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- Failure to detect production of IL-10 by activated human neutrophils
- Nat Immunol 12(11):1017-1018 (2011)
Nature Immunology | Correspondence Failure to detect production of IL-10 by activated human neutrophils * Martin S Davey1, 6 * Nicola Tamassia2, 6 * Marzia Rossato2 * Flavia Bazzoni2 * Federica Calzetti2 * Kirsten Bruderek3 * Marina Sironi4 * Lisa Zimmer5 * Barbara Bottazzi4 * Alberto Mantovani4 * Sven Brandau3 * Bernhard Moser1 * Matthias Eberl1 * Marco A Cassatella2 * Affiliations * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:1017–1018Year published:(2011)DOI:doi:10.1038/ni.2111Published online19 October 2011 To the Editor: We read with great interest the paper by De Santo et al.1 published in the November 2010 issue of Nature Immunology, which has received wide attention because of its potential implications for inflammation and immunotherapy. The authors show that the acute-phase protein serum amyloid A1 (SAA-1) induces substantial secretion of the immunosuppressive cytokine interleukin 10 (IL-10) by human neutrophils (up to 100–400 ng/ml) and that invariant natural killer T cells (iNKT cells) are able to diminish this. The authors conclude that harnessing iNKT cells might "be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses" in patients with melanoma and other patients with high plasma SAA-1 concentrations1. View full text Author information * 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 Primary authors * These authors contributed equally to this work. * Martin S Davey & * Nicola Tamassia Affiliations * Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK. * Martin S Davey, * Bernhard Moser & * Matthias Eberl * Department of Pathology and Diagnostics, Division of General Pathology, University of Verona, Verona, Italy. * Nicola Tamassia, * Marzia Rossato, * Flavia Bazzoni, * Federica Calzetti & * Marco A Cassatella * Department of Otorhinolaryngology, University Hospital Essen, Essen, Germany. * Kirsten Bruderek & * Sven Brandau * Istituto Clinico Humanitas Istituto di Ricovero e Cura a Carattere Scientifico and University of Milan, Milan, Italy. * Marina Sironi, * Barbara Bottazzi & * Alberto Mantovani * Department of Dermatology, University Hospital Essen, Essen, Germany. * Lisa Zimmer Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Matthias Eberl or * Marco A Cassatella Author Details * Martin S Davey Search for this author in: * NPG journals * PubMed * Google Scholar * Nicola Tamassia Search for this author in: * NPG journals * PubMed * Google Scholar * Marzia Rossato Search for this author in: * NPG journals * PubMed * Google Scholar * Flavia Bazzoni Search for this author in: * NPG journals * PubMed * Google Scholar * Federica Calzetti Search for this author in: * NPG journals * PubMed * Google Scholar * Kirsten Bruderek Search for this author in: * NPG journals * PubMed * Google Scholar * Marina Sironi Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa Zimmer Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Bottazzi Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Mantovani Search for this author in: * NPG journals * PubMed * Google Scholar * Sven Brandau Search for this author in: * NPG journals * PubMed * Google Scholar * Bernhard Moser Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Eberl Contact Matthias Eberl Search for this author in: * NPG journals * PubMed * Google Scholar * Marco A Cassatella Contact Marco A Cassatella Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1.1M) Supplementary Figures 1–11 and Methods Additional data - Reply to "Failure to detect production of IL-10 by activated human neutrophils"
- Nat Immunol 12(11):1018-1020 (2011)
Nature Immunology | Correspondence Reply to "Failure to detect production of IL-10 by activated human neutrophils" * Carmela De Santo1 * Mariolina Salio1 * Tao Dong1 * Yoram Reiter2 * Vincenzo Cerundolo1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1018–1020Year published:(2011)DOI:doi:10.1038/ni.2132Published online19 October 2011 Cerundolo et al. respond: In our article, we reported that human neutrophils can secrete IL-10 and that their interaction with iNKT cells can result in less IL-10 secretion1, thereby providing therapeutic opportunities that should be explored in future clinical trials. Davey et al. have described a series of experiments in which they did not detect the secretion of IL-10 by purified neutrophils, and on the basis of such negative results they conclude that human neutrophils are unable to produce IL-10. This is in contrast with results we have been generating routinely for more than 3 years and with other published data demonstrating the ability of human neutrophils2, 3, mouse neutrophils4, 5 and equine neutrophils6 to secrete IL-10, which indicates a common cross-species strategy for down-modulation of the proinflammatory activity of neutrophils. Indeed, a published paper has demonstrated that infiltrating neutrophils in mice infected with Yersinia enterocolitica represent most IL-10-producing cells a! t the site of infection7. As our results were highly reproducible and were not due to the presence of contaminating monocytes (Supplementary Note and Supplementary Figs. 1–3), we can only conclude that the negative results obtained by Davey et al. were caused by their purification protocols and the experimental conditions they used that diminished the ability of their preparations of human neutrophils to secrete IL-10. View full text Author information * 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 Affiliations * Medical Research Council Human Immunology Unit, Nuffield Department of Medicine, John Radcliffe Hospital, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK. * Carmela De Santo, * Mariolina Salio, * Tao Dong & * Vincenzo Cerundolo * Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel. * Yoram Reiter Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vincenzo Cerundolo Author Details * Carmela De Santo Search for this author in: * NPG journals * PubMed * Google Scholar * Mariolina Salio Search for this author in: * NPG journals * PubMed * Google Scholar * Tao Dong Search for this author in: * NPG journals * PubMed * Google Scholar * Yoram Reiter Search for this author in: * NPG journals * PubMed * Google Scholar * Vincenzo Cerundolo Contact Vincenzo Cerundolo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (5.2M) Supplementary Figures 1–9, Note and Methods Additional data - Effective presentations: tips for success
- Nat Immunol 12(11):1021-1023 (2011)
Article preview View full access options Nature Immunology | Commentary Effective presentations: tips for success * Janet P Hafler1Journal name:Nature ImmunologyVolume: 12,Pages:1021–1023Year published:(2011)DOI:doi:10.1038/ni.2119Published online19 October 2011 Presentations are given in a variety of environments, and effective strategies can be used to improve a speaker's presenting skills. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Janet P. Hafler is in the Department of Pediatrics, Yale School of Medicine, New Haven, Connecticut, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Janet P Hafler Author Details * Janet P Hafler Contact Janet P Hafler Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Innate lymphoid cells wield a double-edged sword
- Nat Immunol 12(11):1025-1027 (2011)
Article preview View full access options Nature Immunology | News and Views Innate lymphoid cells wield a double-edged sword * Marsha Wills-Karp1 * Fred D Finkelman2 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1025–1027Year published:(2011)DOI:doi:10.1038/ni.2142Published online19 October 2011 Type 2 cytokine–producing innate lymphoid cells are present in human and mouse lungs, where they contribute to both type 2 immune responses and tissue repair. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Marsha Wills-Karp is in the Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA. * Fred D. Finkelman is in the Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA, and the Department of Medicine, Cincinnati Veterans Affairs Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Marsha Wills-Karp Author Details * Marsha Wills-Karp Contact Marsha Wills-Karp Search for this author in: * NPG journals * PubMed * Google Scholar * Fred D Finkelman Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - TL we meet again
- Nat Immunol 12(11):1027-1028 (2011)
Article preview View full access options Nature Immunology | News and Views TL we meet again * Cathryn Nagler1 * Joanna Wroblewska1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1027–1028Year published:(2011)DOI:doi:10.1038/ni.2138Published online19 October 2011 The MHC class I–like molecule TL selects high-affinity effector memory T cells to man the barriers in the intestinal mucosa. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Cathryn Nagler and Joanna Wroblewska are in the Department of Pathology, Committee on Immunology at the University of Chicago, Chicago, Illinois, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Cathryn Nagler Author Details * Cathryn Nagler Contact Cathryn Nagler Search for this author in: * NPG journals * PubMed * Google Scholar * Joanna Wroblewska Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Don't move: LRRK2 arrests NFAT in the cytoplasm
- Nat Immunol 12(11):1029-1030 (2011)
Article preview View full access options Nature Immunology | News and Views Don't move: LRRK2 arrests NFAT in the cytoplasm * Bana Jabri1 * Luis B Barreiro1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1029–1030Year published:(2011)DOI:doi:10.1038/ni.2139Published online19 October 2011 The kinase LRRK2 is a risk factor for inflammatory bowel disease. New data show that LRRK2 blocks the transport of NFAT to the nucleus and that LRRK2 deficiency results in enhanced susceptibility to experimental colitis in mice. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Bana Jabri is in the Department of Medicine, Department of Pathology and Department of Pediatrics, University of Chicago, Chicago, Illinois, USA. * Luis B Barreiro * Luis B. Barreiro is in the Department of Pediatrics, Faculty of Medicine, Sainte-Justine Hospital Research Centre, University of Montreal, Montreal, Quebec, Canada. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bana Jabri Author Details * Bana Jabri Contact Bana Jabri Search for this author in: * NPG journals * PubMed * Google Scholar * Luis B Barreiro Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - PKC-θ: hitting the bull's eye
- Nat Immunol 12(11):1031-1032 (2011)
Article preview View full access options Nature Immunology | News and Views PKC-θ: hitting the bull's eye * Michael L Dustin1Journal name:Nature ImmunologyVolume: 12,Pages:1031–1032Year published:(2011)DOI:doi:10.1038/ni.2141Published online19 October 2011 How the kinase PKC-θ is targeted to the immunological synapse and is activated once there remains unclear. A targeting motif in PKC-θ and the previously unsuspected kinase GLK identified in two separate papers now explains this. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Immunology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Michael L. Dustin is with the Kimmel Center of Skirball Institute, New York University School of Medicine, New York, New York, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Michael L Dustin Author Details * Michael L Dustin Contact Michael L Dustin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils
- Nat Immunol 12(11):1035-1044 (2011)
Nature Immunology | Review Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils * Stephen J Galli1, 2 * Niels Borregaard3 * Thomas A Wynn4 * Affiliations * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:1035–1044Year published:(2011)DOI:doi:10.1038/ni.2109Published online19 October 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents a common mechanism for modulating innate or adaptive immunity. View full text Figures at a glance * Figure 1: Macrophage populations and functional subsets. Macrophages can be subcategorized into specific populations on the basis of their anatomical location (left) and functional phenotype (right). Tissue-resident macrophages include alveolar macrophages (lungs), histiocytes (interstitial connective tissue), osteoclasts (bone), microglia (brain), intestinal macrophages, Kupffer cells (liver) and so on. Mononuclear phagocyte subpopulations in the circulation can also differentiate into tissue macrophages after entering different anatomical sites; when activated by the appropriate stimuli, these cells differentiate into various subsets with distinct phenotypic and functional characteristics. * Figure 2: Mast-cell populations and patterns of functional activation. Mast cells (MCs) in mice or humans can be subcategorized (left) into populations defined by anatomical location and/or mediator content (such as proteoglycans (heparin versus chondroitin sulfates) or proteases (tryptases, chymases or MC-CPA)). In IgE-associated immune responses to allergens or parasites (top right), the activation of mast cells via crosslinking of IgE bound to high-affinity receptors for IgE (FceRI) on the cell surface by bi- or multivalent antigens results in rapid exocytosis of the cytoplasmic granules (degranulation) and the production of lipid mediators (such as leukotrienes and prostaglandins) and the more sustained secretion of many cytokines, chemokines and growth factors. Although many of these mediators have proinflammatory effects, others can have effects that suppress inflammation or promote tissue remodeling or repair. Signals not dependent on IgE (bottom right) can elicit different patterns of mediator release in mast-cell populations that expre! ss receptors appropriate for such ligands. Microenvironmental factors can influence the phenotype of mast cells that develop under basal conditions in different anatomic sites (left), including those phenotypic features that permit mast cells to respond to various ligands (such as the pattern of expression of receptors for those ligands) or to produce different mediators (right). TLRs are examples of the many pattern-recognition receptors expressed by various populations of mast cells. MCT, mast cell containing mainly tryptase; MCTC, mast cell containing both tryptase and chymase; C3a and C5a, anaphylatoxins of the complement system. * Figure 3: Features shared by 'neutrophil MDSCs' and neutrophils in mice with polymicrobial infection. Growth factors and cytokines generated by tumors and macrophages, as well as bacterial products, modulate the development and phenotype of neutrophils by acting both on developing neutrophils in the bone marrow and locally on neutrophils in tissues. Mast cells (not shown here) also can generate many cytokines and growth factors that can influence neutrophils, including TNF, IL-1b, GM-CSF and IL-6. Reactive oxygen species (ROS) and arginase secreted from activated neutrophils can inhibit T cell function and permit tumor growth. In this setting, such neutrophils constitute 'neutrophil MDSCs'. LPS, lipopolysaccharide. Author information * Abstract * Author information Affiliations * Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. * Stephen J Galli * Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. * Stephen J Galli * Department of Hematology, National University Hospital, University of Copenhagen, Copenhagen, Denmark. * Niels Borregaard * Program in Barrier Immunity and Repair, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA. * Thomas A Wynn Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Stephen J Galli or * Niels Borregaard or * Thomas A Wynn Author Details * Stephen J Galli Contact Stephen J Galli Search for this author in: * NPG journals * PubMed * Google Scholar * Niels Borregaard Contact Niels Borregaard Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas A Wynn Contact Thomas A Wynn Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus
- Nat Immunol 12(11):1045-1054 (2011)
Nature Immunology | Article Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus * Laurel A Monticelli1, 2 * Gregory F Sonnenberg1, 2 * Michael C Abt1, 2 * Theresa Alenghat1, 2 * Carly G K Ziegler1 * Travis A Doering1 * Jill M Angelosanto1 * Brian J Laidlaw1 * Cliff Y Yang3 * Taheri Sathaliyawala4 * Masaru Kubota4 * Damian Turner4 * Joshua M Diamond5 * Ananda W Goldrath3 * Donna L Farber4 * Ronald G Collman5 * E John Wherry1 * David Artis1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:1045–1054Year published:(2011)DOI:doi:10.1038/ni.2131Received02 August 2011Accepted01 September 2011Published online25 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Innate lymphoid cells (ILCs), a heterogeneous cell population, are critical in orchestrating immunity and inflammation in the intestine, but whether ILCs influence immune responses or tissue homeostasis at other mucosal sites remains poorly characterized. Here we identify a population of lung-resident ILCs in mice and humans that expressed the alloantigen Thy-1 (CD90), interleukin 2 (IL-2) receptor α-chain (CD25), IL-7 receptor α-chain (CD127) and the IL-33 receptor subunit T1-ST2. Notably, mouse ILCs accumulated in the lung after infection with influenza virus, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling. These defects were restored by administration of the lung ILC product amphiregulin. Collectively, our results demonstrate a critical role for lung ILCs in restoring airway epithelial integrity and tissue homeostasis after infection with influenza virus. View full text Figures at a glance * Figure 1: ILCs in the lung resemble nuocytes and NHCs in phenotype and cytokine profile. () Identification of lung ILCs in C57BL/6 wild-type (WT) and Rag1−/− mice by flow cytometry as CD90+CD25+CD127+Lin− cells that lack expression of CD3, CD5, NK1.1, CD27 and TCRβ (vertical axes) and B220, CD11b and CD11c (horizontal axes). Numbers adjacent to outlined areas indicate percent cells in each throughout. () Expression of cell surface markers on Lin−CD90+CD25+ lung ILCs in C57BL/6 wild-type and Rag1−/− mice. Gray shading, isotype-matched control antibody. () Absolute number of CD90+CD25+ ILCs in naive wild-type or Rag1−/− lungs. () Flow cytometry analysis of the purity of CD90+CD25+T1-ST2+ wild-type lung ILCs before and after sorting, gated on live Lin− cells. () Enzyme-linked immunosorbent assay of IL-5 and IL-13 in supernatants of CD90+CD25+T1-ST2+ lung ILCs sorted by flow cytometry and cultured for 4 d with IL-2 plus IL-7, with or without IL-33. () Intracellular cytokine staining for IL-22 and IL-17A in Lin−CD90+CD25+ lung ILCs or Lin−CD90+! CD4+ splenic LTi cells obtained from wild-type mice and stimulated for 12 h with recombinant IL-23 (50 ng/ml) and for 4 h with the phorbol ester PMA and ionomycin plus brefeldin A. Numbers in quandrants indicate percent cells in each throughout. () Enzyme-linked immunosorbent assay of IL-17A in supernatants of CD90+CD25+T1-ST2+ lung ILCs or Lin−CD90+CD4+ LTi spleen cells sorted by flow cytometry and cultured for 4 d with IL-2 plus IL-7, with IL-23. () Intracellular cytokine staining of Lin−CD90+CD25+ lung ILCs obtained from wild-type mice treated for 7 d in vivo with 500 ng recombinant IL-33 and stimulated for 4 h ex vivo with PMA and ionomycin plus brefeldin A. *P < 0.05 (unpaired Student's t-test). Data are representative of more than three experiments with at least four mice per genotype (–; mean and s.e.m. in ), three independent experiments (,; mean and s.e.m. of three replicates in , each consisting of ILCs sorted from five pooled lungs), two or more experiments! with three to four mice each (,) or two independent experimen! ts (; mean and s.e.m. of three replicates, each consisting of ILCs sorted from five pooled lungs or spleens). * Figure 2: The development of lung ILCs requires Id2 but is independent of microbial signals. () Expression of Id2 mRNA in Lin−CD90+CD25+ lung ILCs and Lin− CD90+CD4+ splenic LTi cells obtained from C57BL/6 wild-type mice and purified by sorting; results are normalized to those for β-actin and are presented relative to expression in purified B220+ B cells. () Flow cytometry of CD90+CD25+ lung ILCs from wild-type (Id2+/+) or Id2-deficient (Id2−/−) bone marrow chimeras at 10 weeks after reconstitution, gated on live Lin− donor cells. () Expression of Rorc mRNA in cells as in . () Flow cytometry analysis of RORγt expression in Lin−CD90+CD25+ lung ILCs or Lin−CD90+CD4+ LTi cells. () Flow cytometry (left), total frequency (middle) and absolute number (right) of Lin−CD90+CD25+ lung ILCs from conventional C57BL/6 mice (CNV) or germ-free mice (GF). () Cell surface expression of c-Kit, CD127 and T1-ST2 on Lin−CD90+CD25+ lung ILCs from conventional mice or germ-free mice. Gray shading (,), isotype-matched control antibody. Data are representative of two ind! ependent experiments (,; mean and s.e.m. of three replicates, each consisting of spleens or lungs pooled from five mice), three experiments with two to four chimeras per genotype (), more than three experiments with three mice () or two independent experiments (,; mean and s.e.m. of three conventional or germ-free mice in ). * Figure 3: Lin−CD127+CD25+ST2+ ILCs in human lung and airways. () Gating strategy for the identification of CD127+Lin− ILCs (CD3−TCRαβ−CD11c−CD11b−CD19−CD56−) in human lung parenchyma tissue (lower lobe; cadaver tissue) by flow cytometry, gated on live cells. () Expression of CD25 and ST2 on Lin−CD127+ human lung parenchyma cells. FMO, fluorescence-minus-one control. () Gating strategy as in for cells in BAL fluid (from lung transplant recipients). () Expression of CD25 and ST2 on Lin−CD127+ cells in BAL fluid (as in ). Data are representative of one experiment per donor (among four donors (lung) or seven donors (BAL fluid)). * Figure 4: Depletion of CD90+ ILCs during influenza infection results in diminished lung function, compromised epithelial integrity and impaired airway remodeling. (,) Flow cytometry () and frequency () of Lin−CD90+CD25+ ILCs in the lung parenchyma of naive mice (Naive) or mice infected intranasally with 0.5 LD50 PR8 (Inf), assessed 16 d (wild type) or 10 d (Rag1−/−) after infection. () Flow cytometry of Lin−CD90+CD25+ ILCs from the lungs of Rag1−/− mice infected intranasally with 0.5 LD50 PR8 on day 0 and treated intraperitoneally with 200 μg isotype, mAb to NK1.1 (α-NK1.1) or mAb to CD90.2 (α-CD90.2) the day before infection (day –1) and on days 2, 5 and 8 after infection, assessed 10 d after infection. () Quantitative PCR analysis of the viral load of PR8 in wild-type mice infected as in and left untreated (Inf WT) or in Rag1−/− mice infected and treated with isotype (Inf + isotype), mAb to NK1.1 (Inf + α-NK1.1) or mAb to CD90.2 (Inf + α-CD90.2) as in , assessed 10 d after infection and presented as TCID50 per gram of lung tissue; dashed line indicates the limit of detection. () Body temperature of naive Rag1�! ��/− mice and Rag1−/− mice infected and treated as in , assessed 10 d after infection. () Blood oxygen saturation (SpO2) over the course of infection as in . () Total protein in BAL fluid 10 d after infection as in . (–) Hematoxylin-and-eosin staining of lung tissue from Rag1−/− mice infected with PR8 and treated with isotype (), anti-NK1.1 () or anti-CD90.2 () as in , assessed 10 d after infection. Black arrows indicate goblet cell hyperplasia (,) or regions of epithelial shedding or necrosis in the bronchioles (); white arrows indicate epithelial cell hyperplasia (,). Bottom row, enlargement of regions outlined above. Scale bars, 50 μm. () Histological scores of bronchial epithelial degeneration in lung sections from Rag1−/− mice infected and treated as in , obtained 10 d after infection and stained with hematoxylin and eosin. (,) Hematoxylin-and-eosin staining of lung tissue from a naive mouse treated with anti-CD90.2 () and an untreated naive mouse (). ! Scale bars, 50 μm. In ,,, each symbol represents an individua! l mouse; small horizontal lines indicate the mean () or mean and s.e.m. (,). *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired Student's t-test). Data are representative of three or more independent experiments with three to four mice per group (mean and s.e.m. in ,,). * Figure 5: Adoptive transfer of lung-resident ILCs promotes tissue remodeling in mice depleted of ILCs by treatment with anti-CD90.2, but blockade of IL-33R signaling impairs lung function and airway repair. (,) Flow cytometry of endogenous Lin−CD90.2+CD25+ ILCs () and donor Lin−CD90.1+T1-ST2+ ILCs () in the lungs of Rag1−/− mice infected intranasally with 0.5 LD50 PR8 on day 0 and treated with 200 μg isotype or mAb to CD90.2 intraperitoneally the day before infection (day –1) and on days 2, 5 and 8 after infection, either alone (α-CD90.2) or with intravenous injection of 1 × 105 Lin−CD90.1+CD25+T1-ST2+ lung ILCs (sorted by flow cytometry) on days 0 and 5 after infection (α-CD90.2 + ILCs), assessed 10 d after infection. () Body temperature of naive mice and Rag1−/− mice infected as in , and treated with isotype (Inf + isotype) or mAb to CD90.2 without additional cells (Inf + α-CD90.2) or with the injection of ILCs (Inf + α-CD90.2 + ILCs) as in ,, assessed 10 d after infection. () Blood oxygen saturation over the course of infection as in ,. (–) Hematoxylin-and-eosin staining of lung tissue from Rag1−/− mice infected as in , and treated with isotype ()! , anti-CD90.2 alone () or anti-CD90.2 plus ILCs () as in ,, assessed 10 d after infection. Black arrows indicate epithelial cell hyperplasia; gray arrows indicate regions of epithelial shedding and/or necrosis in the bronchioles. Scale bars, 50 μm. (–) Flow cytometry (), frequency () and number () of CD90+CD25+ ILCs in the lungs of C57BL/6 wild-type mice given PBS or 200 μg mAb to IL-33R (α-IL-33R) every 3 d after infection with 0.5 LD50 PR8, assessed 10 d after infection. () Blood oxygen saturation of mice infected and treated as in –. (,) Hematoxylin-and-eosin staining of lung tissue from mice infected with PR8 and treated with PBS () or anti-IL-33R () as in –, assessed 10 d after infection; arrows as in –. Scale bars, 100 μm. NS, not significant. *P < 0.05 and **P < 0.001 (unpaired Student's t-test). Data are representative of two independent experiments with three to four mice per group (–) or three independent experiments with three to four mice per group! (–; mean and s.e.m. in ,,–). * Figure 6: Global gene-expression profiling of lung-resident ILCs shows enrichment for genes encoding molecules that regulate wound-healing pathways. () Heat-map presentation of gene-expression profiles of the top 100 genes with the greatest differences in expression (red, high; blue, low) in Lin−CD90+CD25+ lung ILCs versus Lin−CD90+CD4+ splenic LTi cells sorted by flow cytometry from naive C57BL/6 wild-type mice. () Heat map of key genes from the profiling in . () Functional classification of the gene-expression signatures of lung ILCs and splenic LTi cells (genes with a difference in expression of twofold or more) according to terms from the Gene Ontology project (GO term), analyzed with the DAVID database of functional annotation tools and presented as significance of enrichment (light gray, P > 0.05; black, P < 0.0004). () Gene-set enrichment analysis comparing lung ILC gene-expression signatures with those of a published data set examining the effects of LPS-induced acute lung injury34. Arrow indicates position of Areg in the analysis of the top transcripts in the LPS-treated group that define the 'leading edge' ! (long vertical line, right). Data are representative of one experiment with four biological replicates, each consisting of 1.5 × 104 to 2 × 104 ILCs (six mice per replicate) or LTi cells (ten mice per replicate). * Figure 7: Amphiregulin is produced by lung ILCs and can restore lung function, barrier integrity and remodeling of respiratory tissues after influenza virus–induced damage. () Expression of Areg mRNA in sort-purified Lin−CD90+CD25+T1-ST2+ lung ILCs and Lin−CD90+CD4+ splenic LTi cells, presented relative to expression in splenic LTi cells. () Enzyme-linked immunosorbent assay of amphiregulin in sort-purified lung ILCs stimulated for 4 d with IL-2 and IL-7, with or without IL-33. (,) Expression of Areg mRNA () and amphiregulin protein () in the lungs of naive or PR8-infected Rag1−/− mice at 10 d after infection. Areg mRNA results are presented relative to those of naive lung tissue. () Expression of Areg mRNA in the lungs of naive Rag1−/− mice (Naive) or PR8-infected Rag1−/− mice that received isotype (Inf + istoype) or mAb to CD90.2 (Inf + α-CD90.2), assessed 10 d after infection; results are presented relative to expression in naive lung tissue. () Flow cytometry of lung ILCs from Rag1−/− mice infected intranasally with 0.5 LD50 PR8 and treated with isotype (Isotype), mAb to CD90.2 alone (α-CD90.2) or mAb CD90.2 plus 5–1! 0 μg recombinant mouse amphiregulin administered intraperitoneally every 2 d (α-CD90.2 + AREG). () Body temperature of naive Rag1−/− mice (Naive) or PR8-infected Rag1−/− mice that received isotype (Inf + istoype), mAb to CD90.2 alone (Inf + α-CD90.2) or mAb to CD90.2 plus amphiregulin (Inf + α-CD90.2 + AREG) as in , assessed 10 d after infection (presented as in Fig. 4d,e). () Blood oxygen saturation in mice infected and treated as in . () Protein in BAL fluid of mice infected and treated as in , assessed 10 d after infection. () Hematoxylin-and-eosin staining of lung tissue from Rag1−/− mice infected as in and treated with isotype (), mAb to CD90.2 () or mAb to CD90.2 plus amphiregulin () as in , assessed 10 d after infection (arrows as in Fig. 5e–g). Scale bars, 50 μm. *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired Student's t-test). Data are representative of two independent experiments with three to four mice per group (mean and s.e.m. in –,,). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Microbiology and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Laurel A Monticelli, * Gregory F Sonnenberg, * Michael C Abt, * Theresa Alenghat, * Carly G K Ziegler, * Travis A Doering, * Jill M Angelosanto, * Brian J Laidlaw, * E John Wherry & * David Artis * Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Laurel A Monticelli, * Gregory F Sonnenberg, * Michael C Abt, * Theresa Alenghat & * David Artis * Department of Biology, University of California San Diego, La Jolla, California, USA. * Cliff Y Yang & * Ananda W Goldrath * Department of Surgery and the Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA. * Taheri Sathaliyawala, * Masaru Kubota, * Damian Turner & * Donna L Farber * Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Joshua M Diamond & * Ronald G Collman Contributions L.A.M., G.F.S. and M.C.A. did experiments and analyzed data; T.A. analyzed lung histological specimens; C.G.K.Z. and T.A.D. did microarray analysis; J.M.A. and B.J.L. designed the method for quantifying influenza virus by quantitative RT-PCR; C.Y.Y. and A.W.G. generated and provided Id2-deficient fetal liver chimeras; T.S., M.K., D.T. and D.L.F. collected and processed human lung tissues; J.M.D. and R.G.C. collected and processed human BAL fluid; and L.A.M., E.J.W. and D.A. designed the study, analyzed experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * E John Wherry or * David Artis Author Details * Laurel A Monticelli Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory F Sonnenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Michael C Abt Search for this author in: * NPG journals * PubMed * Google Scholar * Theresa Alenghat Search for this author in: * NPG journals * PubMed * Google Scholar * Carly G K Ziegler Search for this author in: * NPG journals * PubMed * Google Scholar * Travis A Doering Search for this author in: * NPG journals * PubMed * Google Scholar * Jill M Angelosanto Search for this author in: * NPG journals * PubMed * Google Scholar * Brian J Laidlaw Search for this author in: * NPG journals * PubMed * Google Scholar * Cliff Y Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Taheri Sathaliyawala Search for this author in: * NPG journals * PubMed * Google Scholar * Masaru Kubota Search for this author in: * NPG journals * PubMed * Google Scholar * Damian Turner Search for this author in: * NPG journals * PubMed * Google Scholar * Joshua M Diamond Search for this author in: * NPG journals * PubMed * Google Scholar * Ananda W Goldrath Search for this author in: * NPG journals * PubMed * Google Scholar * Donna L Farber Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald G Collman Search for this author in: * NPG journals * PubMed * Google Scholar * E John Wherry Contact E John Wherry Search for this author in: * NPG journals * PubMed * Google Scholar * David Artis Contact David Artis Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6 and Tables 1–2 Additional data - Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161
- Nat Immunol 12(11):1055-1062 (2011)
Nature Immunology | Article Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161 * Jenny M Mjösberg1 * Sara Trifari2, 6 * Natasha K Crellin2, 6 * Charlotte P Peters1 * Cornelis M van Drunen3 * Berber Piet4 * Wytske J Fokkens3 * Tom Cupedo5 * Hergen Spits1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1055–1062Year published:(2011)DOI:doi:10.1038/ni.2104Received11 April 2011Accepted01 August 2011Published online11 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Innate lymphoid cells (ILCs) are emerging as a family of effectors and regulators of innate immunity and tissue remodeling. Interleukin 22 (IL-22)- and IL-17-producing ILCs, which depend on the transcription factor RORγt, express CD127 (IL-7 receptor α-chain) and the natural killer cell marker CD161. Here we describe another lineage-negative CD127+CD161+ ILC population found in humans that expressed the chemoattractant receptor CRTH2. These cells responded in vitro to IL-2 plus IL-25 and IL-33 by producing IL-13. CRTH2+ ILCs were present in fetal and adult lung and gut. In fetal gut, these cells expressed IL-13 but not IL-17 or IL-22. There was enrichment for CRTH2+ ILCs in nasal polyps of chronic rhinosinusitis, a typical type 2 inflammatory disease. Our data identify a unique type of human ILC that provides an innate source of T helper type 2 (TH2) cytokines. View full text Figures at a glance * Figure 1: Lin− lymphocytes in the fetal gut include a CRTH2+CD127+ ILC population. () Flow cytometry analysis of the expression of CD127 and CD117 by two distinct ILC populations in the fetal gut: Lin− cells (left; CD1a−CD3−CD11c−CD14−CD19−CD34−CD123−TCRαβ−TCRγδ−BDCA2−FcεR1−) gated as CD45int (1) or CD45hi (2). Numbers in quadrants indicate percent cells in each throughout. () Flow cytometry characterization of Lin−CD127+CD45int cells (gray lines) and Lin−CD127+CD45hi cells (black lines); light gray shading, isotype-matched control antibody. () Flow cytometry analysis of the expression of CD117 and CRTH2 (left), CD123 (IL-3R; middle) and FcεR1 (right) on CD45hiLin−CD127+ cells (black lines) and peripheral blood monocytes or basophils (gray lines); light gray shading, isotype-matched control antibody. () Flow cytometry analysis of RORγt expression (left) in CD56int peripheral blood NK cells (dashed gray line), fetal gut CD45hiLin−CD127+CRTH2+ ILCs (solid black line) and CD45intLin−CD127+NKp44+ ILCs (solid gray line).! Right, RORC mRNA expression in fetal gut NKp44+ or NKp44− ILCs (gated as CD45intLin−CD127+CD117+), CRTH2+ ILCs (CD117+ or CD117−; gated as CD45hiLin−CD127+) and conventional NK cells (cNK; CD45hiCD127−CD56+), tonsil NKp44+ ILCs and fetal mesenteric lymph node (MLN) NKp44− ILCs. Data are representative of at least three experiments with one to three donors each (– and , left) or are from one experiment with two to five donors (, right; median and range). * Figure 2: CRTH2+ fetal gut ILCs express IL13 transcripts ex vivo. Real-time PCR analysis of the expression of transcripts of IL13, IL22, IL17, IFNG and tumor necrosis factor (TNF) in fetal gut NKp44+ or NKp44− ILCs (gated as CD45intLin−CD127+CD117+), CRTH2+ ILCs (CD117+ or CD117−; purified by flow cytometry and gated as CD45hiLin−CD127+) and conventional NK cells (CD45hiLin−CD127−CD56+), tonsil NKp44+ ILCs and fetal mesenteric lymph node NKp44− ILCs. The lineage 'cocktail' included antibodies to CD1a, CD3, CD4, CD11c, CD14, CD19, CD34, CD123, TCRαβ, TCRγδ, BDCA2 and FcεR1. Data are from one experiment with two to five donors (median and range). * Figure 3: CRTH2+ fetal gut ILCs respond to IL-25 and IL-33 in vitro by producing IL-13. () IL-13 production by fetal gut CD45hiLin−CD127+CRTH2+ ILCs sorted by flow cytometry and cultured for 4 d with IL-2 (10 U/ml) alone (IL-2) or with a combination of IL-2 (10 U/ml) and IL-25 (50 ng/ml; IL-2 + IL-25). () IL-13 production by CD45hiLin−CD127+CRTH2+ ILCs sorted on the basis of CD117 expression and stimulated as in . () Enzyme-linked immunosorbent of IL-13 in supernatants of fetal gut CD117+CRTH2+ and CD117−CRTH2+ ILCs stimulated with IL-2 (10 U/ml) alone (IL-2) or with a combination of IL-2 (10 U/ml) and IL-33 (50 ng/ml; IL-2 + IL-33). Results (–) were normalized for those of 2,000 cells per 200 μl in a 96-plate well (lineage 'cocktail' as in Fig. 2). Data are representative of two experiments with one donor each (,) or one experiment with four donors (). * Figure 4: Analysis of stable cell lines generated from fetal gut CRTH2+ ILCs. () Flow cytometry analysis of expanded fetal gut CD45hiLin− (lineage 'cocktail' as in Fig. 2) CD127+CD117+CRTH2+ ILC lines (black lines); light gray shading, isotype-matched control antibody. () Flow cytometry analysis of fetal gut CRTH2+ ILC lines (black lines) and CD56+ conventional NK cell lines (gray lines) stimulated with PMA and ionomycin and stained for intracellular IL-13, IL-22 or IL-17; light gray shading, unstimulated CRTH2+ ILCs. Far right, expression of IL-22 and IL-13 by fetal gut CD127+CD117+CRTH2+ ILCs stimulated with PMA and ionomycin. Numbers in quadrants (far right) indicate percent cells in each (among total cells). () Expression of AHR mRNA in fetal gut CRTH2+ ILCs (CD117+ or CD117−), blood CD56int conventional NK cells and tonsil NKp44+ ILCs. (,) Secretion of IL-22 from fetal gut CRTH2+ ILCs () or tonsil NKp44+ ILCs () after stimulation with various combinations (horizontal axes) of IL-2, IL-1β (50 ng/ml) and IL-23 (50 ng/ml). () Expression of ST2,! IL17RB, IL17RA and IL23R mRNA in cells as in . () IL-13 response of fetal gut CRTH2+ ILCs to various combinations (horizontal axes) of IL-2, IL-25 and IL-33. Data are representative of three experiments with one donor each (,) or are from one experiment with three to four donors (; median and range), three to four experiments with one donor each (,; mean and s.e.m.), one experiment with three to five donors (; median and range) or three experiments with one donor each (; mean and s.e.m.). * Figure 5: CRTH2+ ILCs are distributed in several fetal and adult tissues and show enrichment in the nasal polyps of patients with chronic rhinosinusitis. () Expression of CD117 and CRTH2 by mononuclear cells isolated from fetal gut, fetal lung, adult gut and adult lung, stained for ILCs (gated as CD45+, Lin− (as in Fig. 1) and CD127+); numbers in quadrants indicate percent cells of the Lin−CD127+ gate. () Flow cytometry analysis of the expression of CD117 and CRTH2 (left and middle) in cells from healthy control nasal tissue (HC) and nasal polyps of chronic rhinosinusitis (CRS), and frequency of CRTH2+ ILCs (right). Each symbol represents a separate donor; small horizontal lines indicate the median. *P < 0.03 (Mann-Whitney two-tailed test). Numbers in quadrants (left) indicate percent cells of the Lin−CD127+ cell gate. () Expression of CD161 by nasal polyp ILCs defined as CD45+, Lin− (as in Fig. 1) and CD127+ (black line); gray shading, isotype-matched control. () Expression of RORγt by nasal polyp ILCs (defined as in ), fetal gut (uninflamed) CRTH2+ ILCs, peripheral blood conventional NK cells and fetal gut (uninfla! med) NKp44+ ILCs. Data are representative of two to five experiments with one patient each (), four experiments with one donor each () or four experiments with one patient or donor each (,). * Figure 6: CRTH2+CD127+CCR6+ innate lymphoid cells are present in peripheral blood. () Flow cytometry analysis of peripheral blood cell samples depleted of most T cells, B cells and monocytes; right, gating showing the presence of a Lin−CD127+CD117+ population (1) and Lin−CD127+CD117− population (4). () Flow cytometry analysis of various markers (horizontal axes) in Lin−CD127+CD117+ and Lin−CD127+CD117− populations as in ; gray shading, isotype-matched control antibody. () Flow cytometry analysis of the expression of CRTH2 and CCR6 in Lin−CD127+CD117+ (top) and Lin−CD127+CD117− (bottom) cells as in . Numbers in quadrants indicate percent cells of gate above plot. () Expression of RORγt in Lin−CD127+CRTH2+ ILCs (black line), conventional CD56+ NK cells (dashed gray line) and IL-17-producing helper T cells (CD3+CD4+CCR4+CCR6+; solid gray line). Data are representative of at least three experiments with one donor each. * Figure 7: CRTH2+ peripheral blood ILCs respond to IL-25 and IL-33 by producing IL-13 protein. Enzyme-linked immunosorbent assay of IL-13 in supernatants of peripheral blood mononuclear cell samples depleted of T cells (with anti-CD3), B cells (with anti-CD19), NK cells (with anti-CD16) and monocytes (with anti-CD14) through the use of magnetic beads, followed by sorting as Lin−CD127+CRTH2+ and Lin−CD127+CRTH2− cells and culture for 4 d with IL-2 (10 U/ml) alone or a combination of IL-2 (10 U/ml) and IL-25 (50 ng/ml; ) or with IL-2 (10 U/ml) alone or a combination of IL-2 (10 U/ml) and IL-33 (50 ng/ml; ); concentrations were normalized to those of 2,000 cells in 200 μl. The lineage 'cocktail' used for sorting included antibodies to CD1a, CD3, CD4, CD11c, CD14, CD19, CD34, CD56, CD94, CD123, TCRαβ, TCRγδ, BDCA2 and FcεR1. Data are from two to six experiments with one donor each (mean and s.e.m.). * Figure 8: Stable cell lines can be generated from CRTH2+ peripheral blood ILCs. () Flow cytometry analysis of peripheral blood Lin− (as in Fig. 2a) CD117+CD127+CRTH2+ ILCs (black lines); gray shading, isotype-matched control antibody. () RORC mRNA expression in blood CRTH2+ cells and freshly isolated fetal gut CRTH2+ ILCs, CD56+ conventional NK cells and NKp44+ ILCs. () Flow cytometry analysis of CD117+CD127+CRTH2+ ILC lines left unstimulated (light gray shading) or stimulated with PMA and ionomycin (black lines) and CD56int NK cell lines (gray lines), stained for intracellular IL-13, IL-22 and IL-17. Far right, CRTH2+ ILCs stimulated with PMA and ionomycin. Numbers in quadrants indicate percent cells in each (among total cells). () Expression of ST2, IL17RB, IL17RA and IL23R mRNA in blood CRTH2+ ILCs (CD117+ or CD117−), blood CD56int conventional NK cells and tonsil NKp44+ ILCs. () IL-13 response of CRTH2+ ILCs to various combinations of IL-2, IL-25 and IL-33 (horizontal axis). () Expression of AHR mRNA in blood CRTH2+ ILCs (CD117+ or CD117−), bl! ood CD56int conventional NK cells and tonsil NKp44+ ILCs. () Secretion of IL-22 from CRTH2+ ILC lines after stimulation with various combinations of IL-2, IL-1β and IL-23 (horizontal axis). Data are representative of three experiments with one donor each (,) or are from one experiment with two to three donors (), one experiment with one to five donors (; median and range), three experiments with one donor each (,; mean and s.e.m.) or one experiment with two to five donors (; median and range). Author information * Abstract * Author information * Supplementary information Affiliations * Tytgat Institute for Liver and Intestinal Research, University of Amsterdam, Amsterdam, The Netherlands. * Jenny M Mjösberg, * Charlotte P Peters & * Hergen Spits * Department of Immunology, Genentech, South San Francisco, California, USA. * Sara Trifari & * Natasha K Crellin * Department of Otorhinolaryngology, University of Amsterdam, Amsterdam, The Netherlands. * Cornelis M van Drunen & * Wytske J Fokkens * Department of Experimental Immunology and Pulmonology of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. * Berber Piet * Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands. * Tom Cupedo * Present address: La Jolla Institute for Allergy and Immunology, La Jolla, California, USA (S.T.), and Pfizer at Rinat, San Francisco, California, USA (N.K.C.). * Sara Trifari & * Natasha K Crellin Contributions J.M.M. designed the study, did experiments, analyzed the data and wrote the manuscript; S.T. designed the study, did experiments, analyzed the data and wrote the manuscript; N.K.C. did experiments and analyzed the data; C.P.P. did experiments, and provided and processed gut tissue; C.M.v.D. and W.J.F. provided inflamed and uninflamed nasal tissue; B.P. provided and processed lung tissue; T.C. designed the study; and H.S. designed the study and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hergen Spits Author Details * Jenny M Mjösberg Search for this author in: * NPG journals * PubMed * Google Scholar * Sara Trifari Search for this author in: * NPG journals * PubMed * Google Scholar * Natasha K Crellin Search for this author in: * NPG journals * PubMed * Google Scholar * Charlotte P Peters Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelis M van Drunen Search for this author in: * NPG journals * PubMed * Google Scholar * Berber Piet Search for this author in: * NPG journals * PubMed * Google Scholar * Wytske J Fokkens Search for this author in: * NPG journals * PubMed * Google Scholar * Tom Cupedo Search for this author in: * NPG journals * PubMed * Google Scholar * Hergen Spits Contact Hergen Spits Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (471K) Supplementary Figures 1–4 and Table 1 Additional data - The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease
- Nat Immunol 12(11):1063-1070 (2011)
Nature Immunology | Article The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease * Zhihua Liu1 * Jinwoo Lee1 * Scott Krummey1 * Wei Lu1 * Huaibin Cai2 * Michael J Lenardo1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1063–1070Year published:(2011)DOI:doi:10.1038/ni.2113Received25 March 2011Accepted19 August 2011Published online09 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Leucine-rich repeat kinase 2 (LRRK2) has been identified by genome-wide association studies as being encoded by a major susceptibility gene for Crohn's disease. Here we found that LRRK2 deficiency conferred enhanced susceptibility to experimental colitis in mice. Mechanistic studies showed that LRRK2 was a potent negative regulator of the transcription factor NFAT and was a component of a complex that included the large noncoding RNA NRON (an NFAT repressor). Furthermore, the risk-associated allele encoding LRRK2 Met2397 identified by a genome-wide association study for Crohn's disease resulted in less LRRK2 protein post-translationally. Severe colitis in LRRK2-deficient mice was associated with enhanced nuclear localization of NFAT1. Thus, our study defines a new step in the control of NFAT activation that involves an immunoregulatory function of LRRK2 and has important implications for inflammatory bowel disease. View full text Figures at a glance * Figure 1: LRRK2 deficiency exacerbates experimental colitis in mice. () Immunoblot analysis of LRRK2 expression in BMDMs, bone marrow DCs (BMDCs) and CD4+ T cells from wild-type (WT) mice; hsp90 (heat-shock protein 90) serves as a loading control. () Mean change in body weight (left; relative to starting weight, set as 100%) and mean clinical score (right) of wild-type mice (n = 5) and Lrrk2−/− mice (n = 5) treated with 3% DSS. () Hematoxylin-and-eosin staining of colonic sections from wild-type and Lrrk2−/− mice on day 8 of treatment of 3% DSS. Blue arrowheads indicate loss of epithelial crypts; open double-headed arrows indicate transmural inflammation and thickened intestinal wall. Scale bars, 200 μm. () Histological scores of colonic sections from wild-type mice (n = 5) and Lrrk2−/− mice (n = 5) on day 8 of treatment of 3% DSS. () IL-12p40 and IL-6 in the serum of wild-type and Lrrk2−/− mice on day 8 of treatment of 3% DSS. () Mean change in body weight (left) and mean clinical scores (right) of irradiated wild-type mice ! (n = 6 per group) reconstituted for 6 weeks with wild-type or Lrrk2−/− bone marrow and treated with 3% DSS. *P < 0.05 and **P < 0.02 (Student's t-test). Data are representative of three (–) or two () independent experiments (error bars (,–), s.e.m.). * Figure 2: LRRK2 inhibits the nuclear function of NFAT1. () Luciferase assay of NFAT (left) and NF-κB (right) in HEK293T cells transfected with vector alone or vector encoding Myc-LRRK2, then stimulated with 0.5 μM ionomycin and the phorbol ester PMA (left) or TNF (right); results are presented relative to those of cells transfected with vector alone and left untreated, set as 1. () Immunoblot analysis (top) of V5-LRRK2 and actin (loading control) in whole-cell lysates of HEK293T cells stably transfected with a tetracycline-regulated V5-LRRK2 construct, left uninduced (–) or induced (+) for 48 h with doxycycline (DOX; 5 μg/ml), and confocal microscopy (bottom) of HEK293T cells transfected with vector encoding NFAT1-GFP (green) as described above and left untreated or treated for 1 h with 1 μM ionomycin (Iono); blue, Hoechst staining. Scale bars, 5 μm. () Quantification of cells with nuclear NFAT1-GFP among cells treated as in , bottom. NC, untreated (no ionomycin). *P < 0.01 (Student's t-test). () Immunoblot analysis of nuc! lear (Nucl) and cytosolic (Cyt) fractions of BMDMs treated for 30 min with ionomycin; hsp90 and PARP (poly(ADP-ribose) polymerase) serve as cytosolic and nuclear markers, respectively, and as loading controls throughout. Numbers below lanes (top blot), densitometry (NFAT1 band/background (far left lane)). () Immunohistochemical analysis of NFAT1 (brown) in paraffin-embedded colon sections of wild-type mice (n = 5) and Lrrk2−/− mice (n = 5) at day 8 after treatment with 3% DSS (as in Fig. 1b); nuclei are counterstained with hematoxylin (blue). Blue arrows indicate cells with cytosolic NFAT1 staining; red arrows indicate cells with nuclear NFAT1 staining. Insets (top right corner, bottom row) are an enlargement of the area outlined at left (~×2 magnification). Scale bars, 25 μm. Data are representative of three independent experiments (,,,; mean and s.e.m. of triplicates in ) or are pooled from three independent experiments with at least 100 cells each (; mean and s.e.m! .). * Figure 3: LRRK2 affects the cytoplasmic sequestration of NFAT1 but not its phosphorylation. () NFAT luciferase activity in HEK293T cells transfected with vector alone or vector encoding wild-type or kinase-dead (KD) LRRK2 and left untreated (0) or stimulated with 0.5 μM ionomycin and 20 or 40 ng/ml PMA; results are presented relative to those of cells transfected with vector alone and left untreated, set as 1. () Immunoblot analysis of whole-cell lysates of HEK293T cells transfected with vector control or vector encoding Myc-LRRK2, along with NFAT1-GFP, 1–15 min after ionomycin was washed out (Chase). NT, untreated; Iono, addition of ionomycin without washout. Open arrowheads, underphosphorylated NFAT1-GFP; filled arrowhead, highly phosphorylated NFAT1-GFP. () Confocal microscopy of stable HEK293T cells transfected with caNFAT1-GFP and doxycycline-inducible V5-LRRK2 (as in Fig. 2b); nuclei are counterstained with Hoechst (blue). Scale bars, 5 μm. () Quantification of cells with predominantly cytoplasmic (Cyt), cytoplasmic and nuclear (Cyt + nucl), or nuclear (N! ucl) distribution of caNFAT1-GFP among 100 GFP+ cells for each condition as in . () Immunoblot analysis of caNFAT1-GFP in cytosolic and nuclear fractions of HEK293T cells transfected with vector control or vector encoding Myc-LRRK2, along with caNFAT1-GFP. Data are representative of at least three independent experiments (mean and s.e.m. of triplicates in ). * Figure 4: LRRK2 regulates NFAT1 by modulating the NRON complex, and degradation of LRRK2 facilitates the translocation of NFAT1 to the nucleus. () Immunoassay of IQGAP1, CSE1L, TNPO1, PPP2R1A, PSMD11 and Myc-LRRK2 in HEK293T cells transfected with plasmid encoding Myc-LRRK2, detected by immunoblot analysis in whole-cell lysates (Input) and after immunoprecipitation (IP) with anti-Myc (α-Myc) or isotype-matched control immunoglobulin (Iso). kDa, kilodaltons.() RT-PCR analysis of NRON RNA in lysates of HEK293T cells expressing doxycycline-inducible V5-LRRK2 (as in Fig. 2b; Input (bottom)) and in lysates of those cells immunoprecipitated with anti-V5 (IP: α-V5; middle), and immunoblot analysis of lysates of those cells immunoprecipitated with anti-V5, probed with anti-V5 (top). bp, base pairs. () Immunoblot analysis of HEK293T cells transfected with plasmid expressing NFAT1-GFP with or without plasmid expressing Myc-LRRK2 (above lanes), with (IP) or without (Input) immunoprecipitation with anti-GFP. Asterisks indicate a small (*) or large (**) change in band density. () Immunoprecipitation and immunoblot analysis of ! THP-1 cells treated as in , probed with antibody to endogenous LRRK2. () Immunoblot analysis of whole-cell lysates of BMDMs treated for 1 h with various amounts of LPS (top) or for 0–60 min with 1 μg/ml of LPS (bottom). () Immunoblot analysis of nuclear and cytosolic fractions of BMDMs left untreated or pretreated for 1 h with LPS (1 μg/ml), followed by treatment for 30 min with ionomycin. Data are representative of three independent experiments. * Figure 5: LRRK2 deficiency enhances NFAT1 activity in BMDMs. () Enzyme-linked immunosorbent assay of IL-12p40, IL-6, TNF and IL-1β in supernatants of wild-type or Lrrk2−/− BMDMs left untreated or stimulated with zymosan (Zym; 100 μg/ml), Pam3CSK4 (Pam; 10 μg/ml), or LPS (25 ng/ml) plus ATP (5 mM; LPS + ATP). () Enzyme-linked immunosorbent assay of IL-12p40 and IL-6 in supernatants of wild-type or Lrrk2−/− BMDMs stimulated with various amounts of zymosan with (+) or without (–) treatment with FK506 (2 μM). () Immunoblot analysis of nuclear and cytosolic fractions of BMDMs treated for 30 min with zymosan. Numbers below lanes (top blot), densitometry (NFAT1 band in nuclear fractions relative to that in untreated cells (far left), set as 1). *P < 0.05 (Student's t-test). Data are representative of at least three independent experiments (mean and s.e.m. of triplicates in ,). * Figure 6: The CD-susceptibility allele M2397 results in LRRK2 protein of lower stability. () Immunoblot analysis (top) of whole-cell lysates of HEK293T cells transfected with increasing amounts (wedges) of a plasmid containing the M2397 or T2397 allele, and densitometry (below) of the results above, presented as the ratio of Myc-LRRK2 to hsp90. *P < 0.05 (Student's t-test). () Immunoblot analysis (top) of whole-cell lysates of HEK293T cells transfected with plasmid expressing Myc-tagged LRRK2 Met2397 or Thr2397 and treated for 0–18 h with cycloheximide (CHX; 100 μg/ml), and densitometry (below) of the results above, presented as the ratio of Myc-LRRK2 to hsp90 relative to that of cells at 0 h. () Immunoblot analysis (top) of LRRK2 and hsp90 in whole-cell lysates of purified peripheral B cells from people homozygous (hom) for the M2397 or T2397 allele, and densitometry (below) of the results above; each symbol represents an individual person; small horizontal lines indicate the mean (± s.e.m.). *P < 0.01 (Student's t-test). () NFAT luciferase assay (below) of ! HEK293T cells transfected with equal amount of vector alone or vector containing the M2397 or T2397 allele and stimulated with PMA (20 ng/ml) and ionomycin (presented as in Fig. 2a), and immunoblot analysis (top) of whole-cell lysates of the transfected cells. *P < 0.05 (Student's t-test). Data are representative of at least five (,, top), two () or three (; mean and s.e.m. of triplicates) independent experiments or are pooled from three independent experiments (,, bottom; mean and s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Zhihua Liu, * Jinwoo Lee, * Scott Krummey, * Wei Lu & * Michael J Lenardo * Unit of Transgenesis, Laboratory of Neurogenetics, National Institute on Aging, US National Institutes of Health, Bethesda, Maryland, USA. * Huaibin Cai Contributions Z.L., J.L., S.K. and W.L. designed and did experiments; Z.L., H.C. and M.J.L. designed experiments and analyzed the data; and Z.L. and M.J.L. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael J Lenardo Author Details * Zhihua Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Jinwoo Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Scott Krummey Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Lu Search for this author in: * NPG journals * PubMed * Google Scholar * Huaibin Cai Search for this author in: * NPG journals * PubMed * Google Scholar * Michael J Lenardo Contact Michael J Lenardo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–12 Additional data - An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation
- Nat Immunol 12(11):1071-1077 (2011)
Nature Immunology | Article An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation * Christoph Wilhelm1 * Keiji Hirota1 * Benjamin Stieglitz2 * Jacques Van Snick3 * Mauro Tolaini1 * Katharina Lahl4, 5 * Tim Sparwasser5 * Helena Helmby6 * Brigitta Stockinger1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1071–1077Year published:(2011)DOI:doi:10.1038/ni.2133Received05 July 2011Accepted06 September 2011Published online09 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin 9 (IL-9) is a cytokine linked to lung inflammation, but its cellular origin and function remain unclear. Here we describe a reporter mouse strain designed to map the fate of cells that have activated IL-9. We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs). IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5. Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses. View full text Figures at a glance * Figure 1: Papain-induced eYFP+ cells do not express lineage markers. () T cells, B cells, dendritic cells (DCs), eosinophils (Eos), macrophages (Mφ) and neutrophils (Neu) in the lungs of IL9CreR26ReYFP mice challenged with papain and PBS on days 0, 3, 6, 14 and analyzed 20 h after last rechallenge (scheme, Supplementary Fig. 9). () Cytokine concentrations in the bronchoalveolar lavage fluid of mice immunized with papain or PBS as in . (,) Flow cytometry of total lung cells from papain-challenged IL9CreR26ReYFP mice as in stained for CD4 () or CD8α, TCRγδ, CD19, Nkp46, Gr-1, CD11c, Siglec F and CD11b () and assessed for eYFP expression. () Absolute number of eYFP+ ILCs in lung of papain-challenged IL9CreR26ReYFP mice. () Flow cytometry analysis of the expression of CD45, Thy-1.2, IL33R, CD25, major histocompatibility complex II (MHCII), Sca-1 and eYFP in papain-challenged IL9CreR26ReYFP mice. Numbers in quadrants indicate percent cells in each throughout. Data represent at least three independent experiments with three mice per group (mean! and s.e.m. in ,,). * Figure 2: IL-9 expression by ILCs is transient. () Flow cytometry of lung cells isolated from IL9CreR26ReYFP mice challenged with papain or PBS at 24 h after the final papain rechallenge, then stimulated with PdBU and ionomycin for 2.5 h and stained for CD4, followed by analysis of eYFP and intracellular IL-13, IL-5, IL-4 and IL-9 in the CD4− lymphocyte fraction. () Flow cytometry analysis of the expression of IL-9 and IL-13 in ILCs (Lin− Thy-1.2+), analyzed either before final papain rechallenge or 6, 12 and 24 h after last papain challenge (scheme, Supplementary Fig. 9). () Frequency of IL-9+ ILCs in lungs at 0–24 h (horizontal axis) after papain rechallenge (0 h, frequency before the final papain rechallenge). *P = 0.009 (t-test). () IL-9 in lung homogenates. *P = 0.004 (t-test). Data represent at least three () or two () independent experiments with three mice per group (mean and s.e.m. in ,). * Figure 3: IL-9 in ILCs is induced by IL-2 but not by IL-25, IL-33 or TSLP. () IL-9 in supernatants of immunomagnetically selected ILCs and cells expressing lineage markers (Lin+) isolated from lungs of papain-challenged B6 mice and stimulated in vitro overnight with PdBU and ionomycin (P+I), IL-2, IL-25, IL-33 or TSLP. (,) IL-9 in supernatants of sorted CD4+ cells, CD25− ILCs, CD25+ ILCs () and sorted eYFP+ ILCs from papain-challenged IL9CreR26ReYFP mice () cultured overnight as in . Ctrl, unstimulated. () Quantitative PCR analysis of transcripts for IL-9 (Il9), IL-4 (Il4), IL-5 (Il5), IL-6 (Il6) and IL-13 (Il13) in sorted CD4+, CD25−, CD25+ and eYFP+ ILCs; mRNA expression was normalized to expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase) and is presented relative to expression in CD4+ cells. Data represent at least three () or two () independent experiments (mean and s.e.m. in ). * Figure 4: Intranasal challenge with IL-33 induces ILCs poised for IL-9 production. () Flow cytometry of lung cells isolated from B6 mice challenged intranasally with PBS, IL-25 or IL-33, stained for surface lineage markers CD4, CD8α, TCRβ, TCRγδ, CD19, Nkp46, Gr-1, CD11c, Ter-119, CD11b (Lin) and Thy-1.2 (top), and of lung cells restimulated for 2.5 h with PdBU and ionomycin, stained for intracellular IL-9 and IL-13 and gated on ILCs (bottom). (,) Absolute number of ILCs (Lin−Thy-1.2+ cells) in the lung () and frequency of IL-13+ ILCs in the lung () after treatment of mice with PBS, IL-25 or IL-33. () Cytokine concentrations in supernatants of immunomagnetically selected ILCs isolated from mice treated with PBS, IL-25 or IL-33 and stimulated overnight in vitro with IL-2. () Flow cytometry of lung cells isolated from IL9CreR26ReYFP mice treated as in , stained for lineage markers and Thy-1.2 (top) and assessed for expression of eYFP and CD45 in ILCs (bottom). Data represent at least two independent experiments with three mice per group (mean and s.e.m! . in ). * Figure 5: IL-9 expression in ILCs depends on IL-2 and the adaptive immune system. () Cytokine concentration in lung homogenates of papain-challenged wild-type (WT), Rag1−/− and Rag2−/−IL2rg−/− mice at 18 h after the final papain rechallenge. *P = 0.001 and **P = 0.0001 (t-test). () IL-9 in supernatants of immunomagnetically selected ILCs isolated from papain-challenged wild-type, Rag1−/− and Rag2−/−IL2rg−/− mice and stimulated overnight in vitro with IL-2. () Cytokine concentrations in lung homogenates of papain-immunized B6 mice treated with neutralizing IL-2-specific antibody (α-IL-2) or isotype-matched control antibody (immunoglobulin G (IgG)), analyzed 18 h after challenge. *P = 0.03 (t-test). () IL-9 in lung homogenates of papain-challenged wild-type and Rag1−/− mice, without additional treatment (IL-2 –) or treated intranasally with 500 ng recombinant mouse IL-2 at 3 h after the final papain rechallenge (IL-2 +), analyzed 18 h after the final papain rechallenge. () Absolute number of ILCs in lung of papain-challenged w! ild-type and Rag1−/− mice left untreated or treated intranasally with IL-2 (treatment regimens, Supplementary Fig. 9). Data represent two independent experiments with four mice per group (mean and s.e.m.). * Figure 6: ILCs are the main source of IL-9. () Flow cytometry of lung cells isolated from papain-challenged B6 mice at 12 h after papain rechallenge, restimulated for 2.5 h with PdBU and ionomycin and stained for lineage markers and intracellular IL-9 and IL-13. () Intracellular expression of IL-9 and IL-13 in gated CD4+ cells or ILCs. (,) Absolute number of ILCs and CD4+ cells () and IL9+IL13+ ILCs or CD4+ cells () in lungs of papain-challenged mice at 12 h after rechallenge. () Flow cytometry of lung cells isolated from Rag2−/−IL2rg−/− mice without additional cells (No transfer) or given CD4+ cells or ILCs and challenged with papain and IL-2 (scheme, Supplementary Fig. 9); lung cells were stained for CD45 and Thy-1.2 (left), or restimulated with PdBU and ionomycin for 2.5 h, stained for intracellular IL-9 and IL-13 and gated on CD45+ Thy-1.2+ cells (right bottom). Right top, absolute number of IL-9+IL-13+ cells. *P = 0.0238 (t-test). () Cytokine concentrations in lung homogenates of papain- and IL-2-challeng! ed Rag2−/−IL2rg−/− mice given no cells (–) or given CD4+ cells or ILCs at 15 h after papain challenge. Data represent at least two independent experiments with three mice per group (mean and s.e.m. in ). * Figure 7: IL-9 promotes cytokine expression in ILCs. () Flow cytometry of lung cells isolated from papain-challenged mice, treated with isotype-matched control antibody or neutralizing antibody to IL-9 at 30 min before the final papain rechallenge, stained for lineage markers and Thy-1.2 (top) and gated on ILCs and stained for Sca-1 and CD25 (bottom). (,) Absolute number of ILCs () and Sca-1+CD25+ ILCs () in lungs of papain-challenged mice at 3 d after rechallenge and antibody treatment. NS, not significant. *P = 0.001 (t-test). () Quantitative PCR analysis of expression of transcripts for IL-9R in sorted naive T cells and B cells and CD25+ ILCs isolated from lungs of papain-challenged mice, presented relative to expression of Hprt1. () Cytokine concentrations in supernatants of sorted CD25+ ILCs stimulated overnight in vitro with or without IL-9. *P = 0.04 and **P = 0.009 (t-test). () Cytokine concentration in lung homogenate and bronchoalveolar lavage fluid (BALF) of papain-challenged wild-type mice treated with IL-9-specifi! c antibody or isotype-matched control antibody (Ctrl) at 3 d after the final papain rechallenge. *P = 0.002 and **P = 0.0002 (t-test). Treatment regimes are in Supplementary Figure 9. Data are representative of at least two independent experiments with four mice each (–,) or pooled cells from two mice per group (,; mean and s.e.m. in ,,,). Author information * Abstract * Author information * Supplementary information Affiliations * Division of Molecular Immunology, Medical Research Council (MRC) National Institute for Medical Research, Mill Hill, UK. * Christoph Wilhelm, * Keiji Hirota, * Mauro Tolaini & * Brigitta Stockinger * Division of Molecular Structure, MRC National Institute for Medical Research, Mill Hill, UK. * Benjamin Stieglitz * Ludwig Institute for Cancer Research, Brussels, Belgium. * Jacques Van Snick * Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. * Katharina Lahl * Twincore, Institut für Infektionsimmunologie, Hannover, Germany. * Katharina Lahl & * Tim Sparwasser * London School of Hygiene and Tropical Medicine, London, UK. * Helena Helmby Contributions C.W. designed and did the experiments and wrote the manuscript; K.H. provided advice and contributed to experiments; B.S. purified neutralizing antibodies; K.L. and T.S. provided help and advice for the BAC construct; J.V.S. provided reagents; M.T. did the BAC injections; H.H. provided advice; and B.S. designed experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Brigitta Stockinger Author Details * Christoph Wilhelm Search for this author in: * NPG journals * PubMed * Google Scholar * Keiji Hirota Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin Stieglitz Search for this author in: * NPG journals * PubMed * Google Scholar * Jacques Van Snick Search for this author in: * NPG journals * PubMed * Google Scholar * Mauro Tolaini Search for this author in: * NPG journals * PubMed * Google Scholar * Katharina Lahl Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Sparwasser Search for this author in: * NPG journals * PubMed * Google Scholar * Helena Helmby Search for this author in: * NPG journals * PubMed * Google Scholar * Brigitta Stockinger Contact Brigitta Stockinger Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–9 and Table 1 Additional data - The carboxypeptidase ACE shapes the MHC class I peptide repertoire
- Nat Immunol 12(11):1078-1085 (2011)
Nature Immunology | Article The carboxypeptidase ACE shapes the MHC class I peptide repertoire * Xiao Z Shen1, 5 * Sandrine Billet1, 5 * Chentao Lin1, 4 * Derick Okwan-Duodu1 * Xu Chen1, 4 * Aron E Lukacher2 * Kenneth E Bernstein1, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1078–1085Year published:(2011)DOI:doi:10.1038/ni.2107Received30 June 2011Accepted15 August 2011Published online02 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell–mediated adaptive immune responses. Aminopeptidases have been linked to the editing of peptides for MHC class I loading, but carboxy-terminal editing is thought to be due to proteasome cleavage. By analysis of wild-type mice and mice genetically deficient in or overexpressing the dipeptidase angiotensin-converting enzyme (ACE), we have now identified ACE as having a physiological role in the processing of peptides for MHC class I. ACE edited the carboxyl terminus of proteasome-produced MHC class I peptides. The lack of ACE exposed new antigens but also abrogated some self antigens. ACE had substantial effects on the surface expression of MHC class I in a haplotype-dependent manner. We propose a revised model of peptide processing for MHC class I by introducing carboxypeptidase activity into the process. View full text Figures at a glance * Figure 1: ACE is upregulated during APC maturation. () Real-time PCR quantification of ACE mRNA in monocytes (Mo), M-CSF-induced macrophages (M-CSF MΦ) and thioglycollate-induced macrophages (TPMs); results are presented relative to expression in monocytes, set as 1. () Abundance of ACE mRNA in TPMs, splenic DCs and skin fibroblasts with (+) or without (–) 4 h of stimulation with IFN-γ (left), and flow cytometry analysis of ACE expression by splenic DCs with (line) or without (shaded) 24 h of stimulation with IFN-γ.() Flow cytometry analysis of cell type and ACE expression of splenocytes from C57BL/6 mice left uninfected (Naive) or 2 d after infection with L. monocytogenes (Listeria). NK, natural killer. *P < 0.05 and **P < 0.005 (two-tailed Student's t-test). Data are pooled from three independent experiments (,; mean and s.e.m.) or are representative of two experiments with a total of five mice per group (). * Figure 2: The effect of ACE on surface expression of MHC class I. () Surface expression of H-2Kb and H-2Db on peritoneal macrophages from Ace+/+ mice, Ace−/− mice and ACE 10 mice (ACE overexpression) or splenic DCs from Ace+/+ mice, Ace−/− mice and Pd mice (ACE overexpression). () Surface expression of H-2Kb and H-2Db on skin-derived fibroblasts from Ace+/+ and Ace−/− mice with (+ IFN-γ) or without (Naive) IFN-γ priming. () Surface expression of H-2Kb on L.Kb cells transfected with an expression construct for ACE (line) or mACE (shaded). () Expression of H-2Kb and H-2Db in splenocytes from C57BL/6 mice with (line) or without (shaded) 6 d of treatment with ramipril. () Expression of H-2Kk and H-2Dk on L929 fibroblasts (n = 3 replicates) and of H-2Kd and H-2Dd on A20 B cells (n = 5 replicates) transfected with an expression construct for ACE (line) or mACE (shaded). Numbers in plots (,) indicate change in expression relative to no treatment () or the difference between transfection with ACE and transfection with mACE (). Data a! re representative of at least three experiments with at least five mice per group (), three experiments with cell lines derived from three mice per group (), three independent experiments (), two experiments with a total of six mice per group () or at least three independent experiments (). * Figure 3: ACE-deficient cells have normal MHC class I–peptide stability but more peptide supply. () Loss of H-2Kb or H-2Db expression in Ace+/+ and Ace−/− peritoneal cells cultured with brefeldin A as assessed by flow cytometry; results are presented relative to those of cells cultured similarly without brefeldin A. () Restoration of H-2Kb and H-2Db in Ace+/+ and Ace−/− peritoneal cells treated with mild acid and then cultured in medium with a pH of 7.4; results are presented relative to those of cells not treated with acid. *P = 0.02 and **P ≤ 0.01 (two-tailed Student's t-test). Data are representative of three experiments with a total of five mice per group () or five experiments with a total of two mice per group (; mean and s.e.m.). * Figure 4: The effect of ACE on the CD8+ T cell repertoire. () IFN-γ-secreting CD8+ T cells among splenocyte populations obtained from female Ace+/+ mice immunized (Imm) with macrophages from male Ace+/+ or ACE 10 mice, expanded in vitro by stimulation with macrophages from male or female mice with ACE expression equivalent to that of the immunizing cells (Boost), followed by restimulation (Restim) with APCs loaded with Smcy or Uty or with macrophages from male or female mice with ACE expression equivalent to that of the immunizing cells (graph), and the CD8+ T cell response (plots: top, immunizing cells; vertical axes, restimulation). Each symbol (graph) represents an individual mouse; small horizontal bars indicate mean (± s.e.m.). Numbers in outlined areas (plots) indicate percent CD8+IFN-γ+ cells throughout. () IFN-γ-secreting CD8+ T cells among splenocyte populations obtained from female Ace+/+ mice immunized with macrophages from male Ace+/+ or Ace−/− mice, then boosted and restimulated as in (presented as in ). () IFN-! γ-secreting CD8+ T cells among cells from Ace−/− mice, with immunization, boosting and restimulation with macrophages from Ace+/+ or Ace−/− mice of the same sex (presented as in ). () TCR Vβ use in splenic CD8+ T cells before and after infection with polyomavirus (PyV), presented as the frequency in Vβ+ T cells among total T cells. *P < 0.05; **P < 0.01 and ***P < 0.005 (two-tailed Student's t-test). Data are representative of two experiments (–), or three experiments with six Ace+/+ mice or two experiments with four Ace−/− mice (; average and s.e.m.). * Figure 5: The effect of ACE in editing self antigens. () IFN-γ+ (CD8+) CTLs among IFN-γ-primed Ace+/+ or Ace−/− skin fibroblasts incubated with CTL clones specific for various minor H (mH) antigens (horizontal axis), assessed by flow cytometry. *P < 0.02; **P < 0.005 (two-tailed Student's t-test). () IFN-γ+ CTLs among Ace+/+ and ACE 10 bone marrow–derived macrophages (APC; horizontal axis) incubated with various CTL clones (top), assessed by flow cytometry. Data are representative of four experiments with cell lines from four mice per group (; mean and s.e.m.) or three independent experiments (). * Figure 6: The effect of ACE on the presentation of viral antigens. () Splenic CD8+ T cell responses to various polyomavirus epitopes (horizontal axes) 8 d after intraperitoneal infection of Ace+/+ and Ace−/− mice with polyomavirus, assessed by flow cytometry analysis of IFN-γ+CD8+ cells (presented as in Fig. 4a). *P = 0.02 and **P < 0.01 (two-tailed Student's t-test). () Surface expression of H-2Db–LT359-368 on Ace+/+ and Ace−/− splenocytes 3 d after infection of mice with PyV.OVA–I, assessed as IL-2 expression by the HLT359 hybridoma (presented as in Fig. 4a). P = 0.063 (two-tailed Student's t-test). () Surface expression of H-2Kb–SKL on splenic CD11b+CD11c− and CD11c+ cells before infection (Naive) or 3 d after infection of Ace+/+ and Ace−/− mice with PyV.OVA–I, assessed with antibody 25-D1.16. Data are representative of three independent experiments with five pairs of mice. * Figure 7: ACE works as a carboxyl dipeptidase on proteasome products. () Efficiency of antigen presentation by L.Kb cells transfected with minigenes expressing SKL, SHL or SKL-TE plus empty vector or construct expressing ACE, assessed by measurement of IL-2 expression by B3Z hybridoma cells incubated together with the L.Kb cells. () SKL presentation by Ace+/+ and ACE 10 macrophages pulsed with SKL-T, SKL-TE or SKL-TE–amide, evaluated with antibody 25-D1.16 and presented as the change in mean fluorescence intensity (ΔMFI), calculated as experimental MFI minus background MFI (in naive cells). () SKL presentation by L.Kb cells transfected with a minigene encoding SKL-TE plus empty vector or a construct expressing wild-type ACE or mACE, evaluated 24 h later. () SKL presentation by TAP-deficient RMA-S cells transfected with a minigene expressing SKL-TE plus empty vector or construct expressing wild-type ACE or ΔACE14. () SKL presentation by L.Kb cells transfected with constructs expressing ovalbumin or SKL-TE, plus empty vector or construct exp! ressing ACE, with (+) or without (−) treatment with epoxomicin (Epox) after transfection, assessed as IL-2 expression by B3Z cells. ND, not detectable. *P < 0.02 and **P < 0.005 (two-tailed Student's t-test). Data are pooled from four independent experiments (), three independent experiments () or at least three independent experiments () or are representative of three independent experiments (,). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Xiao Z Shen & * Sandrine Billet Affiliations * Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, USA. * Xiao Z Shen, * Sandrine Billet, * Chentao Lin, * Derick Okwan-Duodu, * Xu Chen & * Kenneth E Bernstein * Department of Pathology, Emory University School of Medicine, Atlanta, Georgia, USA. * Aron E Lukacher * Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA. * Kenneth E Bernstein * Present address: Department of Immunology, Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, China. * Chentao Lin & * Xu Chen Contributions X.Z.S., study conception and experimental design; X.Z.S. and S.B., experimental input, with assistance from C.L. (RT-PCR), D.O.-D. (L. monocytogenes infection) and X.C. (minigene construction); A.E.L., intellectual advice and reagents for polyomavirus experiments; K.E.B., intellectual advice and project coordination; X.Z.S. and K.E.B., manuscript authorship. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kenneth E Bernstein Author Details * Xiao Z Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Sandrine Billet Search for this author in: * NPG journals * PubMed * Google Scholar * Chentao Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Derick Okwan-Duodu Search for this author in: * NPG journals * PubMed * Google Scholar * Xu Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Aron E Lukacher Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth E Bernstein Contact Kenneth E Bernstein Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (774K) Supplementary Figures 1–11 and Table 1 Additional data - Mucosal memory CD8+ T cells are selected in the periphery by an MHC class I molecule
- Nat Immunol 12(11):1086-1095 (2011)
Nature Immunology | Article Mucosal memory CD8+ T cells are selected in the periphery by an MHC class I molecule * Yujun Huang1, 7 * Yunji Park1, 7 * Yiran Wang-Zhu1 * Alexandre Larange1 * Ramon Arens1 * Iván Bernardo1, 6 * Danyvid Olivares-Villagómez2 * Dietmar Herndler-Brandstetter3 * Ninan Abraham4 * Beatrix Grubeck-Loebenstein3 * Stephen P Schoenberger1 * Luc Van Kaer2 * Mitchell Kronenberg1 * Michael A Teitell5 * Hilde Cheroutre1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1086–1095Year published:(2011)DOI:doi:10.1038/ni.2106Received21 June 2011Accepted15 August 2011Published online02 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The presence of immune memory at pathogen-entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the nonclassical major histocompatibility complex (MHC) class I molecule thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αβ+ T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αβ+ memory T cells. The memory process driven by TL and CD8αα was essential for the generation of CD8αβ+ memory T cells in the intestine and the accumulation of highly antigen-sensitive CD8αβ+ memory T cells that form the first line of defense at the largest entry port for pathogens. View full text Figures at a glance * Figure 1: TL negatively affects the generation of memory cells from CD8αβ+ T cells. () Intracellular staining of interferon-γ (IFN-γ) and cell surface staining of CD8α (left) in or on splenocytes (SPL) and IELs isolated from wild-type (WT) or TL− mice 30 d after oral infection with 1 × 109 ActA− LM-OVA, then restimulated ex vivo with SIINFEKL. Right, pooled data. () Tracking of donor (Ly5.1+) OT-I cells among splenocytes and IELs (left) from Ly5.2+ wild-type or TL− recipients given adoptive transfer of 5 × 104 naive Ly5.1+ CD8+ OT-I cells, then orally infected with 1 × 109 ActA− LM-OVA 1 d later; cells were obtained 2 months after infection. Graph, pooled data. () Tracking of donor OT-I cells among splenocytes and IELs (left) from wild-type or TL-transgenic (TL-Tg) recipients given 1 × 106 naive Ly5.1+ OT-I cells, then infected and assessed as in . () Flow cytometry of memory OT-I cells from the spleens of B6 mice given 5 × 104 Ly5.1+ naive OT-I cells, then immunized intravenously with 5 × 105 SIINFEKL-loaded DCs generated from wild-type or! TL-transgenic bone marrow cells (left; middle, pooled data), or given OT-I cells primed in vitro with APCs that do not express TL (APC) or APCs transfected to express TL (APC-TL; far right); cells were analyzed 2 months after immunization with DCs or transfer of in vitro–activated OT-I cells. () Flow cytometry analysis of TL expression by splenic or mLN DCs sorted on the basis of their expression of CD11c and MHC class II (MHCII). () TL expression by freshly isolated splenic or mLN DCs activated for 1 d with CpG. Isotype, isotype-matched control antibody. Numbers adjacent to outlined areas (–) indicate percent cells in each throughout. Each symbol represents an individual mouse (–); small horizontal lines indicate the mean (and s.e.m.). *P < 0.05, **P < 0.001 and ***P < 0.01 (unpaired t-test). Data are representative of three independent experiments. * Figure 2: TL mediates the death of activated CD8αβ+ T cells. () Tracking of donor cells in the spleens of wild-type or TL-transgenic recipients of 1 × 106 naive Ly5.1+ OT-I cells (top; n = 5 mice per group) or 1 × 106in vitro–activated Ly5.1+ OT-I cells (bottom (Act); n = 8 mice per group), assessed 1 month after transfer. () Tracking of Ly5.2+ donor in the spleens of Ly5.1+ wild-type or TL-transgenic recipients (n = 4 mice per group) of CD8+ T cells (0.5 x 106) sorted from wild-type mice or mice deficient in Fas ligand (FasLgld) or Fas (Faslpr) and preactivated (before transfer) for 3 d in vitro with anti-CD3 and anti-CD28 beads, assessed 1 month after transfer. Data are representative of two independent experiments. * Figure 3: Activation-induced CD8αα rescues CD8αβ+ primary effector T cells from TL-induced cell death. () Death of ΔE8I OT-I cells (2 × 105) labeled with the cytosolic dye CFSE and cultured for 2 d with 4 × 104 SIINFEKL-pulsed DCs from the spleen or mLNs of wild-type or TL− mice, analyzed by annexin V staining. Numbers in quadrants indicate percent cells in each throughout. () Tracking of donor OT-I cells among splenocytes and IELs of Ly5.1+ recipient mice given adoptive transfer of 5 × 104 naive Ly5.2+ wild-type or Ly5.2+ ΔE8I OT-I cells, then orally infected with 1 × 109 ActA− LM-OVA 1 d after transfer, and assessed 2 months after infection. Right, pooled data (as in Fig. 1a–d). () Tracking of donor OT-I cells among splenocytes and IELs of Ly5.1+ recipient mice given adoptive transfer of 5 × 104 naive Ly5.2+ wild-type or Ly5.2+ ΔE8I OT-I cells, then intravenously infected with 2.5 × 105 ActA− LM-OVA 1 d after transfer and assessed 2 months after infection. Right, pooled data (as in Fig. 1a–d). () Expression of CD8β on effector wild-type OT-I or ΔE8I OT-! I cells from recipients treated as in (left) or (right), assessed 7 d after infection. MFI, mean fluorescence intensity. () Expression of CD8β on memory wild-type OT-I or ΔE8I OT-I cells among splenocytes and IELs of recipients treated as in , assessed 2 months after infection. () Tracking of memory OT-I cells among splenocytes and IELs of Ly5.1+ wild-type or Ly5.1+ TL− mice (n = 5 per group) given 5 × 104 naive Ly5.1+Ly5.2+ wild-type OT-I cells and 5 × 104 naive Ly5.2+ ΔE8I OT-I cells (transferred together) and then infected orally with 1 × 109 ActA− LM-OVA 1 d after transfer, assessed 2 months after infection (pooled data). () CD8αα expression, assessed by staining with TL tetramers (TL-Tet), on gated memory wild-type OT-I cells among IELs of wild-type or TL− recipient mice as in , asssessed 2 months after infection. *P < 0.001 and **P < 0.01 (unpaired t-test). Data are representative of three (–,–) or two () independent experiments (mean and s.e.m. in ,! ). * Figure 4: CD8αα expression correlates with the intensity of TCR activation. () Expression of CD8αα (assessed as in Fig. 3g throughout) and IL-7Rα on splenocytes cultured for 3 d in vitro in the presence of graded concentrations (wedge) of soluble anti-CD3 (α-CD3) and anti-CD28 (α-CD28), gated on CD8+ T cells. () Expression of CD8αα and IL-7Rα on OT-I cells cultured for 2 d in vitro with artificial APCs (MEC.B7 adherent fibroblasts, which express the costimulatory molecule B7.1) in the presence of a graded concentration (wedge) of SIINFEKL (with asparagine (N) at position 4 (N4)) or the altered peptide ligands Q4R7 and Q4. () Expression of CD8αα and CD8β on Ly5.1+ CD8+ OT-I cells among splenocytes and IELs from B6 recipient mice (n = 4 per group) given 1 × 105 or 1 × 103 sorted naive Ly5.1+ CD8+ OT-I cells, then infected orally with 1 × 109 ActA− LM-OVA 1 d after transfer and assessed 7 d after infection. () Expression of CD8αα and CD8β on donor OT-I cells among splenocytes and IELs from wild-type recipient mice (n = 5 per group) g! iven 5 × 104 naive Ly5.1+ CD8+ OT-I cells, then infected orally with 2 × 108 wild-type LM-Q4 or LM-N4 1 d after transfer, assessed 7 d after infection. Data are representative of three (,) or two (,) independent experiments. * Figure 5: CD8αα expression marks effector memory CD8αβ+ T cells in humans. () Expression of CD8αα (as in Fig. 3g throughout) on polyclonal human naive CD8+ T cells (TN; CCR7+CD45RA+), recently activated effector-memory CD8+ T cells (TEMRA; CCR7−CD45RA+), TEM cells (CCR7−CD45RA−) and central memory CD8+ T cells (TCM; CCR7+CD45RA−). FSC, forward scatter. Far right, frequency of cells expressing CD8αα among human peripheral blood CD8+ T cells (n = 9 donors). P < 0.001 (unpaired t-test). () Expression of CD8αα by recently activated effector-memory human CD8+ T cells left untreated (TEMRA) or treated with anti-CD8α (TEMRA + α-CD8α) or anti-CD8β (TEMRA + α-CD8β). () Expression of CD8αα by naive CD8+ T cells and CD8+ T cells specific for cytomegalovirus pp65 (CMV(pp65)-specific), left untreated or treated with anti-CD8α (left). Left and middle, staining of human CD8+ T cells with a pentamer of cytomegalovirus pp65 (CMV(pp65) pentamer; left), followed by gating on cells specific for cytomegalovirus pp65 as recently activated effector! -memory CD8+ T cells (middle). Data are representative of three experiments with three donors each (), two independent experiments () or two experiments with three donors each (; two donors among six presented here). * Figure 6: Retinoic acid promotes the affinity-based accumulation of CD8αα+CD8αβ+ T cells in the intestine. () CD8αα expression (as in Fig. 3g throughout) by OT-I cells stimulated for 3 d in vitro by SIINFEKL-loaded DCs from spleen or mLNs of wild-type mice with or without 100 nM retinoic acid (RA; top) or an inhibitor of the retinoic acid receptor (LE135; bottom). (,) CD8αα expression by OT-I cells stimulated for 3 d in vitro by splenic or mLN DCs pulsed with SIINFEKL at high concentration (1 nM SIINFEKLhi) or low concentration (0.01 nM; SIINFEKLlo) in the presence or absence (Medium) of retinoic acid (100 nM; ) or TGF-β (5 ng/ml; ). () CD8αα expression by gated donor OT-I cells among splenocytes and IELs and cells from the mLN and Peyer's patch (PP) of B6 recipient mice (n = 2–3 per group) given adoptive transfer of 0.5 × 106 CD8+ OT-I cells from naive Ly5.1+ OT-I mice, then infected orally with 0.5 × 109 ActA− LM-OVA 1 d after transfer and assessed 5 d after infection. () Expression of CD8αα and CD103 on gated donor OT-I cells among splenocytes and IELs on days 1! 2, 21 and 75 after infection as in . Data are representative of five independent experiments (), more than five independent experiments (,) or at least three independent experiments (,). * Figure 7: Constitutive expression of TL on intestinal epithelial cells mediates the selection of mature memory CD8αβ+ T cells. (,) Tracking of donor Ly5.1+ OT-I cells among splenocytes and IELs from B6 recipient mice (n = 3–4 per group) given adoptive transfer of CD8ααhi or CD8ααlo−neg Ly5.1+ CD8+ OT-I cells (0.5 × 106) cultured for 2 d in the presence of APCs (MEC.B7.SigOVA adherent fibroblasts, which express the H-2Kb-restricted SIINFEKL epitope and B7.1), then sorted as CD8ααhi and CD8ααlo−neg cells and cultured for 3 d in vitro; recipient mice were infected orally with 5 × 108 ActA− LM-OVA1 month after transfer and assessed 3 d () or 5 d () after infection. () Flow cytometry of secondary OT-I memory cells among IELs 45 d after infection as in . () Tracking of memory OT-I cells among splenocytes and IELs from wild-type or TL− recipients of sorted, in vitro–activated Ly5.1+ CD8ααlo−neg OT-I primary effector cells (0.5 × 106) cultured for 3 d; recipient mice were infected orally with 5 × 108 ActA− LM-OVA 1 month after transfer and assessed 4 months after infection (pre! sented as in Fig. 1a–d). (–) Analysis of effector OT-I cells in the peripheral blood (PBMCs; ,) and memory OT-I cells among splenocytes and IELs (,) of Ly5.1+ wild-type or Ly5.1+ TL− recipient mice given 5 × 104 naive CD8+ OT-I cells and infected orally (,) or intravenously (,) with LM-Q4 1 d after transfer, assessed 7 d after infection (effector) or 2 months after infection (memory) and presented as in . () Bacterial load in the liver of Ly5.1+ mice (n = 6) given adoptive transfer of 5 × 104 naive wild-type or ΔE8I OT-I cells, then immunized orally with 1 × 109 ActA− LM-OVA and rechallenged orally with 1 × 1010 wild-type LM-OVA 2 months after the initial immunization and assessed 3 d later. CFU, colony-forming units. NS, not significant; *P < 0.05 (unpaired t-test). Data are representative of at least five (,), three () or two () independent experiments or three independent experiments (–; mean and s.e.m. in ). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yujun Huang & * Yunji Park Affiliations * Division of Developmental Immunology, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA. * Yujun Huang, * Yunji Park, * Yiran Wang-Zhu, * Alexandre Larange, * Ramon Arens, * Iván Bernardo, * Stephen P Schoenberger, * Mitchell Kronenberg & * Hilde Cheroutre * Department of Microbiology & Immunology, Vanderbilt University, School of Medicine, Nashville, Tennessee, USA. * Danyvid Olivares-Villagómez & * Luc Van Kaer * Division of Immunology, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria. * Dietmar Herndler-Brandstetter & * Beatrix Grubeck-Loebenstein * Department of Microbiology and Immunology, Life Sciences Institute, Vancouver, Canada. * Ninan Abraham * Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, USA. * Michael A Teitell * Present address: Clinical Laboratory. Histocompatibility Section, San Pedro Hospital, Logroño, Spain. * Iván Bernardo Contributions Y.H. and Y.P., conceptual development and execution of the studies and preparation of the manuscript; Y.W.-Z., A.L., R.A. and I.B., technical assistance and input into data analyses; D.O.-V. and L.V.K., generation of TL-deficient mice; M.A.T., generation and backcrossing of TL-transgenic mice; D.H.-B. and B.G.-L., experiments with human samples; N.A., mice with mutation in the sequence encoding IL-7Rα Y449XXM; S.P.S., help with in vitro culture experiments; M.K., participation in discussions of the data and preparation of the manuscript; H.C., conception of ideas, generation of TL transgenic mice with the assistance of M.A.T., manuscript authorship and experiment supervision. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hilde Cheroutre Author Details * Yujun Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Yunji Park Search for this author in: * NPG journals * PubMed * Google Scholar * Yiran Wang-Zhu Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandre Larange Search for this author in: * NPG journals * PubMed * Google Scholar * Ramon Arens Search for this author in: * NPG journals * PubMed * Google Scholar * Iván Bernardo Search for this author in: * NPG journals * PubMed * Google Scholar * Danyvid Olivares-Villagómez Search for this author in: * NPG journals * PubMed * Google Scholar * Dietmar Herndler-Brandstetter Search for this author in: * NPG journals * PubMed * Google Scholar * Ninan Abraham Search for this author in: * NPG journals * PubMed * Google Scholar * Beatrix Grubeck-Loebenstein Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen P Schoenberger Search for this author in: * NPG journals * PubMed * Google Scholar * Luc Van Kaer Search for this author in: * NPG journals * PubMed * Google Scholar * Mitchell Kronenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Michael A Teitell Search for this author in: * NPG journals * PubMed * Google Scholar * Hilde Cheroutre Contact Hilde Cheroutre Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (926K) Supplementary Figures 1–6 Additional data - Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes
- Nat Immunol 12(11):1096-1104 (2011)
Nature Immunology | Article Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes * Veronika Lukacs-Kornek1 * Deepali Malhotra1, 2 * Anne L Fletcher1 * Sophie E Acton1 * Kutlu G Elpek1 * Prakriti Tayalia3 * Ai-ris Collier1 * Shannon J Turley1, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1096–1104Year published:(2011)DOI:doi:10.1038/ni.2112Received01 July 2011Accepted18 August 2011Published online18 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) are nonhematopoietic stromal cells of lymphoid organs. They influence the migration and homeostasis of naive T cells; however, their influence on activated T cells remains undescribed. Here we report that FRCs and LECs inhibited T cell proliferation through a tightly regulated mechanism dependent on nitric oxide synthase 2 (NOS2). Expression of NOS2 and production of nitric oxide paralleled the activation of T cells and required a tripartite synergism of interferon-γ, tumor necrosis factor and direct contact with activated T cells. Notably, in vivo expression of NOS2 by FRCs and LECs regulated the size of the activated T cell pool. Our study elucidates an as-yet-unrecognized role for the lymph node stromal niche in controlling T cell responses. View full text Figures at a glance * Figure 1: LNSCs inhibit DC-induced proliferation of CD8+ T cells. () Flow cytometry of LNSC populations expanded in culture for 5 d and stained with anti-CD45, anti-CD31 and anti-gp38. Numbers adjacent to outlined areas indicate percent cells in the gated population (outlined): live cells gated among total cells (left) or CD45− cells among live gated cells (middle); numbers in quadrants indicate percent cells in each throughout (right: live gated, CD45− cells). SSC side scatter; FSC, forward scatter. () Proliferation of CFSE-labeled OT-I T cells (1 × 105; constant number) cultured with OVA-pulsed or unpulsed (control) splenic DCs (1 × 105; constant number) without stromal cells (No stroma) or with the addition of various numbers of unfractionated LNSCs (1 × 105, 2 × 104 or 1 × 104; to achieve a ratio of 1:1, 1:5 or 1:10, respectively, of stromal cells to OT-I T cells), assessed 72 h later by flow cytometry of CFSE dilution. Numbers above bracketed lines indicate percent CSFE+ cells. () Antigen-presentation assay of OT-I T cells cu! ltured as in , presented as division index (left) and frequency of CD25+ OT-I T cells (right). Each symbol represents an individual replicate; small horizontal lines indicate the mean. *P < 0.001 (Student's t-test). Data represent three independent experiments with three replicates each (,) or are pooled from three independent experiments (). * Figure 2: FRCs dampen the proliferation of activated T cells. () Frequency of CD45− cells after purification of stromal cultures expanded ex vivo (>99% CD45− cells; left), and surface expression of CD31 and gp38 on CD45− cells (>95% FRCs; right). Number adjacent to outlined area (left) indicates percent CD45− cells; numbers in quadrants (right) indicate percent of each stromal cell subset among CD45− cells. () Proliferation of CFSE-labeled splenocytes (1 × 106) activated for 48 h in the presence (Act spl + FRC) or absence (Act spl alone) of FRCs (5 × 104). () CFSE profiles of CD8+ or CD4+ T cells from cultures of nonactivated splenocytes without FRCs (Nonact spl) or activated splenocytes with or without FRCs (as in ). () Expression of CD25 and CD69 by activated T cells cultured as in . () Frequency of CD25+ T cells among cells cultured as in . () Frequency of T cells expressing activated caspase-3 and caspase-7 (FLICA+) among cells cultured as in . () Proliferation of splenocytes cultured directly for 48 h with or without f! reshly isolated, sorted FRCs (4 × 104) as in . () Proliferation of purified CD4+ or CD8+ T cells (1 × 105) activated for 48 h by plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of FRCs (5 × 104). In ,,, each symbol represents an individual replicate () or well (,); small horizontal lines indicate the mean. *P < 0.001 and **P < 0.005 (Student's t-test). Data are representative of two to three independent experiments with two to four replicates each (mean and s.d. in ,). * Figure 3: IFNGR1 signaling in FRCs is crucial for suppression. () IFN-γ production by T cells (CFSE+CD4+CD8+) and non-T cells (CFSE+CD4−CD8−) among wild-type FRCs (5 × 104) cultured with wild-type CFSE-labeled splenocytes (1 × 106) and soluble anti-CD3 plus anti-CD28 and evaluated 48 h later by flow cytometry. Numbers in quadrants indicate percent cells in each. () Proliferation of CD4+ or CD8+ T cells among wild-type (WT), Ifng−/− or Ifngr1−/− FRCs (5 × 104) cultured with wild-type or Ifng−/− CFSE-labeled splenocytes (1 × 106) and soluble anti-CD3 plus anti-CD28 and evaluated 48 h later. Vertical dashed lines indicate final peak of the proliferation of activated splenocytes without FRCs. () Division of CD4+ or CD8+ T cells cultured as in . () Proliferation of T cells among Ifngr1−/− CFSE-labeled splenocytes (1 × 106) left nonactivated or activated with soluble anti-CD3 plus anti-CD28 and cultured for 48 h with or without wild-type FRCs (5 × 104). () Division of CD4+ or CD8+ T cells cultured as in . *P < 0.05,! **P < 0.005 and ***P < 0.001 (Student's t-test). Data are representative of two to three independent experiments with three to five replicates each (mean and s.d.). * Figure 4: FRCs use NOS2 to regulate T cell proliferation. () Quantitative RT-PCR analysis of Arg1, Ido1 and Nos2 mRNA in FRC populations expanded ex vivo in the presence of recombinant IFN-γ (50 ng/ml); results are presented relative to those of untreated FRCs (0). (,) Proliferation of CD4+ T cells () or CD8+ T cells () among CFSE-labeled splenocytes (1 × 106) activated with soluble anti-CD3 plus anti-CD28 and cultured for 48 h with or without FRCs (5 × 104) in the presence or absence (Control) of the inhibitors Nor-NOHA (50 μM), 1-MT (5 nM) or L-NMMA (400 μM). () Nitric oxide in supernatants of wild-type or Nos2−/− CFSE-labeled splenocytes (1 × 106) cultured for 48 h with or without wild-type or Nos2−/− FRCs (5 × 104) in the presence of anti-CD3 plus anti-CD28. () Proliferation of CD4+ or CD8+ T cells among wild-type or Nos2−/− CFSE-labeled splenocytes (1 × 106) cultured for 48 h with or without wild-type or Nos2−/− FRCs (5 × 104). () Division of CD4+ or CD8+ T cells cultured as in . *P < 0.05, **P < 0.005! and ***P < 0.001 (Student's t-test). Data are representative of two to three independent experiments with three to five replicates each (mean and s.d.). * Figure 5: TNF and cell contact trigger NOS2 expression in FRCs. () Nitric oxide in supernatants of wild-type FRCs (5 × 104) cultured for 48 h with or without (No stimuli) recombinant IFN-γ (50 ng/ml) or recombinant TNF (50 ng/ml). () Quantitative PCR analysis of Nos2 mRNA expression by wild-type FRCs (5 × 104) incubated for 6, 24 or 48 h with recombinant IFN-γ (50 ng/ml) or recombinant TNF (50 ng/ml); results are presented relative to those of untreated FRCs. () Flow cytometry of NOS2 in Nos2−/− or wild-type FRCs left unstimulated or incubated for 48 h (+ stimuli) as in (left three plots) or with splenocytes activated by soluble anti-CD3 plus anti-CD28 (far right). Numbers in plots indicate percent NOS2+ cells (top) or mean fluorescence intensity (MFI; in parentheses). () Proliferation of wild-type CFSE-labeled splenocytes (1 × 106) left nonactivated or activated as in and cultured for 48 h with or without wild-type FRCs or FRCs doubly deficient in TNFR1 and TNFR2 (Tnfrsf1a−/−Tnfrsf1b−/−; 5 × 104). () Nitric oxide in su! pernatants of cultures as in . () Proliferation of wild-type CFSE-labeled splenocytes (1 × 106) activated as in and cultured with or without wild-type FRCs (5 × 104), with (+) or without (−) a Transwell insert (TW; pore size, 0.4 μm). () Division of T cells cultured as in . *P < 0.001 (Student's t-test). Data are representative of two to three independent experiments with two to four replicates each (mean and s.d.). * Figure 6: LECs inhibit T cell proliferation via NOS2. () Division of splenocytes (1 × 106) activated for 48 h in the presence (LEC) or absence (No LEC) of LEC populations expanded ex vivo (4.5 × 104 LECs). Each symbol represents an individual well; small horizontal lines indicate the mean. () CSFE dilution by wild-type or Ifng−/− splenocytes left nonactivated or activated with anti-CD3 plus anti-CD28 and cultured with or without wild-type LECs as in . () Division of T cells in cultures as in . () Nitric oxide in supernatants of wild-type or Nos2−/− splenocytes cultured for 48 h with or without wild-type or Nos2−/− LECs as in . () CSFE dilution in T cells cultured as in . () Division of T cells among cultures as in . () Nitric oxide in supernatants of wild-type LECs (4.5 × 104) incubated for 48 h with or without recombinant IFN-γ (50 ng/ml) and/or recombinant TNF (50 ng/ml). () NOS2 protein in Nos2−/− or wild-type LECs left unstimulated or incubated for 48 h as in alone (left three plots) or with activated sp! lenocytes (far right). Numbers in plots indicate percent NOS2+ cells. () Nitric oxide in supernatants of splenocytes cultured for 48 h with or without wild-type LECs as in with or without a Transwell insert. () Division of T cells among cultures as in . *P < 0.001 (Student's t-test). Data are representative of two to three independent experiments with three replicates each (mean and s.d.). * Figure 7: NOS2-mediated suppression operates in vivo. () Division of CFSE-labeled OT-I T cells (5 × 105) cultured for 48 h with OVA peptide–loaded DCs or wild-type or Nos2−/− FRCs (5 × 104). () CSFE dilution by T cells cultured alone (OT-I no stimuli) or as in . () Nitric oxide in supernatants of cells cultured for 48 h as in . () Survival of OT-I T cells in SLNs and MLNs collected from iFABP-tOVA and iFABP-tOVA Nos2−/− mice 72 h after injection of 1.5 × 106 CFSE-labeled CD45.1+ congenic OT-I T cells; single-cell suspensions were stained for CD45.1 and CD8, and results are presented as percent among all living cells. () CFSE dilution by OT-I T cells obtained as in . () Flow cytometry analysis of the number of OT-I T cells at division four (Gen 5) and division five (Gen 6) for cells obtained as in ; results are based on the CFSE dilution as in . *P < 0.001 and **P < 0.05 (Student's t-test). Data are representative of two to three independent experiments with three to four replicates in each (mean and s.d.). * Figure 8: NOS2 is expressed by FRCs and LECs in vivo. Microscopy of cryosections of SLNs () and MLNs () from iFABP-tOVA mice given no cells (iFABP-tOVA (no OT-I)) or 1.5 × 106 CD45.1+ congenic OT-I T cells (iFABP-tOVA + OT-I), assessed 48 h later by staining for gp38, CD31 and NOS2. Bottom row (), enlargement of area outlined in middle row, far left. Scale bars, 50 μm (top and middle rows) or 20 μm (bottom row, ). Data are representative of two independent experiments with two mice per group and two molds per organ. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. * Veronika Lukacs-Kornek, * Deepali Malhotra, * Anne L Fletcher, * Sophie E Acton, * Kutlu G Elpek, * Ai-ris Collier & * Shannon J Turley * Division of Medical Sciences, Harvard Medical School, Boston, Massachusetts, USA. * Deepali Malhotra * School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA. * Prakriti Tayalia * Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA. * Shannon J Turley Contributions V.L.-K. designed and did most of the experiments, analyzed and interpreted data and wrote the manuscript; D.M. did individual experiments and discussed and interpreted results; A.L.F. edited the manuscript; A.L.F., S.E.A., K.G.E., P.T. and A.C. discussed and interpreted results and provided technical help for the experiments; and S.J.T. directed the study, analyzed and interpreted results and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shannon J Turley Author Details * Veronika Lukacs-Kornek Search for this author in: * NPG journals * PubMed * Google Scholar * Deepali Malhotra Search for this author in: * NPG journals * PubMed * Google Scholar * Anne L Fletcher Search for this author in: * NPG journals * PubMed * Google Scholar * Sophie E Acton Search for this author in: * NPG journals * PubMed * Google Scholar * Kutlu G Elpek Search for this author in: * NPG journals * PubMed * Google Scholar * Prakriti Tayalia Search for this author in: * NPG journals * PubMed * Google Scholar * Ai-ris Collier Search for this author in: * NPG journals * PubMed * Google Scholar * Shannon J Turley Contact Shannon J Turley Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (786K) Supplementary Figures 1–5 and Methods Additional data - A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28
- Nat Immunol 12(11):1105-1112 (2011)
Nature Immunology | Article A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28 * Kok-Fai Kong1 * Tadashi Yokosuka2, 3 * Ann J Canonigo-Balancio1 * Noah Isakov4 * Takashi Saito2, 5 * Amnon Altman1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1105–1112Year published:(2011)DOI:doi:10.1038/ni.2120Received04 April 2011Accepted29 August 2011Published online02 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (TH2 cells) and interleukin 17–producing helper T cells (TH17 cells) but not of T helper type 1 cells (TH1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC! -θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell–mediated inflammatory diseases. View full text Figures at a glance * Figure 1: Requirement of PKC-θ V3 for localization to the immunological synapse, cSMAC and signaling. () Immunofluorescence imaging of Prkcq−/− OT-II CD4+ T cells infected with retrovirus expressing GFP-tagged wild-type (WT) PKC-θ, PKC-θ–ΔV3 or PKC-θ+δV3 (PKC-GFP; green) and mixed (1:1 ratio) with APCs labeled with the cell-tracking dye CMAC (APC (CMAC); blue), preincubated with (+OVA) or without (–OVA) OVA peptide; fixed conjugates were stained with anti-talin plus secondary Alexa Fluor 647–coupled antibody (Talin + AF647; red). DIC, differential interference contrast. Original magnification, ×63. () Quantification of the localization of PKC-θ in (IS) or outside (No IS) the immunological synapse and cSMAC in T cell–APC conjugates as described in (n = ~40), limited to conjugates that had reorganized their talin and had detectable PKC-θ. *P < 0.05 (one-way analysis of variance (ANOVA)). () Luciferase activity in MCC-specific T hybridoma cells transfected with empty vector or vector encoding PKC-θ tagged with the Xpress epitope, together with a luciferase ! reporter for the CD28 response element RE/AP and a β-galactosidase (β-Gal) reporter, then incubated for 6 h with DCEK fibroblasts expressing I-Ek and B7-1 in the presence (+ MCC) or absence (No stim) of MCC peptide. Results are presented in relative luciferase units (RLU), relative to β-Gal activity. Below, immunoblot analysis (IB) of PKC-θ expression, probed with anti-Xpress. *P < 0.05 (one-way ANOVA). (,) Expression of CD69 () or CD25 and PKC-θ () in GFP+ CD4+ T cells sorted from Rag1−/− mice reconstituted with Prkcq−/− bone marrow cells transduced with empty vector, wild-type PKC-θ or PKC-θ+δV3, left unstimulated or stimulated overnight with anti-CD3 plus anti-CD28. Numbers above bracketed lines indicate percent positive cells. (,) IL-2 production () and proliferation () of GFP+ CD4+ T cells isolated as in , or T cells isolated from control, unreconstituted Prkcq−/− mice (KO), and left unstimulated or stimulated for 48 h with anti-CD3 plus anti-CD28. ! Data are representative of six experiments (,) or two experime! nts (,) or are from three () or two (,) experiments (mean and s.e.m. in ,,). * Figure 2: The PKC-θ V3 domain is required and sufficient for CD28 interaction. () Association of PKC-θ V3 with CD28 in Prkcq−/− CD4+ T cells infected with retrovirus expressing empty vector or vectors for PKC-θ or PKC-δ (above lanes), then restimulated for 5 min with anti-CD3 plus anti-CD28, followed by immunoblot analysis of lysates directly (Lysates) or after immunoprecipitation (IP) with anti-CD28. () Imaging of Jurkat T cells cotransfected with Myc-tagged PKC-θ V3 plus cyan fluorescent protein–tagged CD28 (CD28-CFP) incubated at a ratio of 1:1 with Raji B cells pulsed with staphylococcus enterotoxin E; fixed conjugates were stained with rabbit anti-Myc plus secondary Alexa Fluor 488–coupled antibody (AF488). Original magnification, ×63. Data are representative of three experiments () or are from four experiments (). * Figure 3: A proline-rich motif in the V3 domain of PKC-θ determines its localization to the immunological synapse and interacts with CD28. () PKC-θ localization in CD4+ T cells from OT-II transgenic mice infected with retrovirus expressing GFP-tagged wild-type PKC-θ, PKC-δ+θPR or wild-type PKC-δ (green), then stimulated, fixed, stained and analyzed as in Figure 1a. Original magnification, ×63. () Quantitative analysis of the results in (as in Fig. 1a). *P < 0.05 (one-way ANOVA). () PKC-CD28 association in Prkcq−/− CD4+ T cells infected with retrovirus expressing wild-type PKC-θ, PKC-δ+θPR or wild-type PKC-δ, then restimulated for 5 min with anti-CD3 plus anti-CD28 (left) or left unstimulated (right). * indicates position of the immunoprecipitating antibody heavy chain. () Luciferase activity in MCC-specific T hybridoma cells cotransfected with the vectors in a together with reporter plasmids for RE/AP and β-Gal, then stimulated and assessed in Figure 1c. *P < 0.05 (one-way ANOVA). Data are representative of six experiments (,; mean and s.e.m. in ) or three experiments () or are from two experimen! ts (; mean and s.e.m. of triplicates). * Figure 4: Importance of the proline-rich motif in the V3 domain of PKC-θ for localization to the immunological synapse and CD28 interaction. () Quantification of PKC-θ localization in Prkcq−/− OT-II CD4+ T cells infected with retrovirus expressing GFP-tagged wild-type or mutant PKC-θ (images, Supplementary Fig. 5). *P < 0.05 (one-way ANOVA). () PKC-θ–CD28 association in Prkcq−/− CD4+ T cells infected with retrovirus expressing wild-type or mutant PKC-θ and restimulated for 5 min with anti-CD3 plus anti-CD28. () Luciferase activity in MCC-specific T hybridoma cells transfected with empty vector or expression vector for wild-type or mutant PKC-θ, together with reporter plasmids for RE/AP and β-Gal, then stimulated and assessed as in Figure 1c. *P < 0.05 (one-way ANOVA). (–) Expression of CD69 and CD25 () and proliferation () and production of IL-2 () in T cells isolated from control, unreconstituted Prkcq−/− mice (KO) or from Rag1−/− bone marrow chimeras reconstituted with Prkcq−/− bone marrow cells transduced with retrovirus expressing empty vector or expression vector for wild-type o! r mutant PKC-θ; T cells from Prkcq−/− mice or sorted GFP+ CD4+ T cells from chimeras were left unstimulated or stimulated overnight () or for 48 h (,) with anti-CD3 plus anti-CD28. Data are representative of five (), four () or two () experiments or are from two experiments (–; mean and s.e.m. in ,,). * Figure 5: The interaction between CD28 and the V3 domain of PKC-θ is Lck dependent. Immunoassay (bottom) of the PKC-θ–Lck–CD28 association in Lck-deficient JCam1.6 Jurkat cells cotransfected with Myc-tagged PKC-θ V3 plus expression vector for wild-type or mutant Lck (top), then stimulated for 5 min with anti-CD3 and anti-CD28, followed by immunoblot analysis of Lck and endogenous CD28 in whole-cell lysates (WCL) or lysates immunoprecipitated with anti-Myc. W97A, inactivated SH3 domain; R154K, inactivated SH2 domain; Cat, catalytic. Asterisks (top) indicate point substitutions. Data are from three experiments. * Figure 6: The V3 domain interferes with PKC-θ-mediated signaling and T cell differentiation. () Localization of PKC-θ and V3 in OT-II CD4+ T cells infected with retrovirus expressing Myc-tagged V3, V3 with substitution of all four proline residues with alanine (V3-4PA) or V3 with deletion of the entire proline-rich motif (V3-ΔPR; green), stimulated with OVA-pulsed, CMAC-labeled APCs (blue), and fixed; conjugates were stained with anti-Myc plus a secondary Alexa Fluor 555–coupled antibody (AF555; orange) and with anti–PKC-θ plus a secondary Alexa Fluor 647–coupled antibody (AF647; red) and analyzed as in Figure 1a. Original magnification, ×63. () Quantitative analysis of the results in (as in Fig. 1b). *P < 0.05 (one-way ANOVA). () Luciferase activity in MCC-specific T hybridoma cells transfected with PKC-θ and/or V3 vectors (below graph) plus reporter plasmids for RE/AP-luciferase and β-Gal, then stimulated and analyzed as in Figure 1c. () Helper T cell differentiation of naive CD4+ T cells from B6 mice stimulated with anti-CD3 plus anti-CD28, cultured u! nder TH1-, TH2- or TH17-polarizing conditions (left margin) and transduced by retrovirus with empty vector or vectors for PKC-θ V3 (above plots), analyzed by intracellular staining 8 h after restimulation with anti-CD3 plus anti-CD28. Numbers in quadrants indicate percent cells in each. Right, frequency of cytokine-producing cells (cumulative data); each symbol represents an individual replicate, and small horizontal lines indicate the mean. Data are representative of five experiments (,) or three experiments (; mean and s.e.m.) or are from six experiments (). * Figure 7: V3 inhibits TH2- but not TH1-mediated lung inflammation. (–) Infiltration of mononuclear cells into bronchoalveolar lavage fluid () and cytokine expression by cells (,) from naive B6 mice given OT-II CD4+ T cells stimulated with anti-CD3 plus anti-CD28 and cultured under TH2-polarizing conditions, then transduced with PKC-θ V3 vectors via retrovirus (as in Fig. 6), sorted as GFP+ populations and adoptively transferred into recipient mice that were challenged with OVA and analyzed 1 d later. (,) Infiltration () and cytokine expression () by naive B6 mice treated as in –, except the transferred cells were cultured under TH1-polarizing conditions. Data are from two experiments (mean and s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA. * Kok-Fai Kong, * Ann J Canonigo-Balancio & * Amnon Altman * Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan. * Tadashi Yokosuka & * Takashi Saito * Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan. * Tadashi Yokosuka * The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Be'er Sheva, Israel. * Noah Isakov * Laboratory of Cell Signaling, World Premiere International Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan. * Takashi Saito Contributions K.-F.K. and A.A. designed the experiments and wrote the manuscript; K.-F.K. generated and analyzed data; T.S. and T.Y. provided expertise in cell imaging; T.S. participated in discussion of the data; T.Y. generated the PKC-GFP and CD28–cyan fluorescent protein fusion vectors; N.I. participated actively in discussions leading to this work and in experimental design; and A.J.C.-B. did various experiments and animal work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Amnon Altman Author Details * Kok-Fai Kong Search for this author in: * NPG journals * PubMed * Google Scholar * Tadashi Yokosuka Search for this author in: * NPG journals * PubMed * Google Scholar * Ann J Canonigo-Balancio Search for this author in: * NPG journals * PubMed * Google Scholar * Noah Isakov Search for this author in: * NPG journals * PubMed * Google Scholar * Takashi Saito Search for this author in: * NPG journals * PubMed * Google Scholar * Amnon Altman Contact Amnon Altman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–9. Additional data - The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells
- Nat Immunol 12(11):1113-1118 (2011)
Nature Immunology | Article The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells * Huai-Chia Chuang1 * Joung-Liang Lan2, 3, 5 * Der-Yuan Chen2, 3, 5 * Chia-Yu Yang1, 5 * Yi-Ming Chen2, 3 * Ju-Pi Li1 * Ching-Yu Huang1 * Pao-En Liu2 * Xiaohong Wang4 * Tse-Hua Tan1, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:1113–1118Year published:(2011)DOI:doi:10.1038/ni.2121Received14 June 2011Accepted30 August 2011Published online09 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Protein kinase C-θ (PKC-θ) is required for activation of the transcription factor NF-κB induced by signaling via the T cell antigen receptor (TCR); however, the direct activator of PKC-θ is unknown. We report that the kinase GLK (MAP4K3) directly activated PKC-θ during TCR signaling. TCR signaling activated GLK by inducing its direct interaction with the upstream adaptor SLP-76. GLK-deficient mice had impaired immune responses and were resistant to experimental autoimmune encephalomyelitis. Consistent with that, people with systemic lupus erythematosus had considerable enhanced GLK expression and activation of PKC-θ and the kinase IKK in T cells, and the frequency of GLK-overexpressing T cells was directly correlated with disease severity. Thus, GLK is a direct activator of PKC-θ, and activation of GLK–PKC-θ–IKK could be used as new diagnostic biomarkers and therapeutic targets for systemic lupus erythematosus. View full text Figures at a glance * Figure 1: GLK directly interacts with and phosphorylates PKC-θ at Thr538. () Luciferase activity of an NF-κB reporter in Jurkat T cells transfected with vector alone, GLK-specific siRNA or plasmid encoding GLK. *P < 0.01 (two-tailed t-test). () Immunoprecipitation (IP) of endogenous GLK together with PKC-θ in lysates of mouse primary T cells after stimulation with anti-CD3 (α-CD3), analyzed by immunoblot (IB). NS, normal serum; p-, phosphorylated; Pre-IP, immunoblot analysis before immunoprecipitation. () In vitro binding assays of purified Flag-GLK and glutathione S-transferase (GST)–PKC-θ proteins. ppt, precipitation. Tubulin serves as a loading control throughout. () Immunoblot analysis of GLK expression and GLK-induced PKC-θ phosphorylation in in vitro kinase assays with Flag-GLK and Myc–PKC-θ (as the substrate) isolated from transfected HEK293T cells; far right, immunoblot analysis of PKC-θ in lysates of Jurkat cells (control). () Immunoblot analysis of GLK expression and GLK-induced PKC-θ phosphorylation in in vitro kinase assays! with purified Flag-GLK and GST–PKC-θ. Data are representative of three independent experiments (error bars (), s.d. of triplicate samples). * Figure 2: SLP-76 is a direct upstream regulator of GLK. () Coimmunoprecipitation of endogenous GLK with SLP-76 or PKC-θ in lysates of mouse primary T cells after CD3 stimulation. () In vitro binding assays of purified Flag-GLK and Flag–SLP-76 proteins. (,) In vitro kinase assays of Flag-GLK in lysates of J14 cells () or Jurkat T cells () transfected with empty vector or plasmid encoding GLK with or without SLP-76 shRNA, then left untreated or treated with anti-CD3. () Immunoblot analysis of the phosphorylation of SLP-76 at Ser376 (arrow) in lysates of HEK293T cells transfected with empty vector or plasmid encoding SLP-76 plus plasmid encoding GLK or HPK1. Data are representative of three independent experiments. * Figure 3: GLK-deficient primary T cells are defective in PKC-θ–IKK activation and in proliferation. (,) Immunoblot analysis of GLK and phosphorylated (p-) and total PKCθ, IKK, Erk and PDK1 in lysates of purified wild-type (WT) or GLK-deficient (GLK-KO) T cells after stimulation with CD3 alone () or CD3 plus CD28 (). () Flow cytometry of the calcium indicator Fluo-4 in purified mouse T cells stimulated with anti-CD3 and then with ionomycin (Ion). (,) [3H]thymidine incorporation () and CFSE dilution () by wild-type or GLK-deficient CD3+ T cells treated for 72 h with anti-CD3 or PMA plus ionomycin (P + I). () [3H]thymidine incorporation in wild-type and GLK-deficient T cells transfected with empty vector (encoding green fluorescent protein alone) or vector encoding GLK tagged with green fluorescent protein. Data are representative of three independent experiments (error bars (,), s.e.m.). * Figure 4: In vivo T cell–dependent immune responses are impaired in GLK-deficient mice. (,) CFSE dilution () and enzyme-linked immunosorbent assay (ELISA) of IFNγ, IL-2 and IL-4 () in culture supernatants of KLH-restimulated lymph node cells isolated from KLH-immunized mice and restimulated for 3 d in vitro with KLH. (,) ELISA of nitrophenol-specific antibody (NP-specific Ab) in the serum of mice (n = 6 per group) 14 d after primary immunization () or 7 d after secondary immunization (); results are presented relative to those of normal serum from a wild-type mouse. () EAE induction in GLK-deficient mice and their wild-type littermates (n = 7 per group) of F5 on the B6 background (~97%), presented as clinical scores on a scale of 1 to 5. () Flow cytometry of infiltrating TH17 cells (CD45 gated) from the brains and spinal cords of myelin oligodendrocyte glycoprotein (MOG)-immunized mice on day 14. () ELISA of IL-17 in serum from MOG-immunized mice (n = 6 per group). () Flow cytometry of IL-17A-producing CD4+ T cells among in vitro–differentiated TH17 cells. (! ) Suppression of CFSE-labeled CD3+ T cells by wild-type or GLK-deficient Treg cells, presented as CFSE dilution in responding T cells cultured at a ratio of 3:1 (left) or 9:1 (right) with Treg cells plus anti-CD3-coated beads. Numbers in or adjacent to outlined areas indicate percent cells in each throughout. *P < 0.05 and **P < 0.001 (two-tailed t-test). Data are from two (,,,,) or three (,,,) independent experiments (mean ± s.e.m.). * Figure 5: GLK expression and phosphorylation of PKC-θ at Thr538 are induced in T cells from people with SLE. () Flow cytometry analysis of GLK-expressing (GLK+) lymphocytes from PBLs of people with SLE (n = 49) and unaffected controls (HC; n = 35); the results from one person with SLE (SLEDAI, 20) are presented here. () Positive correlation and significant regression between SLEDAI and the frequency of GLK-expressing T cells from all people with SLE (Pearson correlation coefficient r = 0.773; simple linear regression, y = −0.886 + 0.3901x; regression correlation coefficient, adjusted r2 = 0.597, P = 1.08 × 10−17). () Correlation and significant regression between SLEDAI and the frequency of GLK+ T cells from people with SLE with high frequency of GLK+ cells (≥21%; n = 16; Pearson correlation coefficient r = 0.807; simple linear regression, y = 5.2085 + 0.2757x; regression correlation coefficient, adjusted r2 = 0.626, P = 0.000159). () Immunoblot analysis of GLK and phosphorylated PKC-θ and IKK in lysates of PBLs from people with SLE (n = 5; randomly sampled) and healthy con! trols (n = 5). Below lanes, SLEDAI and densitometry results, presented relative to tubulin. () Flow cytometry analysis of phosphorylated PKCθ or IKK among (CD3-gated) PBLs of with a person with SLE and a healthy control (). Data are representative of 35 (), at least three () or at least 16 () independent experiments. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Joung-Liang Lan, * Der-Yuan Chen & * Chia-Yu Yang Affiliations * Immunology Research Center, National Health Research Institutes, Zhunan, Taiwan. * Huai-Chia Chuang, * Chia-Yu Yang, * Ju-Pi Li, * Ching-Yu Huang & * Tse-Hua Tan * Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan. * Joung-Liang Lan, * Der-Yuan Chen, * Yi-Ming Chen & * Pao-En Liu * Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan. * Joung-Liang Lan, * Der-Yuan Chen & * Yi-Ming Chen * Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA. * Xiaohong Wang & * Tse-Hua Tan Contributions H.-C.C. designed and did experiments, analyzed and interpreted data and wrote the manuscript; J.-L.L. and D.-Y.C. provided samples from affected people, analyzed clinical data and coordinated clinical research; C.-Y.Y., J.-P.L. and P.-E.L. did experiments; Y.-M.C. provided samples from affected people and analyzed clinical data; C.-Y.H. assisted in generation of the GLK-deficient mice; X.W. assisted in the experimental design and manuscript writing; and T.-H.T. conceived of the study, supervised experiments and composed the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tse-Hua Tan Author Details * Huai-Chia Chuang Search for this author in: * NPG journals * PubMed * Google Scholar * Joung-Liang Lan Search for this author in: * NPG journals * PubMed * Google Scholar * Der-Yuan Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Chia-Yu Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Yi-Ming Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Ju-Pi Li Search for this author in: * NPG journals * PubMed * Google Scholar * Ching-Yu Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Pao-En Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaohong Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Tse-Hua Tan Contact Tse-Hua Tan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–8. Additional data - Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments
- Nat Immunol 12(11):1119-1126 (2011)
Nature Immunology | Article Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments * Akanksha Chaturvedi1 * Rebecca Martz1 * David Dorward2 * Michael Waisberg1 * Susan K Pierce1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:1119–1126Year published:(2011)DOI:doi:10.1038/ni.2116Received16 May 2011Accepted23 August 2011Published online02 October 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Binding of antigen to the B cell antigen receptor (BCR) triggers both BCR signaling and endocytosis. How endocytosis regulates BCR signaling remains unknown. Here we report that BCR signaling was not extinguished by endocytosis of BCRs; instead, BCR signaling initiated at the plasma membrane continued as the BCR trafficked intracellularly with the sequential phosphorylation of kinases. Blocking the endocytosis of BCRs resulted in the recruitment of both proximal and downstream kinases to the plasma membrane, where mitogen-activated protein kinases (MAPKs) were hyperphosphorylated and the kinase Akt and its downstream target Foxo were hypophosphorylated, which led to the dysregulation of gene transcription controlled by these pathways. Thus, the cellular location of the BCR serves to compartmentalize kinase activation to regulate the outcome of signaling. View full text Figures at a glance * Figure 1: Spatial distribution of proximal and downstream phosphorylated kinases after BCR crosslinking. () TEM of mouse splenic B cells incubated for 15 min on ice with rat anti–mouse IgM conjugated to magnetic beads, then fixed directly (0 min) or incubated for 2–45 min (above images) at 37 °C, fixed and imaged. Arrows indicate BCRs (dense black dots). Scale bars, 500 nm. () Merged confocal images of Alexa Fluor 488 (green) and Cy5 (red) with differential interference contrast images (top rows) of mouse splenic B cells fixed, made permeable and either stained with Cy5–anti-IgM (to detect the BCR) and antibodies specific for p-Lyn, p-c-Raf, or p-Jnk followed by Alexa Fluor 488–conjugated secondary antibodies (0 min) or treated with Cy5–anti-IgM (to label and crosslink the BCR) and incubated for 0.5–30 min (above images) at 37 °C, then fixed, made permeable and stained with antibodies specific for p-Lyn, p-c-Raf, or p-Jnk followed by Alexa Fluor 488–conjugated secondary antibodies. Scale bars, 5 μm. Below each image, analysis of the intensity of staining for ki! nase phosphorylation (Alexa Fluor 488; green) and the BCR (Cy5; red) for cells marked with an asterisk (red arrows indicate plane of image): horizontal axis, arrow length (~6 μm); vertical axis, fluorescence intensity in a range from 0 to 4,095 on a 12-bit scale (similar analyses for p-Erk and p-p38, Supplementary Fig. 1). Data are representative of two experiments with 10–15 grids each () or eight separate experiments with 100 cells (). * Figure 2: Colocalization of proximal and downstream phosphorylated kinases with the BCR. Pearson's colocalization coefficient for phosphorylated kinases and BCRs in images of cells treated as described in Figure 1b, calculated by intensity correlation analysis of Alexa Fluor 488 and Cy5. Each symbol represents an individual cell; small horizontal lines indicate the mean (± s.e.m.). Data are from three independent experiments with approximately 15–22 cells total. * Figure 3: Colocalization of phosphorylated kinases with TfR and LAMP-1 after BCR crosslinking. () Confocal images merged with differential interference contrast images of mouse splenic B cells incubated for 7 or 15 min (right and left margins) at 37 °C with anti-IgM, then fixed, made permeable and stained with anti-TfR or anti-LAMP-1 followed by Alexa Fluor 488–conjugated secondary antibodies (green) and with antibodies specific for phosphorylated kinases followed by secondary antibodies conjugated to Alexa Fluor 647 (red). Scale bars, 5 μm. Right of each image, intensity analysis of Alexa Fluor 488 (green) and Alexa Fluor 647 (red) for cells as indicated by an asterisk as in Figure 1b. () Pearson's colocalization coefficient for phosphorylated kinases and TfR or LAMP-1 (as in Fig. 2). Data are representative of three separate experiments with 50 cells each. * Figure 4: Temporal and spatial distribution of the phosphorylation of kinases in the BCR signaling pathway. TEM of mouse splenic B cells incubated for 0–45 min (above images) with anti-IgM, then fixed, made permeable and stained with antibodies specific for p-Syk, p-Erk, p-p38 or p-Jnk, followed by horseradish peroxidase–conjugated secondary antibodies and development with 3,3-diaminobenzidine tetrahydrochloride. Arrows indicate phosphorylated kinases (dense black areas); M, mitochondria; N, nucleus. Scale bars, 500 nm. Data are representative of two experiments with 10–15 grids per time point in each. * Figure 5: Dynasore blocks the internalization of BCRs. (,) TEM of mouse splenic B cells, treated for 1 h with the vehicle DMSO alone (– DS) or 100 μM dynasore in DMSO (+ DS), then incubated for 15 min on ice in serum-free RPMI medium with rat anti–mouse IgM conjugated to magnetic beads, followed by incubation for 10 min () or 45 min () at 37 °C. White arrows indicate BCRs (dense black dots). Scale bars, 500 nm. () Internalization of BCRs in B220+ mouse splenic B cells incubated with for 1 h in RPMI medium with DMSO alone or 100 μM dynasore in DMSO, then fixed directly (0 min) or incubated for various times (horizontal axis) at 37 °C with biotin-labeled anti-IgM, fixed and stained with streptavidin–fluorescein isothiocyanate and B220-phycoerythrin. Data are representative of two independent experiments with 10–15 grids per time point in each (,) or are from one of two independent experiments (). * Figure 6: The subcellular distribution of phosphorylated kinases in dynasore-treated B cells. Intensity analyses (as in Fig. 1b) of Alexa Fluor 488 (green) and Cy5 (red) in mouse splenic B cells either incubated for 1 h in RPMI medium with DMSO alone or dynasore in DMSO and immediately fixed, made permeable and stained with Cy5–anti-IgM (to detect the BCR) and antibodies specific for p-Lyn, p-c-Raf, or p-Jnk followed by Alexa Fluor 488–conjugated secondary antibodies (far left; 0 min), or incubated for 0.5–60 min (above plots) in RPMI medium with DMSO alone or dynasore in DMSO, with Cy5–anti-IgM, then fixed, made permeable and stained with and antibodies specific for p-Lyn, p-c-Raf, or p-Jnk followed by Alexa Fluor 488–conjugated secondary antibodies (cell images, Supplementary Fig. 4). Data are representative of five different experiments with ~80 cells total. * Figure 7: Inhibiting endocytosis of the BCR alters the amount of phosphorylation of kinases in the BCR signaling pathway. Kinase phosphorylation in mouse splenic B cells left untreated (0) or incubated for 2–90 min with anti-IgM in the presence (+ DS) or absence (– DS) of dynasore, then made soluble and analyzed by immunoblot with antibodies specific for p-Syk, p-MEK1, p-Erk, p-p38, p-Akt or p-Jnk; blots were then stripped and reprobed with anti-tubulin (Supplementary Fig. 7) and results are presented relative to the intensity of tubulin. Data are from three separate experiments (average and s.e.m.). * Figure 8: Blocking endocytosis of the BCR blocks signals downstream of Akt and dysregulates transcription. () Immunoblot analysis of mouse splenic B cells left untreated (0) or incubated for 2–60 min (above lanes) in RPMI medium with anti-IgM in the presence of DMSO alone or dynasore in DMSO, probed with antibodies specific for p-Akt, phosphorylated Foxo1 and Foxo3a (p-Foxo1-Foxo3a) or phosphorylated 4E-BP1 (p-4E-BP1); blots were then stripped and reprobed with anti-tubulin (loading control). (,) Quantitative PCR analysis of the change in expression of genes encoding 80 transcription factors () or 90 molecules involved in B cell activation and the Akt pathway () in mouse splenic B cells left untreated, treated for 2 h with dynasore alone (+ DS) or incubated for 1 h with anti-IgM in the presence (+ DS + anti-IgM) or absence (+ anti-IgM) of pretreatment for 1 h dynasore; results are presented as 'volcano plots' with P value versus expression relative to that in untreated cells. Shaded areas indicate genes with a difference twofold or more in expression (log2 fold change ≥ 1.0 o! r ≤ −1.0) and a P value of ≥ 0.05. Additional information, Supplementary Data Set. Data are representative of four separate experiments () or are from four independent experiments (,). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA. * Akanksha Chaturvedi, * Rebecca Martz, * Michael Waisberg & * Susan K Pierce * Microscopy Unit, Rocky Mountain Laboratories, Research Technologies Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA. * David Dorward Contributions A.C. designed and did the experiments, analyzed data and wrote the manuscript; R.M. did experiments and analyzed the data; D.D. did TEM and interpreted the images; M.W. designed transcription analysis and analyzed the quantitative PCR data; and S.K.P. designed experiments, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Akanksha Chaturvedi or * Susan K Pierce Author Details * Akanksha Chaturvedi Contact Akanksha Chaturvedi Search for this author in: * NPG journals * PubMed * Google Scholar * Rebecca Martz Search for this author in: * NPG journals * PubMed * Google Scholar * David Dorward Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Waisberg Search for this author in: * NPG journals * PubMed * Google Scholar * Susan K Pierce Contact Susan K Pierce Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–7 Excel files * Supplementary Data Set (86K) Ct values of complete data sets for Fig. 8b and 8c. Additional data
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