertise; T.S.X., K.A.F. and S.R.P. supervised experiments and analyzed data; and A.G.B. designed and supervised the study, analyzed data and cowrote the manuscript. M.B. and K.A.H. contributed equally to this work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Andrew G Bowie (agbowie@tcd.ie) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–8 and Supplementary Methods Additional data - The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1
- Nat Immunol 11(11):1005-1013 (2010)
Nature Immunology | Article The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1 * Nan Yan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ashton D Regalado-Magdos1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bart Stiggelbout1 Search for this author in: * NPG journals * PubMed * Google Scholar * Min Ae Lee-Kirsch2 Search for this author in: * NPG journals * PubMed * Google Scholar * Judy Lieberman1lieberman@idi.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:1005–1013Year published:(2010)DOI:doi:10.1038/ni.1941Received27 July 2010Accepted27 August 2010Published online26 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Viral infection triggers innate immune sensors to produce type I interferon. However, infection of T cells and macrophages with human immunodeficiency virus (HIV) does not trip those alarms. How HIV avoids activating nucleic acid sensors is unknown. Here we found that the cytosolic exonuclease TREX1 suppressed interferon triggered by HIV. In Trex1−/− mouse cells and human CD4+ T cells and macrophages in which TREX1 was inhibited by RNA-mediated interference, cytosolic HIV DNA accumulated and HIV infection induced type I interferon that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate interferon expression via a pathway dependent on the kinase TBK1, the adaptor STING and the transcription factor IRF3. HIV-stimulated interferon production in cells deficient in TREX1 did not involve known nucleic acid sensors. View full text Figures at a glance * Figure 1: TREX1 deficiency inhibits HIV replication and activates IFN-β in response to HIV infection. () Luciferase (Luc) activity in wild-type (WT) and Trex1−/− primary MEFs infected with VSV-G-pseudotyped HIV with luciferase reporter expression driven by the HIV LTR (as described in Results). (–) Quantitative RT-PCR analysis (,) and ELISA () of cytokine induction in MEFs left uninfected (HIV −) or infected as described in (HIV +) and left untreated (−) or treated with inhibitors of reverse transcription (RT) or integrase (IN), assessed 22 h after infection. Results are presented in arbitrary units (AU). (,) Luciferase activity () and late reverse transcripts () in primary MEFs infected as in and left untreated (HIV) or treated with inhibitors of reverse transcription (HIV RTin) or integrase (HIV INin), each added at the same time as HIV, assessed 48 h () or 10 h () after infection. () IFN-β induction in MEFs infected with HIV (far left) or equivalent amounts (based on p24 ELISA) of virus-like particles (VLP) or HIV inactivated by heating for 5 min at 95 °C (Hea! t-inact HIV); results are presented in arbitrary units (AU). () HIV autointegration in primary MEFs infected and treated with inhibitors as in ,. Data are representative of at least three independent experiments (error bars, s.d.). * Figure 2: HIV-stimulated expression of interferon is IRF3 dependent. () Quantitative RT-PCR analysis of IFN-β induction in wild-type (WT), Trex1−/− (KO) and Trex1−/−Irf3−/− (DKO) primary MEFs infected with VSV-G-pseudotyped HIV-GFP, assessed 22 h after infection; results are presented in arbitrary units. () Luciferase activity of primary MEFs infected with HIV-luciferase, assessed 48 h after infection. () HIV autointegration in primary MEFs infected as in . () Epifluorescence microscopy of the translocation of IRF3 to the nucleus in wild-type and Trex1−/− MEFs left uninfected (– HIV) or infected with HIV (+ HIV) and stained 22 h later for IRF3 (red) and with the DNA-intercalating dye DAPI (blue). Original magnification, ×63. () Flow cytometry analysis of GFP expression in wild-type and Trex1−/− MEFs infected as in , assessed 24 h after infection. Data are representative of three experiments (,), at least three independent experiments (; error bars (–), s.d.) or two experiments (), or are from one of three independent! experiments (). * Figure 3: Cytosolic HIV DNA in Trex1−/− cells is the trigger for interferon expression. () Quantitative PCR analysis of HIV DNA and quantitative RT-PCR analysis of IFN-β mRNA in wild-type and Trex1−/− cells infected with HIV-GFP, presented relative to the peak value in Trex1−/− cells. () Quantitative RT-PCR analysis of HIV genomic RNA (gRNA) at 2 h and 5 h after infection as in , presented relative to the value in wild-type cells at 2 h. (,) Quantitative RT-PCR analysis of IFN-β mRNA () and quantitative PCR analysis of cytosolic HIV DNA () in wild-type and Trex1−/− MEFs infected with HIV at an MOI of 0.5, 2 or 8; results relative to those of wild-type MEFs infected at an MOI of 0.5. () Two-step semiquantitative PCR assay9 of integrated DNA in MEFs infected as in ,. () Luciferase activity of wild-type MEFs treated with conditioned medium from wild-type, Trex1−/− or Trex1−/−Irf3−/− cells left uninfected (HIV −) or infected with HIV-GFP (HIV +) (below graph) and then infected with HIV-luciferase, assessed 48 h after infection and presen! ted relative to the activity of cells incubated with medium alone (far left). () Luciferase activity of wild-type and Trex1−/− MEFs treated with medium alone (far left; MEF −, mIFN-β nAb −) or with conditioned medium (preincubated with (+) or without (−) neutralizing antibody to mouse IFN-β (mIFN-β nAb)) and infected with HIV-luciferase. () Luciferase activity of wild-type MEFs infected with HIV-luciferase and treated with various concentrations (horizontal axis) of purified recombinant mouse IFN-β (mIFN-β). *P < 0.01 (Student's t-test). Data are representative of three independent experiments (error bars, s.d.). * Figure 4: Recognition of HIV products of reverse transcription by enzymatically active TREX1 suppresses interferon induction. () Synthetic nucleic acids used in as generated during reverse transcription. () Quantitative RT-PCR analysis of IFN-β expression by wild-type and Trex1−/− cells mock-transfected (Mock) or transfected with the synthetic nucleic acids in and assessed 6 h later. () Quantitative PCR analysis of cytosolic DNA in wild-type and Trex1−/− MEFs (left) or wild-type and TREX1-mutant (TREX1(D18N)) human fibroblasts (right) infected with HIV or transfected with ssDNA and dsDNA oligonucleotides of Gag-encoding sequence (100 base pairs), assessed with primers for sequence encoding Gag 10 h after infection or 3 h after transfection. *P < 0.01 (Student's t-test). () Immunoprecipitation (IP), with anti-Flag or immunoglobulin G (IgG), of cytosolic lysates of HeLa-CD4 cells expressing Flag-tagged TREX1, left uninfected (HIV −) or infected for 10 h with HIV IIIB (HIV +), followed by quantitative PCR (qPCR) or quantitative RT-PCR (qRT-PCR), respectively, of DNA and RNA extracted from t! he immunoprecipitates. *P < 0.05 (Student's t-test). () Luciferase activity (left) of wild-type and Trex1−/− MEFs left untransfected (−) or transfected to express GFP alone (GFP) or GFP-tagged TREX1, either wild-type (GFP-TREX1) or an enzymatically inactive mutant (GFP-D18N), and then infected with HIV-luciferase, assessed 48 h after infection. *P < 0.01 (Student's t-test). Right, immunoblot analysis of TREX1, GFP and tubulin (Tub; loading control). GFP-TREX1 FL, full-length TREX1 (wild type or D18N) fused to GFP. () Quantitative PCR analysis of HIV DNA in wild-type and Trex1−/− MEFs treated as in ; results are presented relative to those of mock-transfected wild-type MEFs. *P < 0.01 (Student's t-test). Data are representative of three independent experiments (,,; error bars, s.d.) or two independent experiments (,; error bars, s.d. of triplicates). * Figure 5: TREX1-specific siRNA treatment induces IFN-α and IFN-β and inhibits HIV replication in primary human immune cells. () Experimental procedure: human MDMs were transfected with control siRNA or siRNA specific for TREX1 alone or TREX1 plus IRF3 and infected 3 d later with HIV (BaL strain). () Quantitative RT-PCR analysis of TREX1 and IRF3 mRNA in MDMs from two different donors (1 and 2), treated as outlined in and assessed 2 d after siRNA transfection; results are relative to those of cells treated with control (Ctrl) siRNA. () Cytosolic HIV DNA in MDMs treated as outlined in , presented relative to results of cells treated with control siRNA. () ELISA of p24 released into culture supernatants of MDMs treated as outlined in , assessed on days 4–10 after infection. ND, not determined (not measured on day 1). () Quantitative RT-PCR analysis of IFN-α and IFN-β mRNA in MDMs left uninfected (left) or treated as outlined in , assessed on days 1, 4 or 7 after infection and presented relative to the control values on day 1. Flow cytometry analysis of p24 immunostaining indicated that 20% of cel! ls were infected by day 7 (data not shown). *P < 0.01 (Student's t-test). () TREX1 mRNA in CD4+ T cells positively selected from peripheral blood mononuclear cells, transfected with no siRNA (Mock), control siRNA (600 pmol) or TREX1-specific siRNA (200, 400 or 600 pmol per 3 × 106 cells) and infected 3 d later with HIV IIIB; mRNA measured 2 d after transfection is presented relative to that in cells treated with control siRNA. () Cytosolic HIV DNA in the cells in , assessed 10 h after infection. (,) IFN-α mRNA () and IFN-β mRNA () in the cells in , measured 22 h after infection and presented relative to results obtained with control cells. (–) Flow cytometry analysis of HIV IIIB replication in CD4+ T cells treated with control or TREX1-specific siRNA and then left uninfected (No HIV) or infected with HIV IIIB, and assessed 3 d after infection as percent p24+ cells (; numbers above bracketed lines), frequency of p24+ cells () and the mean fluorescence intensity (MFI) of! p24 (). FITC, fluorescein isothiocyanate. *P < 0.05 (Student'! s t-test). Data are representative of two experiments ((–); error bars, s.d. () or average and s.d. of two replicates from two independent donors (,)) or 2 experiments (; error bars, s.d.) or are from two independent experiments (–; error bars, s.d. of six replicates). * Figure 6: HIV-stimulated interferon induction requires IRF3, TBK1 and STING. () Quantitative RT-PCR analysis of genes related to innate immunity in Trex1−/− MEFs 48 h after transfection with control or gene-specific siRNA. Ddx58 encodes RIG-I; Tmem173 encodes STING. *P < 0.01 (Student's t-test). () Quantitative RT-PCR analysis of IFN-β mRNA in wild-type and Trex1−/− MEFs left untransfected (siRNA −) or transfected with control or gene-specific siRNA (siRNA +), and left uninfected (HIV −) or infected with VSV-G-pseudotyped HIV (HIV +), assessed 24 h after infection; results are presented relative to those of untransfected wild-type MEFs. *P < 0.01 (Student's t-test). () Quantitative PCR analysis of cytosolic HIV DNA in Trex1−/− MEFs transfected with control or gene-specific siRNA, assessed 10 h after infection with HIV-GFP; results are presented relative to those of infected Trex1−/− MEFs transfected with control siRNA. Data are representative of three independent experiments (error bars, s.d.). Author information * Abstract * Author information * Supplementary information Affiliations * Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Boston, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA. * Nan Yan, * Ashton D Regalado-Magdos, * Bart Stiggelbout & * Judy Lieberman * Children's Hospital, Technical University Dresden, Dresden, Germany. * Min Ae Lee-Kirsch Contributions N.Y. conceived of the study, designed and did most experiments and helped write the paper; A.D.R.-M. and B.S. helped do the experiments; M.A.L.-K. provided human cell lines and scientific advice; and J.L. conceived of and supervised the study and helped write the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Judy Lieberman (lieberman@idi.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5 and Supplementary Tables 1–2 Additional data - IL-37 is a fundamental inhibitor of innate immunity
- Nat Immunol 11(11):1014-1022 (2010)
Nature Immunology | Article IL-37 is a fundamental inhibitor of innate immunity * Marcel F Nold1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia A Nold-Petry1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jarod A Zepp1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brent E Palmer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philip Bufler3 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles A Dinarello1cdinarello@mac.com Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:1014–1022Year published:(2010)DOI:doi:10.1038/ni.1944Received12 July 2010Accepted09 September 2010Published online10 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The function of interleukin 37 (IL-37; formerly IL-1 family member 7) has remained elusive. Expression of IL-37 in macrophages or epithelial cells almost completely suppressed production of pro-inflammatory cytokines, whereas the abundance of these cytokines increased with silencing of endogenous IL-37 in human blood cells. Anti-inflammatory cytokines were unaffected. Mice with transgenic expression of IL-37 were protected from lipopolysaccharide-induced shock, and showed markedly improved lung and kidney function and reduced liver damage after treatment with lipopolysaccharide. Transgenic mice had lower concentrations of circulating and tissue cytokines (72–95% less) than wild-type mice and showed less dendritic cell activation. IL-37 interacted intracellularly with Smad3 and IL-37-expressing cells and transgenic mice showed less cytokine suppression when endogenous Smad3 was depleted. IL-37 thus emerges as a natural suppressor of innate inflammatory and immune responses. View full text Figures at a glance * Figure 1: Production and silencing of endogenous IL-37 in human PBMCs. () Left, immunoblot analysis of PBMC lysates 20 h after treatment with substances shown; blot was probed with anti-IL-37. One of four donors is shown. Concentrations (ng/ml) are: IL-10, 25; IL-1β, 10; IL-12, 20; IL-18, 25; IL-32γ, 5; IFN-γ, 25; TNF, 20; TGF-β1, 20; LPS, as indicated; Pam3CSK4 (Pam), 10; CpG, 1,000; IL-4, 20; GM-CSF, 40. Right, immunoblots with non-pretreated anti-IL-37 (left) and with anti-IL-37 mixed with 1 μg/ml recombinant human IL-37b (right). () immunoblot of lysates from two donors 24 h after incubation with 1 μg/ml of LPS. Twenty hours earlier, PBMCs were transfected with 100 nM of siIL-37 or scrambled siRNA. Two of five similar blots are shown. (–) Concentrations of secreted IL-1β, IL-6 and IL-10 in transfected PBMCs left untreated or treated 24 h after transfection with 1 μg/ml LPS or 10 ng/ml Pam3CSK4 and assessed 24 h after treatment. Mean ± s.e.m. shown. (n = 7; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 for siIL-37 compa! red to scrambled.) The numbers above the bars indicate fold changes. () Results of densitometric analysis as normalized density per mm2 (± s.e.m.) of PBMCs transfected with either 100 nM siIL-37 (open bars) or scrambled siRNA (closed bars) and stimulated with 1 μg/ml LPS. After 24 h, pairs of siIL-37 and scrambled siRNA-treated cells from three donors were assessed by cytokine protein array. *P < 0.05; **P < 0.01. Numbers indicate percent decrease or fold increase. Blue, mean increase >2-fold; green, mean increase 1.5- to 2-fold; black, small changes. () Immunohistochemical staining of IL-37 in synovial tissue from a person with active rheumatoid arthritis. () Staining of the same tissue with a rabbit IgG control antibody. The panels are representative of five similar pairs of images. * Figure 2: Effect of TLR-induced IL-37b on cytokine production in RAW cells. () Immunoblots of RAW-IL-37 cells. Cells were incubated without (ctrl) or with increasing concentrations of LPS (left) or TLR ligands (right) for 24 h. Each blot represents one of four independent experiments. TLR ligands (in μg/ml) are below lanes: Pam, Pam3CSK4; M, MALP-2; CpG, CpG-ODN. (–) Mean absolute concentrations of TNF and MIP-2 in supernatants (,) or IL-1α in lysates () of LPS-stimulated cells, presented relative to total protein in lysates. Error bars, s.e.m., n = 8. *P < 0.05; **P < 0.01; ***P < 0.001 for RAW-IL-37 compared to mock-transfected. IL-37b was absent in lysates of mock-transfected cells (data not shown). () Protein array analysis of supernatants from RAW-IL-37 (open bars) and mock-transfected cells (closed bars) stimulated with LPS (100 ng/ml) for 24 h. Mean densities of RAW-IL-37 versus mock-transfected cells were obtained from three independent experiments. Results are shown as density per mm2 ± s.e.m.; *P < 0.05; **P < 0.01. Red, mean reductio! n >67%; orange mean reduction 33–67%; black, little change. * Figure 3: Cytokine production in THP-1 and A549 cells transfected with the pIRES-IL-37b plasmid or mock-transfected with pIRES lacking IL-37b. (,) IL-1β () and TNF concentrations () in undifferentiated, transiently transfected THP-1 cells stimulated with 1 μg/ml LPS, 25 ng/ml IL-1β or vehicle. () Absolute TNF concentrations in PMA-differentiated, transfected THP-1 cells stimulated with LPS, IL-1β or vehicle. (–) n = 6; *P < 0.05; **P < 0.01; ***P < 0.001 for mock-transfected versus IL-37b–expressing cells. () Immunoblot of IL-37b in non-transfected (no) or transfected A549 lung epithelial cells; IL-37b-ex, IL-37b expression plasmid; mock, empty vector. One representative of four similar blots is shown. (,) IL-6 and IL-1α concentrations in IL-1β–stimulated (10 ng/ml) A549 cells after transfection with mock plasmid or IL-37b construct. Bars show absolute cytokine concentrations (mean ± s.e.m.); n = 5; ns, not significant; **P < 0.01; ***P < 0.001. * Figure 4: Smad3 and IL-37. () Confocal microscopy of IL-37b-transfected A549 cells treated with 10 ng/ml IL-1β or vehicle. Localization of IL-37b/FLAG (Cy3, yellow), phospho-Smad3 (FITC, green), cytoskeleton (Alexa Fluor 633—wheat germ agglutinin (Alexa633-WGA), red) and nuclei (DAPI, blue, shown only in overlays) are shown. Bright yellow in the overlay images indicates colocalization. See Supplementary Figure 3 for mock-transfected cells. () Anti-FLAG immunoprecipitation of IL-37b-transfected A549 cells stimulated with IL-1β (10 ng/ml) followed by non-reducing PAGE and immunoblotting with anti-Smad3. (,) IL-6 () and IL-1β () concentrations in culture supernatants of RAW cell clones () or transfected and differentiated THP-1 macrophages () treated first with SIS3 (in μM), followed 30 min later by the addition of LPS and analyzed 24 h later. () Mean ± s.e.m. percent changes from LPS (100 ng/ml) alone, n = 6; *P < 0.05; **P < 0.01 for RAW-IL-37 versus mock-transfected cells. () LPS and SIS3 were ! used at 1,000 ng/ml and 2 μM, respectively. The graph depicts absolute IL-1β concentrations ± s.e.m., n = 4; numbers indicate fold-increases induced by SIS3 treatment. ns, not significant; **P = 0.001 for increases in IL-1β production with SIS3 versus without. () Mean IL-8 (± s.e.m.) in THP-1 cells stably transfected with shRNA to Smad3 (shSmad3) or scrambled (eLV) after transfection with the IL-37b construct or the control vector and stimulation with 25 ng/ml IL-1β or 1,000 ng/ml LPS (n = 4; **P < 0.01; ***P < 0.001 for mock-transfected versus IL-37b–expressing cells). * Figure 5: Amelioration of endotoxic shock in mice transgenic for IL-37b. () Immunoprecipitation with anti-FLAG and anti-IL-37 and immunoblotting of lysates of splenocytes from several F1 generation IL-37tg and four PCR-negative littermates (neg). Splenocytes were incubated with or without LPS (1 μg/ml) for 20 h. Data from one representative negative and three IL-37tg heterozygous (het) mice are shown. () IL-37b immunoblotting of peripheral blood taken from IL-37tg heterozygotes 24 h after they were injected with 10 mg/kg LPS or vehicle. Data shown from two representative pairs of vehicle- or LPS-injected mice. (–) Quantification (mean ± s.e.m.) of endotoxic shock measures in 23 IL-37tg heterozygous, 10 homozygous and 25 neg-WT mice injected with 10 mg/kg LPS. *P < 0.05; **P < 0.01; ***P < 0.001 for neg-WT compared to IL-37tg mice from one-way ANOVA or one-way ANOVA on ranks. The dash-dot lines indicate mean values (± s.e.m.) in vehicle-injected IL-37tg and neg-WT mice (between which no difference was observed). n = 5 heterozygotes, 3 homozyg! otes and 5 neg-WT. () Body temperature measured at the indicated time points. (–) Blood gases (–), electrolytes (,) and liver enzymes (,) measured after 24 h. * Figure 6: Production of LPS-induced cytokines in IL-37tg mice. IL-37tg and neg-WT (as in Fig. 5) mice were injected with 10 mg/kg LPS or vehicle. (–) After 24 h, plasma (–) and lungs (,) were analyzed by electrochemiluminescence assay or ELISA (–) or protein array (). () Protein array analysis of plasma of three IL-37tg heterozygous (open bars) and four neg-WT (closed bars) mice. Results depicted as normalized density per mm2 ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001. Numbers indicate percent decrease or fold increase. Red, mean decrease >67%; orange, mean decrease 33–67%; black, small changes (33% decrease to 50% increase or 1.5-fold increase); green, 1.5- to 2-fold increase; blue, >2-fold increase. (–) Absolute values ± s.e.m. of cytokines in 23 IL-37tg heterozygotes, 10 homozygotes and 25 neg-WT mice. *P < 0.05; **P < 0.01; ***P < 0.001 for IL-37tg heterozygote or homozygote versus neg-WT mice. For individual P-values and statistical tests performed, see Supplementary Table 2. * Figure 7: Cytokines and DC activation in IL-37tg and neg-WT spleens and whole blood. (–) Spleens were harvested from the same mice as in Figure 6 (24 h after injection of 10 mg/kg LPS or vehicle) and either lysed and analyzed for cytokine () or incubated with collagenase IV, stained and subjected to flow cytometry (,). () Electrochemiluminescence and ELISA measurements of cytokine concentration (± s.e.m.) normalized to total protein (t.p.). *P < 0.05; **P < 0.01; ***P < 0.001. (,) Expression of CD86 and MHC II in CD11c+ DCs from viable splenocytes; n = 11 LPS and four vehicle neg-WT, eight LPS and five vehicle IL-37tg heterozygotes (het), and nine LPS and five vehicle IL-37tg homozygotes (hom). **P < 0.01 for neg-WT vs IL-37tg hom. () Statistical assessment of DC activation shown as per cent CD86+MHC II+ DCs among total DCs in mice injected with LPS (left) or vehicle (right). Horizontal lines show means. () Exemplary scatter plot of one LPS-injected mouse from each strain. Top right of each plot, percent of CD86/MHC II double-positive cells. (,) Absolute ! cytokine amounts in whole blood from IL-37tg heterozygotes and neg-WT mice stimulated as indicated (concentrations in ng/ml: LPS, 1,000; IL-12, 20; IL-1β, 20; TNF, 20; IL-18, 50) and incubated for 20 h. Data show mean ± s.e.m. in picograms per million white blood cells (WBCs). n = 6 IL-37tg and 8 neg-WT mice; *P < 0.05; **P < 0.01; ***P < 0.001 for neg-WT versus IL-37tg mice. * Figure 8: Silencing of Smad3 reduces the activity of IL-37 in vivo. () Production of Smad3 in lungs from IL-37tg and neg-WT mice that had inhaled 3 μg/g of scrambled siRNA (scr) or siRNA to Smad3 (siSm3) then after 20 h inhaled 3 μg/g LPS. Lungs were harvested after another 18 h and phospho-Smad3 was assessed by immunoblotting. Four representative pairs of mice are shown. (,) Absolute concentrations of each cytokine in lungs from mice as in per mg total protein (t.p.) ± s.e.m. n = 8 for each group. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 for IL-37tg + scrambled versus neg-WT + scrambled; §P < 0.05 for neg-WT + scrambled versus neg-WT + siSmad3; and †P < 0.05; ‡P < 0.01 for IL-37tg + scrambled versus IL-37tg + siSmad3. Numbers indicate fold increases conferred by treatment with siSmad3 (comparing scrambled to siSmad3 in neg-WT and IL-37tg). P-values are listed in Supplementary Table 3. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA. * Marcel F Nold, * Claudia A Nold-Petry, * Jarod A Zepp, * Brent E Palmer & * Charles A Dinarello * The Ritchie Centre, Monash Institute of Medical Research, Monash University, Melbourne, Victoria, Australia. * Marcel F Nold & * Claudia A Nold-Petry * Children's Hospital, Ludwig-Maximilians University, Munich, Germany. * Philip Bufler Contributions M.F.N., C.A.N.-P., P.B. and C.A.D. designed the study, analyzed the data and wrote the manuscript. M.F.N., C.A.N.-P., J.A.Z. and P.B. did experiments. B.E.P. was in charge of the flow-cytometry analysis. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Charles A Dinarello (cdinarello@mac.com) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7 and Supplementary Tables 1–3 Additional data - The structural basis for intramembrane assembly of an activating immunoreceptor complex
- Nat Immunol 11(11):1023-1029 (2010)
Nature Immunology | Article The structural basis for intramembrane assembly of an activating immunoreceptor complex * Matthew E Call1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Kai W Wucherpfennig2, 3kai_wucherpfennig@dfci.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * James J Chou1chou@cmcd.hms.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 11 ,Pages:1023–1029Year published:(2010)DOI:doi:10.1038/ni.1943Received24 June 2010Accepted09 September 2010Published online03 October 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Many receptors that activate cells of the immune system are multisubunit membrane protein complexes in which ligand recognition and signaling functions are contributed by separate protein modules. Receptors and signaling subunits assemble through contacts among basic and acidic residues in their transmembrane domains to form the functional complexes. Here we report the nuclear magnetic resonance (NMR) structure of the membrane-embedded, heterotrimeric assembly formed by association of the DAP12 signaling module with the natural killer (NK) cell–activating receptor NKG2C. The main intramembrane contact site is formed by a complex electrostatic network involving five hydrophilic transmembrane residues. Functional mutagenesis demonstrated that similar polar intramembrane motifs are also important for assembly of the NK cell–activating NKG2D-DAP10 complex and the T cell antigen receptor (TCR)–invariant signaling protein CD3 complex. This structural motif therefore lies at ! the core of the molecular organization of many activating immunoreceptors. View full text Figures at a glance * Figure 1: Construct design and labeling strategy. () Assembly of DAP12 with the NKG2C-CD94 heterodimer in the membrane (left); assembly of DAP12TM with NKG2CTM (middle; NKG2C ectodomain and CD94 omitted as they do not participate in the transmembrane assembly); and covalent peptide construct representing the membrane-embedded portion of the trimolecular complex (right). (–) The tr-HSQC spectra of trimer samples segmentally labeled with 15N-2H on the DAP12TM (D12TM)-only strand () or on the DAP12TM-NKG2CTM (2CTM) strand (), and of the DAP12TM homodimer alone (), recorded for samples prepared in 250 mM tetradecylphosphocholine with 25 mM SDS in 20 mM phosphate buffer, pH 6.8, and recorded at 600 MHz and 303 K. Top row, separate view of glycine region. Amino acid positions are noted with single-letter designations and position number. Data are representative of at least three experiments each. * Figure 2: Structure of the DAP12TM-DAP12TM-NKG2CTM complex. () One structure from the ensemble of the fifteen trimer structures of lowest energy (helical portions only), showing surface features of the DAP12TM dimer (all NKG2CTM (orange ribbon) side chains omitted for clarity); amino acid and position included for regions of interest (bundle views, sample NOE strips and assigned methyl spectra, Supplementary Figs. 2 and 3). C term, carboxyl terminus; N term, amino terminus. (,) In vitro translation–based assay of the effects of DAP12 transmembrane substitutions on its assembly with NKG2C () or KIR2DS3 (), assessed with 35S-labeled proteins extracted in 0.5% digitonin and immunoprecipitated with monoclonal antibody to the hemagglutinin tag (DAP12). Numbers below lanes indicate assembly efficiency, calculated as the ratio of receptor to DAP12 dimer and presented relative to assembly efficiency with wild-type DAP12 (set as 100%). WT, wild-type; T20A, substitution of alanine for threonine at position 20; A24F, substitution of phenylala! nine for alanine at position 24; 3GL and 3GF, triple substitution of leucine (3GL) or phenylalanine (3GF) for Gly7, Gly11 and Gly15; D12-D, DAP12 dimer; D12-M, DAP12 monomer. Data are from at least two experiments with similar results (mean). (,) Two views of one structure from the ensemble of fifteen DAP12TM dimer structures (helical portions only, from Gly7 to Leu30): red, side-chain oxygen atoms; yellow, glycine residues at positions 7, 11 and 15, as in (bundle views, sample NOE strips and assigned methyl spectra, Supplementary Figs. 2 and 3). () In vitro translation analysis of DAP12 transmembrane substitutions in homodimer formation as in ,. IAVA, double substitution of alanine for isoleucine at position 12 and alanine for valine at position 13; D16A, substitution of alanine for aspartic acid at position 16; DATA, double substitution of alanine for aspartic acid at position 16 and alanine for threonine at position 20. Numbers below lanes indicate assembly efficiency, c! alculated as the ratio of DAP12 dimer to DAP12 monomer and pre! sented relative to assembly efficiency with wild-type DAP12 (set as 100%). Data are from at least two experiments with similar results (mean). () Alignment of human DAP12 and NKG2C transmembrane sequences; numbers indicate amino acid positions in the engineered NMR constructs. Letter color and underlining: red, electronegative; blue, electropositive. * Figure 3: Structural comparison of ζζTM and DAP12TM receptor-binding sites. () En face view of ribbon structures showing the relative locations of disulfide bonds and side chains that participate in the receptor-binding sites in the ζζTM-ζζTM dimer (blue ribbons; Protein Data Bank, 2HAC) and DAP12TM-DAP12TM dimer (green ribbons); all other side chains omitted for clarity. Numbers below indicate χ1 values for aspartic acid side chains. (,) Enlargement of the receptor-binding sites of homodimers of ζζTM () and DAP12TM (); green dotted lines indicate putative hydrogen bonds. * Figure 4: Structure of the electrostatic network at the DAP12TM-DAP12TM-NKG2CTM binding site. () Bundle of five selected ribbon structures of the DAP12TM-DAP12TM-NKG2CTM covalent trimer showing possible configurations of the critical binding site (all other side chains omitted for clarity). (,) Enlarged views of the electrostatic network at Asp16-Thr20-Lys52, presented in the same orientation as in () and presented in an axial view from above (); green dotted lines indicate putative hydrogen bonds. * Figure 5: A similar electrostatic network governs the assembly of NKG2C-DAP12, NKG2D-DAP10 and TCR-CD3 complexes. (–) Sequence alignment of vertebrate DAP12TM () and DAP10TM () and human and mouse CD3TM (); red, acidic and hydroxyl-bearing residues involved in receptor binding; green, conserved and semiconserved aromatic residues that suggest further similarities in helix packing. Homo, Homo sapiens; Mus, Mus musculus; Bos, Bos taurus; Xeno, Xenopus laevis; Danio, Danio rerio; Fugu, Fugu rubripes. (–) In vitro analysis of the effect of substitution of alanine for serine or threonine in the transmembrane domains on the assembly of DAP10 with NKG2D (), TCRα with CD3δ-CD3ε () or TCRβ with CD3γ-CD3ε (), assessed as described above (Fig. 2), with monoclonal antibody to hemagglutinin (DAP10; ) or to CD3 (UCH-T1; ,) used for immunoprecipitation. () Formation of disulfide-linked dimers of wild-type DAP10 (D10: WT) and mutant DAP10 with serine-to-alanine substitution (D10: SA), assessed in nonreducing conditions (without dithiothreitol (–DTT); left) or reducing conditions (because ox! idized NKG2D runs as a diffuse band that is difficult to quantify; +DTT; right). (,) δ(WT), ε(WT) or γ(WT), wild-type CD3δ, CD3ε or CD3γ; δ(TA) or ε(TA), mutant CD3δ or CD3ε with threonine-to-alanine substitution; γ(SA), mutant CDγ with serine-to-alanine substitution. Numbers above lanes indicate assembly efficiency, calculated as the ratio of DAP10 dimer to DAP10 monomer (, left), NKG2D to total DAP10 (, right) or TCR to CD3ε () and presented relative to assembly efficiency with wild-type DAP12 (set as 100%). Data are from at least three experiments with similar results (mean). * Figure 6: Proposed model for intramembrane immunoreceptor complex assembly. () Dimeric signaling modules (CD3, DAP10 or DAP12; green coils) exist as metastable intermediates in the endoplasmic reticulum membrane, whereas newly synthesized receptor subunits (orange coils) are either incorporated into complexes or rapidly degraded4, 27. The electrostatic network in the signaling dimer (red shading) shows a symmetrical electron distribution and may have multiple opportunities to assemble with basic residues (blue shading) from receptor subunits. K(R), lysine or arginine; T(S), threonine or serine. () Once a receptor subunit has associated with an available binding site, an asymmetric redistribution of electronegativity in the network (color gradients) may render the opposite side of the signaling module unable to bind a second receptor. This trimeric assembly therefore represents the fundamental structural unit that organizes immunoreceptor complexes. () In a step that is either subsequent to or simultaneous and cooperative with that in , receptors tha! t form dimers (such as TCR, NKG2D and the mouse Ly49 family) combine two or more trimeric units to form the final complex. The relative orientation of the individual trimeric units cannot be determined from structural or biochemical data available at present. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2L34 * 2L35 * 2L34 * 2L35 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. * Matthew E Call & * James J Chou * Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. * Kai W Wucherpfennig * Program in Immunology, Harvard Medical School, Boston, Massachusetts, USA. * Kai W Wucherpfennig * Present address: Division of Structural Biology, the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. * Matthew E Call Contributions M.E.C., K.W.W. and J.J.C. conceived of the study; M.E.C. designed and did all biochemical experiments, produced transmembrane peptide constructs and prepared NMR samples; M.E.C. and J.J.C. collected and analyzed NMR data and calculated structures; M.E.C. wrote the paper; and all authors contributed to editing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * James J Chou (chou@cmcd.hms.harvard.edu) or * Kai W Wucherpfennig (kai_wucherpfennig@dfci.harvard.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–3 Additional data - IL-12 initiates tumor rejection via lymphoid tissue–inducer cells bearing the natural cytotoxicity receptor NKp46
- Nat Immunol 11(11):1030-1038 (2010)
Nature Immunology | Article IL-12 initiates tumor rejection via lymphoid tissue–inducer cells bearing the natural cytotoxicity receptor NKp46 * Maya Eisenring1 Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes vom Berg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Glen Kristiansen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth Saller1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Burkhard Becher1burkhard.becher@neuroimm.uzh.ch Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:1030–1038Year published:(2010)DOI:doi:10.1038/ni.1947Received20 April 2010Accepted13 September 2010Published online10 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The potent tumoricidal activity of interleukin 12 (IL-12) is thought to be mediated by the activation and polarization of natural killer (NK) cells and T helper type 1 (TH1) cells, respectively. By systematic analysis of the IL-12-induced immune response to subcutaneous melanoma (B16), we found that tumor suppression was mediated independently of T lymphocytes or NK cells. IL-12 initiated local antitumor immunity by stimulating a subset of NKp46+ lymphoid tissue–inducer (LTi) cells dependent on the transcription factor RORγt. The presence of these NKp46+ LTi cells induced upregulation of adhesion molecules in the tumor vasculature and resulted in more leukocyte invasion. Thus, this innate cell type is responsive to IL-12 and is a powerful mediator of tumor suppression. View full text Figures at a glance * Figure 1: IL-12 acts locally in a paracrine manner. () Repression of subcutaneous tumors in wild-type (WT) mice given subcutaneous injection of 2 × 105 B16.F10, B16–IL-12 or B16 cells (n = 6 mice per group). () Repression of tumors in wild-type mice given subcutaneous injection of a mixture of B16 cells and B16–IL-12 cells at a ratio of 1:1, 2:1 or 10:1 (n = 5 mice per group). () Repression of tumors in wild-type or Il12rb2−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 6 mice per group). () Tumor repression in wild-type mice given subcutaneous injection of 2 × 105 B16 cells and treated intraperitoneally (i.p.) with PBS or recombinant IL-12 (rIL-12) on days 1, 3, 5 and 9 after tumor injection (n = 3 mice per group). () Tumor repression in wild-type mice given subcutaneous injection of 2 × 105 B16 cells in the right abdomen and 2 × 105 B16–IL-12 cells in the left contralateral abdomen (n = 6 mice per group). () Tumor repression in wild-type mice given subcutaneous injection of 2 ×! 105 B16 cells and intratumoral (i.t.) injection of PBS or recombinant IL-12 on days 7, 9, 12, 14, 16 and 19 after tumor injection (n = 3 mice per group). Additional data, Supplementary Figure 1. NS, not significant. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of four (), three (,) or two (,,) experiments (mean and s.e.m.). * Figure 2: IL-12 elicits the recruitment of leukocytes into the tumor mass. () Immunohistochemistry of frozen tumor sections obtained from wild-type mice 3 weeks after challenge with B16 or B16–IL-12 cells and stained with anti-CD4 (CD4), anti-CD8 (CD8) and anti–asialo GM1 (NK). Scale bar, 50 μm. () Cytofluorometry of tumor-invading leukocytes in mice treated as in , assessed in the entire tumor mass after exclusion of cellular debris, dead cells and duplets, and presented as tumor-infiltrating CD45+ leukocytes relative to CD45− melanoma cells. Each symbol represents an individual mouse; long horizontal lines indicate the mean (short lines, s.e.m.). (–) Cytofluorometry of tumor-invading leukocytes in mice treated as in , presented as the frequency of infiltrating CD8+ cells (), CD4+ cells (), CD11c+ cells (), CD11b+ (), NK1.1+ cells (), 1A8+ cells () and CD19+ cells (), gated on CD45+ cells, in B16 tumors relative to that in B16–IL-12 tumors. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of two experim! ents with at least four mice per group (mean and s.e.m. in –). * Figure 3: IL-12-mediated repression of subcutaneous tumor acts independently of T cells, B cells, NKT cells and cNK cells. () Tumor repression in wild-type and Rag1−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 6 mice per group). () Tumor repression in Rag1−/− and anti-NK1.1 (α-NK1.1)-treated Rag1−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 3 mice per group; additional data, Supplementary Fig. 2). () Tumor repression in Rag1−/− and anti-GM1 (α-GM1)-treated Rag1−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 5 mice per group; additional data, Supplementary Fig. 2). () Tumor repression in wild-type and Il15ra−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 6 mice per group). () Lung metastasis in Rag1−/− mice given intravenous injection of 1 × 105 B16 or B16–IL-12 cells (n = 6 mice per group), followed by depletion of NK cells with anti-NK1.1 (three times per week); metastases were counted after 21 d (additional data, Supplementar! y Figure 2). *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of four () or two (–) experiments (mean and s.e.m.). * Figure 4: Adoptive transfer of leukocytes bearing IL-12R reestablishes tumor suppression in IL12rb2−/− mice. () Tumor repression in Il12rb2−/− mice given subcutaneous coinjection of 2 × 105 B16–IL-12 cells and 2 × 105 splenocytes (sp) from wild-type, Rag1−/− or Il12rb2−/− mice (n ≥ 5 mice per group). () Tumor suppression in wild-type and Rag2−/−Il2rg−/− mice given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 6 mice per group). () Tumor suppression in Rag2−/−I12rg−/−− mice given subcutaneous coinjection of 2 × 105 B16–IL-12 cells and 2 × 105 splenocytes from wild-type, Rag1−/− or Rag2−/−Il2rg−/− mice (n ≥ 6 mice per group). *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of at least three (,) or four () experiments (mean and s.e.m.). * Figure 5: IL-12 induces the invasion of NKp46+ cells into the tumor mass. () Immunohistochemistry of frozen tumor sections obtained from wild-type, Rag2−/−Il2rg−/− and Il15ra−/− mice 3 weeks after challenge with B16 or B16–IL-12 cells and stained with anti-NKp46 (n ≥ 6 mice per group). Arrowheads indicate NKp46+ cells. Scale bar, 50 μm. () Tumor suppression in Il12rb2−/− mice given subcutaneous coinjection of 2 × 105 B16–IL-12 cells and 6 × 103 sorted NKp46+NK1.1loDX5lo cells obtained from Rag1−/− or Il12rb2−/− mice (n ≥ 6 mice per group); cells were sorted by flow cytometry with a high-yield pre-sort for NKp46+ cells followed by a low-pressure, high-purity sort for NKp46+NK1.1loDX5lo cells. () Tumor suppression in Il12rb2−/− mice given subcutaneous coinjection of 2 × 105 B16–IL-12 cells and 6 × 103 sorted NKp46+NK1.1hiDX5hi cNK cells or NKp46+NK1.1loDX5lo cells obtained from Rag1−/− mice (n ≥ 6 mice per group); cells were sorted by flow cytometry with a high-yield pre-sort for NKp46+ cells followed! by a low-pressure, high-purity sort for NKp46+NK1.1loDX5lo cells or NKp46+NK1.1hiDX5hi cells. *P < 0.01 and **P < 0.001 (Student's t-test). Data are representative of at least two experiments (mean and s.e.m. in ,). * Figure 6: NKp46+ LTi cells suppress tumor growth in an IL-12R- and RORγt-dependent way. () Flow cytometry analysis of NKp46 and eYFP in tumor cells from Rorc-eYFP mice 3 weeks after inoculation with B16 cells (left) or B16–IL-12 cells (right), gated on live, singlet CD45+ and CD3− cells. Numbers above outlined areas indicate percent cells in each. () Cryosections from the tumor periphery of tumor-bearing Rorc-eYFP mice 3 weeks after inoculation with B16 cells (left) or B16–IL-12 cells (right), stained with anti-GFP-YFP (green) and anti-NKp46 (red), detected with fluorescent secondary antibodies, and counterstained with the DNA-intercalating dye DAPI. Arrowheads indicate regions enlarged in insets (bottom left). Scale bars, 100 μm (main images) or 10 μm (insets). () Tumor suppression in Il12rb2−/− mice given subcutaneous coinjection of 2 × 105 B16–IL-12 cells at a ratio of 1:1 with unfractionated splenocytes from Rag1−/−, Il12rb2−/− or Rorc−/− mice (n ≥ 6 mice per group). () Tumor suppression in Il12rb2−/− mice given subcutaneous! coinjection of 2 × 105 B16–IL-12 cells with 2 × 105 splenocytes from wild-type mice, 2 × 105 splenocytes from Il12rb2−/− mice or 6 × 103 sorted NKp46+Rorc-eYFP+CD3− cells from Rorc-eYFP mice (n ≥ 4 mice per group); cells were sorted with a high-yield pre-sort for eYFP+ cells followed by a low-pressure, high-purity sort for NKp46+eYFP+CD3− cells. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of two (,,) or three () experiments (mean and s.e.m. in ,). * Figure 7: The role of effector cytokines in IL-12-mediated tumor suppression. (,) Tumor suppression in wild-type mice, Il22−/− mice () or Il17a−/− mice () given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 6 mice per group). (,) Tumor suppression in wild-type mice, IFN-γ-deficient mice (Ifng−/−; ) or IFN-γ receptor–deficient mice (Ifngr1−/−; ) given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 5 mice per group). () Tumor suppression in wild-type mice and perforin-deficient mice (Prf1−/−) given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n = 3 mice per group). Additional data, Supplementary Figure 3. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). Data are representative of at least two individual experiments (mean and s.e.m.). * Figure 8: LTi cells alter the tumor microvasculature. (,) Microscopy of tumors obtained from wild-type or Rag2−/−Il2rg−/− mice injected with B16 or B16–IL-12 cells, stained for CD31 (green), ICAM (red; ) or VCAM (red; ) and counterstained with DAPI (blue). Left (merged images), overview of tumor margins; arrowheads indicate regions enlarged in insets (bottom left). Right, red single-channel images. Scale bars, 50 μm (main images) or 10 μm (insets). Data are representative of at least two experiments. () Tumor suppression in wild-type and lymphotoxin-β receptor–deficient mice (Ltbr−/−) given subcutaneous injection of 2 × 105 B16 or B16–IL-12 cells (n ≥ 3 mice per group) *P < 0.001 (Student's t-test). Data are representative of two experiments (mean and s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland. * Maya Eisenring, * Johannes vom Berg, * Elisabeth Saller & * Burkhard Becher * Institute of Clinical Pathology, Department of Pathology, University Hospital of Zurich, Zurich, Switzerland. * Glen Kristiansen * Present address: Institut für Medizinische & Molekulare Diagnostik, Zurich, Switzerland. * Elisabeth Saller Contributions M.E. and E.S. designed and did the experiments and analyzed the data; J.v.B. did experiments and analyzed the histology; G.K. provided advice for the histological analysis; B.B. designed the experiments, supervised and funded the study; B.B. and M.E. wrote the report. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Burkhard Becher (burkhard.becher@neuroimm.uzh.ch) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (392K) Supplementary Figures 1–3 Additional data - Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A
- Nat Immunol 11(11):1039-1046 (2010)
Nature Immunology | Article Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A * Carmela De Santo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ramon Arscott1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Booth1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ioannis Karydis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret Jones2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ruth Asher3 Search for this author in: * NPG journals * PubMed * Google Scholar * Mariolina Salio1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Middleton4 Search for this author in: * NPG journals * PubMed * Google Scholar * Vincenzo Cerundolo1vincenzo.cerundolo@imm.ox.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:1039–1046Year published:(2010)DOI:doi:10.1038/ni.1942Received25 June 2010Accepted08 September 2010Published online03 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Neutrophils are the main effector cells during inflammation, but they can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms that modulate their plasticity remain unclear. We now show that systemic serum amyloid A 1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory interleukin 10 (IL-10)-secreting neutrophils but also promoted the interaction of invariant natural killer T cells (iNKT cells) with those neutrophils, a process that limited their suppressive activity by diminishing the production of IL-10 and enhancing the production of IL-12. Because SAA-1-producing melanomas promoted differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses. View full text Figures at a glance * Figure 1: Proliferation of immunosuppressive CD11b+CD15+ cells in patients with melanoma. () Population expansion of melan-A(26–35) tetramer–specific CD8+ T cells from PBMCs or total leukocytes of one HLA-A2+ patient with melanoma and one healthy donor. Numbers adjacent to outlined areas indicate percent tetramer-positive CD8+ cells. () Frequency (left) and forward scatter (FSC) and side scatter (SSC; right) of CD11b+CD15+ cells purified from one patient with melanoma and one healthy donor. Numbers adjacent to outlined areas (left) indicate percent CD11b+CD15+ cells; red dots (right) indicate back gating of CD11b+CD15+ cells. () Intracellular staining, with anti-IL-10 and anti-IL-8, of CD11b+CD15+ cells purified from a patient with melanoma and one healthy donor. Numbers in top right quadrants indicate percent CD15+ cells stained with antibody. () Cumulative IL-10 secretion from CD11b+CD15+ cells from seven healthy donors and ten patients with melanoma. () Population expansion of melan-A(26–35) tetramer–specific CD8+ T cells from total leukocytes of one H! LA-A2+ patient with melanoma, depleted (−CD11b+CD15+ cells) or not depleted (+CD11b+CD15+ cells) of CD11b+CD15+ cells, and frequency of melan-A(26–35) tetramer–specific CD8+ T cells after the addition of 10% purified CD11b+CD15+ cells to CD11b+CD15+ populations depleted of total leukocytes with (+IL-10R-blocking Ab) or without (−IL-10R-blocking Ab) blocking antibody to the IL-10 receptor. Data are representative of five independent experiments with ten patients with melanoma and five healthy donors (), more than ten independent experiments with forty patients with melanoma and thirty healthy donors (), five independent experiments with five patients with melanoma and five healthy donors (), five independent experiments with ten patients with melanoma and seven healthy donors (; error bars, s.d.) or three independent experiments with three patients with melanoma and three healthy donors (). * Figure 2: SAA-1 is present in plasma and primary tumors of patients with melanoma. () ELISA of SAA-1 in plasma from 40 patients with melanoma and 30 healthy donors (each symbol identifies a different patient). () Immunohistochemistry of serial sections of primary melanoma. Top left, melanin location (arrow); top right, staining with anti-SAA-1; bottom left, staining with antibody to human CD68; bottom right, double staining with anti-SAA-1 and anti-CD68. Original magnification, ×20. Data are representative of two experiments () or six experiments with five patients with melanoma (). * Figure 3: SAA-1 induces IL-10 production from human neutrophils. () IL-10 secretion by CD11b+CD15+ cells purified from healthy donors, in response to increasing concentrations of SAA-1. () IL-10 secretion by CD11b+CD15+ cells from healthy donors, treated with various combinations of SAA-1, Pam3Cys or formyl peptide (FMLP) in the presence or absence of blocking anti-FPR2 and anti-TLR2. () Proliferation of alloreactive T cells stimulated by allogeneic DCs plus graded numbers of CD11b+CD15+ cells from third-party healthy donors left untreated or pretreated with SAA-1 (100% proliferation corresponds to 7 × 104 c.p.m.). () Release of IL-10 from CD11b+CD15+ cells purified from a patient with melanoma and a healthy donor, preincubated or not with the PI(3) kinase inhibitor LY294002, the Erk inhibitor U1026 and the p38 inhibitor SB203580 and then left untreated or treated with SAA-1. () Flow cytometry of CD11b+CD15+ cells obtained from patients with melanoma and left untreated or incubated with soluble CD40L (+CD40L), assessed by intracellular s! taining with antibodies specific for phosphorylated (p-) kinases. Ab, antibody. Data are representative of five independent experiments (–; error bars, s.d.) or three independent experiments (). * Figure 4: Treatment of human neutrophils with SAA-1 promotes crosstalk with iNKT cells. () Intracellular anti-IFN-γ staining of CD1d+α-GalCer tetramer–positive human iNKT cells cultured together with untreated (left), SAA-1-treated (middle) or α-GalCer-treated (right) CD11b+CD15+ cells from a patient with melanoma without (top) or with (bottom) CD1d-blocking antibody. Numbers in top right quadrants indicate percent IFN-γ+ tetramer-positive cells. () Release of IL-10 from CD11b+CD15+ cells from a patient with melanoma, left untreated or treated with SAA-1 and incubated with iNKT cells with or without CD1d- or CD40-blocking antibodies. () Release of IL-10 and IL-12 from α-GalCer-pulsed or unpulsed CD11b+CD15+ cells from a patient with melanoma, incubated at various ratios (horizontal axes) with iNKT cells. () Frequency of melan-A(26–35) tetramer–specific CD8+ T cells among total leukocytes obtained from a patient with melanoma and incubated with autologous melan-A(26–35) peptide–pulsed DCs and α-GalCer-pulsed autologous CD11b+CD15+ cells with or w! ithout autologous iNKT cells (left four plots), without (left two plots) or with blocking anti-CD1d (+CD1d-blocking Ab) or anti-CD40 (+CD40-blocking Ab). Far right, population expansion of melan-A(26–35)-specific CD8+ T cells after the addition of soluble CD40L to CD11b+CD15+ cells in the absence of α-GalCer and autologous iNKT cells. Numbers adjacent to outlined areas indicate percent tetramer-postive CD8+ cells. Data are representative of three (,,) or two () experiments (error bars (,), s.d.). * Figure 5: Population expansion of immunosuppressive IL-10-secreting neutrophils in Jα18−/− mice injected with SAA-1. () Frequency of CD11b+Ly6G+ cells in the blood of wild-type (WT) and Jα18−/− mice (n = 6 per genotype) injected subcutaneously daily for 5 d with SAA-1; blood samples were collected before injection (day 0) and at day 5 and day 8 (3 d after the final SAA-1 injection). Numbers under outlined areas indicate percent CD11b+Ly6G+ cells. () Cumulative frequency of CD11b+Ly6G+ cells in wild-type and Jα18−/− mice (n = 6) injected with SAA-1 as described in . () Flow cytometry of circulating CD11b+Ly6G+ cells from wild-type or Jα18−/− mice injected subcutaneously daily for 5 d with SAA-1, stained ex vivo with anti-IL-10 or isotype-matched control antibody (rat immunoglobulin G2b (IgG2b)). () Cumulative frequency of IL-10+CD11b+Ly6G+ cells in wild-type and Jα18−/− mice (n = 6 per genotype) injected with SAA-1 as described in . () Proliferation of OT-I splenocytes labeled with the cytosolic dye CFSE and pulsed with the ovalbumin peptide SIINFEKL in the presence of 1! 0% CD11b+Ly6G+ cells sorted from the blood of SAA-1-injected wild-type or Jα18−/− mice at day 5, analyzed at day 4. Data are representative of three (–) or two () experiments (error bars (,,), s.d.). * Figure 6: Population expansion of immunosuppressive IL-10-secreting Cd1d−/− neutrophils in SAA-1-injected Cd1d+/+Cd1d−/− mixed–bone marrow chimeras. () Expression of CD11b and Ly6G by cells from Cd1d+/+Cd1d−/− chimeric mice injected subcutaneously daily for 5 d with SAA-1 (120 μg per kg body weight), in blood samples collected on days 0, 5 and 8 after the initial injection, stained with anti-CD11b and anti-Ly6G and gated on CD45.1+ (Cd1d+/+) cells (top) or CD45.2+ (Cd1d−/−) cells (bottom). Numbers in quadrants indicate percent CD11b+Ly6G− cells (top left) or CD11b+Ly6G+ cells (top right). () Flow cytometry of CD11b+Ly6G+ cells derived from CD45.1+ (Cd1d+/+) bone marrow (top) or CD45.2+ (Cd1d−/−) bone marrow (bottom) stained intracellularly with anti-IL-10. () Proliferation of CFSE-labeled unpulsed or SIINFEKL-pulsed OT-I splenocytes with or without CD11b+Ly6G+ cells from CD45.1+ (Cd1d+/+) cells (left) or CD45.2+ (Cd1d−/−) cells (right) sorted from the blood of SAA-1 injected chimeric mice at day 5 and assessed at day 4. Data are representative of two experiments with three mice each (,) or two experime! nts (). Author information * Abstract * Author information * Supplementary information Affiliations * Medical Research Council Human Immunology Unit, Nuffield Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. * Carmela De Santo, * Ramon Arscott, * Sarah Booth, * Ioannis Karydis, * Mariolina Salio & * Vincenzo Cerundolo * Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK. * Margaret Jones * Department of Cellular Pathology, John Radcliffe Hospital, University of Oxford, Oxford, UK. * Ruth Asher * Department of Medical Oncology, Oxford Cancer and Haematology Centre, Churchill Hospital, University of Oxford, Oxford, UK. * Mark Middleton Contributions C.D.S. designed and did the experiments, prepared the figures and contributed to the writing of the manuscript; R. Arscott did specific experiments; I.K. and M.M. obtained consent from patients with melanoma and collected blood samples; S.B., M.J. and R. Asher did tissue staining of melanoma sections; M.S. provided reagents and contributed to the writing of the manuscript; and V.C. designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vincenzo Cerundolo (vincenzo.cerundolo@imm.ox.ac.uk) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (816K) Supplementary Figures 1–7 Additional data - The S1P1-mTOR axis directs the reciprocal differentiation of TH1 and Treg cells
- Nat Immunol 11(11):1047-1056 (2010)
Nature Immunology | Article The S1P1-mTOR axis directs the reciprocal differentiation of TH1 and Treg cells * Guangwei Liu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kai Yang1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Samir Burns1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sharad Shrestha1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hongbo Chi1hongbo.chi@stjude.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:1047–1056Year published:(2010)DOI:doi:10.1038/ni.1939Received27 July 2010Accepted23 August 2010Published online19 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Naive CD4+ T cells differentiate into diverse effector and regulatory lineages to orchestrate immunity and tolerance. Here we found that the differentiation of proinflammatory T helper type 1 (TH1) cells and anti-inflammatory Foxp3+ regulatory T cells (Treg cells) was reciprocally regulated by S1P1, a receptor for the bioactive lipid sphingosine 1-phosphate (S1P). S1P1 inhibited the generation of extrathymic and natural Treg cells while driving TH1 development in a reciprocal manner and disrupted immune homeostasis. S1P1 signaled through the kinase mTOR and antagonized the function of transforming growth factor-β mainly by attenuating sustained activity of the signal transducer Smad3. S1P1 function was dependent on endogenous sphingosine kinase activity. Notably, two seemingly unrelated immunosuppressants, FTY720 and rapamycin, targeted the same S1P1 and mTOR pathway to regulate the dichotomy between TH1 cells and Treg cells. Our studies establish an S1P1-mTOR axis that con! trols T cell lineage specification. View full text Figures at a glance * Figure 1: S1P1 inhibits de novo generation of Foxp3+ iTreg cells. () Expression of Foxp3 and CD25 in CD4+ T cells (left) and the proportion and absolute number of Foxp3+CD4+ T cells (right) in OT-II Rag1−/− and S1P1-Tg OT-II Rag1−/− mice given OVA in the drinking water for 5 d. Numbers in outlined areas (left) indicate percent Foxp3+ cells. Each symbol represents an individual mouse (right); small horizontal lines indicate the mean. () Expression of Foxp3, LAG-3 and Gpr83 mRNA in CD4+ T cells isolated from MLNs of the mice in (n = 3 per genotype), presented relative to that of wild-type cells. () Distribution of Foxp3+ and CD3+ cells in the lamina propria of the mice in . Green arrows indicate cells positive for Foxp3 expression. α-, antibody to. Original magnification, ×200. () Foxp3 expression in naive OT-II Foxp3gfp and S1P1-Tg OT-II Foxp3gfp T cells stimulated for 5 d with MLN-derived CD103+ DCs and either OVA peptide or anti-CD3. Numbers in outlined areas indicate percent Foxp3+ cells. () Foxp3 expression (left) in wild-type! (WT) and S1P1-Tg naive T cells activated with anti-CD3 and anti-CD28 in the presence of TGF-β (5 ng/ml). Numbers above bracketed lines indicate percent Foxp3+ cells. Right, proportion and absolute number of Foxp3+ and Foxp3− cells (n = 3 mice per genotype). () Kinetics of Foxp3 mRNA expression in wild-type and S1P1-Tg naive cells activated for various times with anti-CD3 and anti-CD28 in the presence (+) or absence (−) of TGF-β (5 ng/ml), presented relative to the expression in wild-type cells not treated with TGF-β. P values (,,), Student's t-test. Data represent three to four independent experiments (error bars (,), s.e.m. of triplicates). * Figure 2: S1P1 is needed to restrain the generation and maintenance of Foxp3+ iTreg cells. () Foxp3 expression 4 weeks after the transfer of Foxp3− CD4+ single-positive thymocytes from wild-type mice and S1pr1flox/floxCd4-Cre mice into Rag1−/− recipient mice. PLN, peripheral lymph node. () Foxp3 expression (left) in wild-type and S1pr1CreER naive T cells left untreated (−) or treated with 4-OHT (+) and then activated in the presence of TGF-β for iTreg differentiation. Numbers above bracketed lines indicate percent Foxp3+ cells. Right, proportion and absolute number of Foxp3+ and Foxp3− T cells. () Foxp3 expression in naive wild-type and S1pr1CreER T cells treated with 4-OHT and then activated by CD103+ DCs and anti-CD3, without any exogenous cytokines. () Foxp3 expression in mature iTreg cells differentiated from naive wild-type and S1pr1CreER T cells, sorted as Foxp3+ (GFP+) cells, treated with 4-OHT and cultured with IL-2 and analyzed 4–5 d later. Numbers adjacent to or in outlined areas (,,) indicate percent Foxp3+CD4+ cells. Data represent three i! ndependent experiments. * Figure 3: S1P1 drives the differentiation of TH1 cells. () Expression of IFN-γ and IL-17 (left) and proportion of IFN-γ+ CD4+ T cells (right) in OT-II Rag1−/− and S1P1-Tg OT-II Rag1−/− mice fed OVA in their drinking water for 5 d. Numbers in quadrants indicate percent IFN-γ+IL-17− cells (top left), IFN-γ+IL-17+ cells (top right) or IFN-γ−IL-17+ cells (bottom right). () Quantitative RT-PCR analysis of the expression of T-bet and IFN-γ in CD4+ T cells from the mice in (n = 3 per genotype), presented relative to the expression in wild-type cells. () Proportion of IFN-γ+ CD4+ T cells among gated donor cells from OT-II Foxp3gfp and S1P1-Tg OT-II Foxp3gfp mice transferred into naive wild-type mice that were subsequently immunized intravenously with OVA. () IFN-γ expression (left) in total CD4+ and CD8+ T cells from MLNs of wild-type and S1P1-Tg mice, detected by intracellular staining 5 h after stimulation phorbol 12-myristate 13-acetate and ionomycin. Numbers in outlined areas indicate percent IFN-γ+ cells. Right,! quantitative RT-PCR analysis of IFN-γ expression after 24 h of stimulation with anti-CD3 and anti-CD28, relative to the expression in wild-type cells (n = 3–4 mice per genotype). Each symbol represents an individual mouse (,); small horizontal lines indicate the mean. P values, Student's t-test. Data represent four independent experiments (error bars (,), s.e.m. of at least three replicates). * Figure 4: S1P1 regulates reciprocal TH1 and iTreg differentiation and immune homeostasis in vivo. (,) Expression of IFN-γ and Foxp3 in wild-type cells () and S1P1-Tg cells () differentiated under TH0 or TH1 conditions in the presence or absence of TGF-β. () Ratio of IFN-γ+ cells to Foxp3+ cells for wild-type and S1P1-Tg cells differentiated under TH1 conditions in the presence of TGF-β. () Expression of IFN-γ and Foxp3 (left) and ratio of IFN-γ+ cells to Foxp3+ cells (right) for untreated or 4-OHT-treated wild-type and S1pr1CreER cells differentiated as in . (,) IFN-γ+ and Foxp3+ populations of wild-type and S1P1-Tg cells () and 4-OHT-treated wild-type and S1pr1CreER cells () activated by CD103+ DCs and OVA or anti-CD3 without exogenous cytokines. () Change in body weight of Rag1−/− recipient mice given wild-type or S1P1-Tg naive T cells in combination with wild-type (CD45.1+) Treg cells. () Intestinal histology of the mice in . Original magnification, ×100. () Proportion of Foxp3+ and IFN-γ+ CD4+ T cells derived from naive T cell donors for the mice in . Nu! mbers in quadrants (,,–) indicate percent IFN-γ+Foxp3− cells (top left), IFN-γ+Foxp3+ cells (top right) or IFN-γ−Foxp3+ cells (bottom right). Each symbol represents an individual mouse (,,); small horizontal lines indicate the mean. P values (,,), Student's t-test. Data represent three independent experiments (error bars (), s.e.m. of five replicates). * Figure 5: S1P1 mediates iTreg and TH1 differentiation via discrete mechanisms. () Foxp3 expression in wild-type and Ifng−/− naive T cells transduced with control retrovirus (RV) or S1P1-expressing retrovirus (S1P1-RV) and activated in the presence of TGF-β for iTreg differentiation. () Foxp3 expression in naive Ifng−/− and S1P1-Tg Ifng−/− T cells differentiated in the presence of various concentrations of TGF-β. Numbers above bracketed lines (,) indicate percent Foxp3+ cells. () IFN-γ expression in wild-type and S1P1-Tg cells activated under TH0 conditions and transduced with control retrovirus (RV) or Foxp3-expressing retrovirus (Foxp3-RV) linked to a GFP reporter; gated GFP+ cells are presented here. Numbers adjacent to outlined areas indicate percent IFN-γ+GFP+ cells. () IFN-γ expression in Foxp3-deficient cells from sublethally irradiated Rag1−/− mice given a 1:1 mixture of wild-type (CD45.2+) and scurfy (CD45.1+CD45.2+) bone marrow cells; 6 weeks after reconstitution, naive T cells from the two donor populations were purified,! activated under TH0 conditions and transduced with control or S1P1-expressing retrovirus linked to a Thy-1.1 reporter; gated Thy-1.1+ cells are presented here. Numbers adjacent to outlined areas indicate percent CD45.1+CD45.2+ (scurfy) cells (top left) or CD45.1−CD45.2+ (wild-type) cells (bottom left), or IFN-γ+Thy-1.1+ cells (right four plots). () IFN-γ expression in Foxp3-deficient cells after naive T cells from OT-II and S1P1-Tg OT-II mice bred onto the scurfy background were activated by OVA and irradiated splenic antigen-presenting cells. Numbers adjacent to outlined areas indicate percent IFN-γ+ cells. Data represent three independent experiments. * Figure 6: S1P1 attenuates TGF-β-Smad3 signaling. () Foxp3 expression in wild-type, CD4-dnTGFβRII and S1P1-Tg cells activated by CD103+ DCs and anti-CD3. Numbers in quadrants indicate percent IFN-γ+Foxp3− cells (top left), IFN-γ+Foxp3+ cells (top right) or IFN-γ−Foxp3+ cells (bottom right). () Nuclear translocation of Smad3 after 2 d of stimulation of wild-type and S1P1-Tg cells with TGF-β, assessed by immunoblot analysis of total Smad3 and phosphorylated Smad3 (p-Smad3). Numbers below lanes indicate band intensity relative to that of loading controls Sp1 (Nucleus) or p38 (Cytosol). () Foxp3 expression in wild-type and S1P1-Tg cells transduced with control retrovirus (RV) or constitutively active Smad3 retrovirus (Smad3CA-RV) and activated in the presence of TGF-β for iTreg differentiation. () Foxp3 expression in untreated (−) or 4-OHT-treated (+) wild-type and S1pr1CreER cells activated in the presence of TGF-β and the Smad3 inhibitor SIS3 (5 μM). Numbers above bracketed lines (,) indicate percent Foxp3+ cell! s. Data represent three independent experiments. * Figure 7: S1P1-mTOR axis targeted by FTY720 and rapamycin. () Kinetics of mTOR activation in wild-type and S1P1-Tg cells stimulated for various times (above lanes) with anti-CD3, anti-CD28 and IL-2 in the presence (+) or absence (−) of TGF-β. () Activation of Smad3 in wild-type and S1P1-Tg cells treated with FTY720 and rapamycin and activated for 2 d (above lanes). Numbers below lanes (,) indicate band intensity relative to that of the loading control β-actin. (,) Expression of Foxp3 () and IFN-γ () in OT-II Rag1−/− and S1P1-Tg OT-II Rag1−/− mice fed OVA in their drinking water for 5 d, accompanied by no treatment (Vehicle) or daily treatment with FTY720 (1 mg per kg body weight) or rapamycin (3 mg per kg body weight). Numbers adjacent to outlined areas indicate percent Foxp3+ cells () or IFN-γ+ cells (). () Foxp3 expression in thymic CD4+ single-positive cells from wild-type and S1P1-Tg mice left untreated (Vehicle) or treated daily with FTY720 and rapamycin for a total of 5 d. Numbers in outlined areas indicate perce! nt Foxp3+ cells. () Induction of Foxp3 in wild-type and S1pr1flox/floxCd4-Cre thymic Treg precursor cells stimulated overnight with IL-2 (50 U/ml) in the presence of SIS3 or vehicle. Numbers above bracketed lines indicate percent Foxp3+ cells. Data represent three independent experiments. * Figure 8: Sphingosine kinase activity regulates T cell differentiation. () Foxp3 expression in wild-type cells treated with vehicle alone (left) or various concentrations of S1P (above plots) in serum-free medium. Numbers above bracketed lines indicate percent Foxp3+ cells. () Expression of SphK1 and SphK2 mRNA in cells activated in the presence (5 ng/ml) or absence (none) of TGF-β, presented relative to the expression in naive T cells, set as 1. () Foxp3 expression in naive T cells pretreated with vehicle, DMS or SKI and activated for 5 d under iTreg conditions. Numbers above bracketed lines indicate percent Foxp3+ cells. () Expression of Foxp3 and CD25 in wild-type and S1P1-Tg naive T cells pretreated with vehicle, DMS (0.25 μM) or SKI (2.5 μM) and activated for 5 d under iTreg conditions. Numbers in quadrants indicate percent Foxp3+CD25+ cells (top right) or Foxp3−CD25+ cells (bottom right). Data represent three independent experiments. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Guangwei Liu & * Kai Yang Affiliations * Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Guangwei Liu, * Kai Yang, * Samir Burns, * Sharad Shrestha & * Hongbo Chi Contributions G.L. designed and did in vivo and cellular experiments and contributed to the writing of the manuscript; K.Y. designed and did biochemical analyses and cellular and molecular experiments; S.B. did in vivo and cellular experiments and gene-expression analysis; S.S. contributed to cell isolation and gene expression analysis and managed the mouse colony; and H.C. designed experiments, wrote the manuscript and provided overall direction. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hongbo Chi (hongbo.chi@stjude.org) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (996K) Supplementary Figures 1–13 Additional data - The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes
- Nat Immunol 11(11):1057-1062 (2010)
Nature Immunology | Article The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes * Anna-Barbara Stittrich1 Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Haftmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Evridiki Sgouroudis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anja Andrea Kühl2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ahmed Nabil Hegazy1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabel Panse1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Rene Riedel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Flossdorf4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Dong1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Franziska Fuhrmann1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Gitta Anne Heinz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhuo Fang7 Search for this author in: * NPG journals * PubMed * Google Scholar * Na Li7 Search for this author in: * NPG journals * PubMed * Google Scholar * Ute Bissels8 Search for this author in: * NPG journals * PubMed * Google Scholar * Farahnaz Hatam1 Search for this author in: * NPG journals * PubMed * Google Scholar * Angelina Jahn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ben Hammoud1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mareen Matz9 Search for this author in: * NPG journals * PubMed * Google Scholar * Felix-Michael Schulze10 Search for this author in: * NPG journals * PubMed * Google Scholar * Ria Baumgrass1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Bosio8 Search for this author in: * NPG journals * PubMed * Google Scholar * Hans-Joachim Mollenkopf11 Search for this author in: * NPG journals * PubMed * Google Scholar * Joachim Grün1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Thiel5 Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Chen7 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Höfer4 Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Loddenkemper2 Search for this author in: * NPG journals * PubMed * Google Scholar * Max Löhning1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hyun-Dong Chang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nikolaus Rajewsky7 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Radbruch1, 12 Search for this author in: * NPG journals * PubMed * Google Scholar * Mir-Farzin Mashreghi1, 12mashreghi@drfz.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11 ,Pages:1057–1062Year published:(2010)DOI:doi:10.1038/ni.1945Received13 August 2010Accepted09 September 2010Published online10 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor–mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell–mediated immune responses and open new therapeutic possibilities. View full text Figures at a glance * Figure 1: Induction of miR-182 after activation of primary naive helper T lymphocytes. () Quantitative PCR analysis of the expression of miR-182 and miR-183 in TH1, TH2 and TH17 lymphocytes activated in vitro with anti-CD3, anti-CD28 and irradiated splenocytes and differentiated for 6 d under their respective polarizing conditions, or in ex vivo naive helper T lymphocytes analyzed on day 0 (far right); results are presented relative to the expression of U6 small nuclear RNA. Data are from one experiment representative of two independent experiments. () Quantitative PCR analysis of the kinetics of miR-182 expression in helper T lymphocytes activated in vitro with anti-CD3 and anti-CD28 and differentiated into the TH1, TH0, TH2 and TH17 subsets; results are presented relative to U6 expression. No activation, naive helper T lymphocytes cultivated without anti-CD3 or anti-CD28. Data are from three independent experiments (error bars, s.d. of experiment pools). * Figure 2: Foxo1 is a target of miR-182. () Reporter gene expression in helper T lymphocytes restimulated every 6 d with anti-CD3, anti-CD28 and irradiated splenocytes and transduced with a reporter vector for the miR-183-binding site (miR-183 bs), the overlapping binding sites for miR-96 and miR-182 (miR-96–miR-182 bs) or all three predicted binding sites (miR-183–miR-96–miR-182 bs) or an empty reporter vector (w/o miR bs; control), assessed by flow cytometry of the mean fluorescence intensity (MFI) of human CD4, followed by normalized to the mean fluorescence intensity of the empty control vector. Data are representative of two experiments with similar results. () Reporter gene expression in helper T lymphocytes stimulated with anti-CD3, anti-CD28 and irradiated splenocytes and transduced with empty reporter vector (Control) or reporter vector for overlapping binding sites for miR-96 and miR-182 (miR-96–miR-182 bs) or mutated binding sites for miR-96 and miR-182 (miR-96–miR-182(mut) bs), assessed by flo! w cytometry of the mean fluorescence intensity of human CD4 on day 3 after activation. *P < 0.005 (Student's t-test). Data are from three independent experiments (error bars, s.d. of experiment pools). * Figure 3: Inactivation of Foxo1 by phosphorylation occurs rapidly after activation and ceases after 66 h. () Quantitative PCR analysis of the expression kinetics of Foxo1 mRNA (left axis) and miR-182 (right axis) in naive and activated helper T lymphocytes; results are presented relative to the expression of U6 (for miR-182) or Hprt1 (encoding mouse hypoxanthine guanine phosphoribosyl transferase; for Foxo1). () Quantitative PCR analysis of miR-182 expression in activated helper T lymphocytes on day 4 after treatment with an miR-182-specific antagomir (antagomir-182) or control scrambled antagomir (antagomir-scr); results are presented relative to U6 expression. *P < 0.001 (Student's t-test). () Quantitative PCR analysis of Foxo1 mRNA expression in activated helper T lymphocytes on day 4 after treatment with the antagomirs in ; results are normalized to Hprt1 and presented relative to those obtained with scrambled antagomir. () Immunoblot and densitometry analysis of the expression of Foxo1 protein in activated helper T lymphocytes on day 4 after treatment with the antagomirs in! . () Immunoblot and densitometry analysis of the kinetics of the ratio of total Foxo1 to phosphorylated Foxo1 (p-Foxo1) in helper T lymphocytes activated by anti-CD3 and anti-CD28. () Immunoblot analysis of the kinetics of the expression of total Foxo1 protein in helper T lymphocytes activated by anti-CD3 and anti-CD28. Data are representative of four experiments () or are from six (), two (,) or four (,) independent experiments (error bars (,,), s.d. of experiment pools). * Figure 4: IL-2 induces miR-182 expression. () Quantitative PCR analysis of miR-182 expression in TH0 lymphocytes activated for 48 h with plate-bound anti-CD3 and anti-CD28 and cultured with recombinant IL-2 (concentration, horizontal axis); results are presented relative to U6 expression. () Expression of miR-182 in TH0 lymphocytes activated for 48 h under standard conditions (Control) or with anti-IL-2 and anti-IL-2Rα (α-IL-2 + α-IL-2Rα), and in naive helper T cells (Naive); results are presented relative to U6 expression. *P < 0.05 (Student's t-test). () Chromatin-immunoprecipitation analysis of the binding of STAT5 to a predicted STAT5-binding site (STAT bs) at the miR-182 locus in TH0 cells activated for 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to input DNA. Negative, intronic sequence of the Foxo1 locus with no putative STAT-binding site 0.7 kilobases upstream or downstream (negative control); positive, known STAT5-binding site of the Cd25 promoter region (positive control! ). () Quantitative PCR analysis of miR-182 expression in TH0 lymphocytes treated with a chromone-based STAT5 inhibitor (compound 6) or vehicle (DMSO (dimethyl sulfoxide; control)); results are presented relative to U6 expression. () Chromatin-immunoprecipitation analysis of the kinetics of histone 4 acetylation at the miR-182 locus in TH1, TH2, TH17 and naive helper T lymphocytes; results are presented relative to U6 expression. Data are from one experiment representative of two independent experiments () or are from three () or five () independent experiments (error bars, s.d. of experiment pools) or one experiment (,). * Figure 5: Inhibition of miR-182 results in less T cell population expansion in vitro and in vivo. () Flow cytometry of viable cells in helper T cells activated with anti-CD3 and anti-CD28 and treated with antagomir-182 or control scrambled antagomir. *P < 0.05 (Student's t-test). () Frequency of dead cells among helper T cells activated with anti-CD3 and anti-CD28 and treated with the antagomirs in , assessed by flow cytometry analysis of propidium iodide staining. *P < 0.05, day four, and **P < 0.005, day three (Student's t-test). () CFSE-dilution analysis of CD4+ helper T lymphocytes after 3 d of treatment in vitro with the antagomirs in . () Flow cytometry of viable helper T cells recovered from lymph nodes and spleens of BALB/c mice given adoptive transfer of antagomir-treated and CFSE-labeled CD4+ helper T lymphocytes from DO11.10 mice, analyzed on day 4 after immunization with OVA peptide. *P < 0.005 (Student's t-test). () CFSE-dilution analysis of helper T cells recovered from lymph nodes and spleen of BALB/c mice given adoptive transfer of antagomir-treated and C! FSE-labeled CD4+ helper T lymphocytes from DO11.10 mice, analyzed by flow cytometry on day 2 after immunization with OVA peptide. Data are from three independent experiments (,; error bars, s.d. of experiment pools), three independent experiments (), one experiment with three mice (scrambled antagomir) or four mice (antagomir-182; ; error bars, s.e.m.), or one experiment with three samples per group (). * Figure 6: Inhibition of miR-182 results in lower severity of OVA-induced arthritis in mice. () Knee swelling of mice deficient in recombination-activating gene 1, immunized with OVA and given adoptive transfer of OT-II cells left untreated (Control) or treated with antagomir-182. P < 0.001 (two-way analysis of variance). () Absolute number of viable CD4+ lymphocytes in pooled popliteal and inguinal lymph nodes from mice deficient in recombination-activating gene 1 and given adoptive transfer of OT-II cells left untreated or treated with antagomir-182. *P < 0.05 (Student's t-test). () Knee swelling of OVA-immunized BALB/c mice given adoptive transfer of DO11.10 cells left untreated or treated with antagomir-182 after challenge with OVA peptide. P < 0.001 (two-way analysis of variance). () Histological scores of arthritis-induced knee joints after transfer of DO11.10 helper T cells left untreated or treated with antagomir-182 into BALB/c mice. Each symbol represents an individual mouse; small horizontal lines indicate the median. *P < 0.005 (Student's t-test). () Hem! atoxylin and eosin staining of knee joint sections showing fibroblast proliferation, mononuclear cell infiltration, bone and cartilage destruction and the formation of pannus in mice treated with antagomir-182 or control scrambled antagomir (histological score, 18 (control) or 13 (antagomir-182); maximum score, 21). Scale bars, 100 μm. Data are from one experiment with three (,), five to seven () or five () mice per group (error bars, s.e.m.) or five samples per group (). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Andreas Radbruch & * Mir-Farzin Mashreghi Affiliations * Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany. * Anna-Barbara Stittrich, * Claudia Haftmann, * Evridiki Sgouroudis, * Ahmed Nabil Hegazy, * Isabel Panse, * Rene Riedel, * Jun Dong, * Franziska Fuhrmann, * Gitta Anne Heinz, * Farahnaz Hatam, * Angelina Jahn, * Ben Hammoud, * Ria Baumgrass, * Joachim Grün, * Max Löhning, * Hyun-Dong Chang, * Andreas Radbruch & * Mir-Farzin Mashreghi * Charité Research Center for ImmunoSciences–Institute of Pathology, Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany. * Anja Andrea Kühl & * Christoph Loddenkemper * Experimental Immunology, Department of Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Campus Charité Mitte, Berlin, Germany. * Ahmed Nabil Hegazy, * Isabel Panse & * Max Löhning * German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg, Germany. * Michael Flossdorf & * Thomas Höfer * Berlin-Brandenburg Center for Regenerative Therapies, Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany. * Jun Dong & * Andreas Thiel * Robert Koch Institute, Berlin, Germany. * Franziska Fuhrmann * Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. * Zhuo Fang, * Na Li, * Wei Chen & * Nikolaus Rajewsky * Miltenyi Biotec, Bergisch-Gladbach, Germany. * Ute Bissels & * Andreas Bosio * Department of Nephrology, Charité Universitätsmedizin Berlin, Campus Charité Mitte, Berlin, Germany. * Mareen Matz * Institut für Immunologie und Transfusionsmedizin, Universität Greifswald, Greifswald, Germany. * Felix-Michael Schulze * Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany. * Hans-Joachim Mollenkopf Contributions A.-B.S. designed and did experiments, analyzed data and wrote the manuscript; C.H. did experiments and analyzed data; E.S. designed and did the OVA-induced arthritis experiments; A.A.K. and C.L. did histology; A.N.H., I.P. and M.L. designed and analyzed LCMV experiments; R.R. did the OVA-induced arthritis experiments; M.F. and T.H. analyzed CFSE measurements; J.D. and A.T. did human T cell experiments; F.F. provided cells; G.A.H. did inhibitory experiments; N.L., Z.F., U.B., A.B., J.G., H.-J.M., W.C. and N.R. did miRNA screening experiments and/or analyzed them; N.R., M.L. and Z.F. discussed the results and commented on the manuscript; F.H. revised the manuscript; A.J. constructed scrambled overexpression vector; B.H., M.M., F.-M.S. and R.B. provided technical support and conceptual advice; H.-D.C. designed experiments and supervised research; A.R. designed study, supervised research and wrote the manuscript; and M.-F.M. designed the study, analyzed data and wrote the manusc! ript. Competing financial interests U.B. and A.B. are employees of Miltenyi Biotec. Corresponding author Correspondence to: * Mir-Farzin Mashreghi (mashreghi@drfz.de) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 and Supplementary Table 1 Additional data
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