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Latest Articles Include:

• Dogmas, paradigms and proving hypotheses
  - Nat Immunol 11(6):455 (2010)
  Strong hypotheses stand the test of time because of rigorous experimentation by authors and the scientific community.
• 20 years of gene therapy for SCID
  - Nat Immunol 11(6):457-460 (2010)
  Severe combined immunodeficiency conditions are devastating disorders of adaptive immunity. Although these diseases were initially treated by transplantation of allogeneic hematopoietic stem cells, the past 20 years has shown that these conditions are correctable by gene therapy.
• Multitasking in the medulla
  - Nat Immunol 11(6):461-462 (2010)
  Medullary thymic epithelial cells maintain tolerance by expressing peripheral self antigens. New data show that they also present these antigens, which leads to the deletion of conventional CD4+ T cells and the induction of regulatory T cells.
• Leukocyte chemotaxis: from lysosomes to motility
  - Nat Immunol 11(6):463-464 (2010)
  Chemoattractants direct the extravasation of leukocytes to the site of immune response. New data highlight the role of synaptotagmins and Rab proteins in leukocyte chemotaxis.
• Give and take in the germinal center
  - Nat Immunol 11(6):464-466 (2010)
  B cell–T cell interactions in germinal centers are needed to generate high-affinity antibodies. PD-1 signaling is now shown to influence the quality of germinal center responses.
• Catenin' on to nucleic acid sensing
  - Nat Immunol 11(6):466-468 (2010)
  Many pathogens induce a type I interferon response via a pathway dependent on the kinase TBK1 and transcription factor IRF3. However, LRRFIP1, a cytosolic sensor of DNA and RNA, triggers interferon production by a β-catenin-dependent signal.
• Research Highlights
  - Nat Immunol 11(6):469 (2010)
  
• The dual nature of TH17 cells: shifting the focus to function
  - Nat Immunol 11(6):471-476 (2010)
  Nature Immunology | Perspective The dual nature of TH17 cells: shifting the focus to function * William O'Connor Jr1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lauren A Zenewicz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard A Flavell1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature ImmunologyVolume:11,Pages:471–476Year published:(2010)DOI:doi:10.1038/ni.1882Published online18 May 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin 17 (IL-17)-producing helper T cells (TH17 cells) have been broadly linked to the pathogenesis of multiple autoimmune diseases. In the few short years since the discovery of TH17 cells, new paradigms about their prominence in chronic inflammation and human autoimmunity have emerged. Recent findings that TH17 cells might be capable of regulatory functions and that the associated effector molecules IL-17 and IL-22 aid in restricting tissue destruction during inflammatory episodes illuminate the complexities of IL-17 and TH17 biology. In this Perspective we highlight critical differences between IL-17 itself and TH17 cells and discuss the protective nature of IL-17 and TH17 cells. View full text Author information * Abstract * Author information Affiliations * Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA. * William O'Connor Jr, * Lauren A Zenewicz & * Richard A Flavell * Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA. * Richard A Flavell Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * William O'Connor Jr (william.oconnor@yale.edu) or * Richard A Flavell (richard.flavell@yale.edu) Additional data
• Induction of an IL7-R+c-Kithi myelolymphoid progenitor critically dependent on IFN-γ signaling during acute malaria
  Belyaev NN Brown DE Diaz AI Rae A Jarra W Thompson J Langhorne J Potocnik AJ - Nat Immunol 11(6):477-485 (2010)
  Nature Immunology | Article Induction of an IL7-R+c-Kithi myelolymphoid progenitor critically dependent on IFN-γ signaling during acute malaria * Nikolai N Belyaev1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas E Brown1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ana-Isabel Garcia Diaz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Aaron Rae3 Search for this author in: * NPG journals * PubMed * Google Scholar * William Jarra2 Search for this author in: * NPG journals * PubMed * Google Scholar * Joanne Thompson4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jean Langhorne2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandre J Potocnik1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:477–485Year published:(2010)DOI:doi:10.1038/ni.1869Received01 February 2010Accepted23 March 2010Published online02 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Although the relationship between hematopoietic stem cells and progenitor populations has been investigated extensively under steady-state conditions, the dynamic response of the hematopoietic compartment during acute infection is largely unknown. Here we show that after infection of mice with Plasmodium chabaudi, a c-Kithi progenitor subset positive for interleukin 7 receptor-α (IL-7Rα) emerged that had both lymphoid and myeloid potential in vitro. After being transferred into uninfected alymphoid or malaria-infected hosts, IL-7Rα+c-Kithi progenitors generated mainly myeloid cells that contributed to the clearance of infected erythrocytes in infected hosts. The generation of these infection-induced progenitors was critically dependent on interferon-γ (IFN-γ) signaling in hematopoietic progenitors. Thus, IFN-γ is a key modulator of hematopoiesis and innate and adaptive immunity during acute malaria infection. View full text Figures at a glance * Figure 1: Changes in bone marrow hematopoiesis during acute infection with P. chabaudi. () Course of malaria infection in C57BL/6 mice inoculated with blood stages of P. chabaudi. Data are from one representative of twelve independent experiments with 12 mice per time point (mean ± s.e.m.). () Frequency of clonogenic myeloerythroid bone marrow progenitors during acute malaria infection. CFU-GM, CFU activity of granulocyte-macrophage colonies; CFU-E, CFU activity of erythroid colonies; BFU-E, burst-forming unit activity of erythroid colonies; G, granulocyte; Mac, macrophage; GM, granulocyte-macrophage. Data are from seven experiments (granulocyte-macrophage) or five experiments (erythroid) with four to five samples per experiment and time point (mean ± s.e.m., left; mean, right). () Analytical flow cytometry of bone marrow cells from infected and control mice, costained with antibodies to lineage markers and dead cells (Lin (7-amino-actinomycin D (7-AAD)), Sca-1, c-Kit, FcRγII/III and CD34); outlined areas indicate gating (numbers in or above outlined areas i! ndicate percent cells in each). Data are from one representative of eight independent experiments with five mice per time point. () Absolute number of Lin−c-KithiCD34+FcRγII/III+ cells per femur during malaria infection. *P ≤ 0.01 (Mann-Whitney U-test). Data are from twelve independent experiments with at least three mice per time point each (mean and s.e.m.). * Figure 2: Transient emergence of Lin−IL-7Rα+c-Kithi progenitors during acute infection with P. chabaudi. () Lymphoid potential of uninfected and malaria-infected bone marrow on OP9 (B cell potential) and OP9-Dll1 (T cell potential) stromal cells under limiting-dilution conditions. Data are representative of two independent experiments (n = 9 mice). () Flow cytometry of Lin−IL-7Rα+ cells before and after infection with P. chabaudi. FSC, forward scatter. Data are representative of 21 independent experiments (n = 98 mice). () Absolute number of CLPs (Lin−IL-7Rα+c-KitloSca-1lo) and infection-induced Lin−IL-7Rα+c-Kithi progenitors per femur during malaria infection. *P ≤ 0 0.01 (Mann-Whitney U-test). Data are from five independent experiments with at least three mice per time point (mean and s.e.m.). () Expression of surface antigens on Lin−IL-7Rα+c-Kithi infection-induced bone marrow progenitors at day 11 after infection. Black lines, antigen staining on infection-induced progenitors; red lines, antigen staining on uninfected CLPs; gray-filled histograms, isotype-matc! hed control antibody. Data are representative of three independent experiments (n = 16 mice). * Figure 3: Infection-induced Lin−IL-7Rα+c-Kithi cells are bipotent myelolymphoid progenitors. () In vitro clonogenic culture of sorted uninfected hematopoietic progenitors and malaria-infected Lin−IL-7Rα+c-Kithi cells. CFU-M, CFU activity of macrophage colonies. Data are from three independent experiments (mean ± s.e.m.). () Coculture of sorted uninfected hematopoietic progenitors and malaria-infected Lin−IL-7Rα+c-Kithi cells on OP9 stromal cells (n ≥ 144 colonies per subset). Lymphoid, CD19+; Myelolymphoid, CD19+CD11b+Gr-1+; Myeloid, CD11b+Gr-1+. *P ≤ 0.05 and **P ≤ 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.d.). () Flow cytometry analysis of B cell or myeloid cell growth (OP9 and S17) or T cell growth (OP9-Dll1) by single-cell cultures of infection-induced Lin−IL-7Rα+c-Kithi cells after 14 d of culture (Supplementary Fig. 3a,b). Plating efficiency was ≥75%. Data are from five experiments (mean ± s.d. of at least 48 individual cultures per experiment and cytokine). () Expression of mRNA transcripts in sorted he! matopoietic progenitors, presented relative to expression of Rplp0 (which encodes the 60S acidic ribosomal protein P0). Data are from at least two independent experiments. * Figure 4: Infection-induced Lin−IL-7Rα+c-Kithi cells differentiate in vivo into functional myeloid cells. () Flow cytometry of bone marrow (BM) and spleen from hosts deficient in RAG-2 and γc 14 d after transplantation of infection-induced Lin−IL-7Rα+c-Kithi cells. Numbers above outlined areas indicate percent cells in each. Data are representative of three independent experiments (n = 11 mice). () Specific phagocytosis of P. chabaudi–infected erythrocytes by splenic CD11b+ myeloid cells purified by flow cytometry from hosts deficient in RAG-2 and γc transplanted with infection-induced Lin−IL-7Rα+c-Kithi cells and then cultured with erythrocytes in the presence (+) or absence (–) of cytochalasin. Above plots, ratio of infected erythrocytes (iRBC) to CD11b+ cells. Numbers adjacent to outlined areas indicate percent cells in each. Data are representative of two independent experiments. * Figure 5: Contribution of Lin−IL-7Rα+c-Kithi cells to the clearance of malaria-infected erythrocytes in vivo. () Parasitemia and anemia in malaria-infected mice transplanted with various hematopoietic subsets (key) on day 2 of infection. *P ≤ 0.05 and **P ≤ 0.01 (Mann-Whitney U-test). Data are from three independent experiments for each subset with four to seven mice per group (mean and s.e.m.). () Analysis of splenocytes of mice transplanted with CMPs, LMPPs or infection-induced Lin−IL-7Rα+c-Kithi progenitors on day 2 of infection. Numbers below outlined areas indicate percent cells in each. Data are representative of two independent experiments with six to nine mice per group. () Parasitemia and anemia in mice infected with GFP-transfected P. chabaudi and transplanted with infection-induced Lin−IL-7Rα+c-Kithi cells on day 2 of infection. *P ≤ 0.05 and **P ≤ 0.01 (Mann-Whitney U-test). Data are from two independent experiments with five to seven mice per group (mean ± s.e.m.). () Flow cytometry of Lin−IL-7Rα+c-Kithi eYFP+ cell–derived cells obtained from the spl! een of GFP-transfected P. chabaudi–infected hosts on day 11 of infection. Numbers above outlined areas and bracketed lines indicate percent cells in each. Data are representative of two independent experiments (n = 6 mice). * Figure 6: Generation of infection-induced Lin−IL-7Rα+c-Kithi cells in Myd88-, Trif-, Tlr9- and Ifngr1-null mutants. () Flow cytometry of infection-induced Lin−IL-7Rα+c-Kithi cells (c-Kithi) or CLPs (c-KitloSca-1lo) in Lin−IL-7Rα+ bone marrow. Numbers above and below outlined areas indicate percent cells in each. Data are representative of three independent experiments with five mice per mutant strain. () Absolute number of infection-induced Lin−L-7Rα+c-Kithi progenitors per femur in infected and control (uninfected) mice. B6, C57BL/6. NS, not significant; *P ≤ 0.01 (Mann-Whitney U-test). Data are from two to four independent experiments per mouse strain with at least five mice per time point in each (mean and s.e.m.). () Flow cytometry of Lin− bone marrow cells in uninfected and infected Ifngr1−/− mice. Numbers above outlined areas indicate percent cells in each. Data are representative of two independent experiments (n = 10 mice). * Figure 7: Critical dependence of infection-induced Lin−IL-7Rα+c-Kithi cells on IFN-γ signaling. () Expression of IFN-γR on CMPs, GMPs, megakaryocyte-erythrocyte progenitors (MEP) and infection-induced LSK subsets at day 7 after infection with P. chabaudi. () Expression of IFN-γR on LMPPs (LSK Flk-2+), CLPs (Lin−IL-7Rα+Flk-2+) and infection-induced LSK Flk-2+ or Lin−IL-7Rα+c-Kithi cells at day 11 after infection with P. chabaudi. In ,: gray-filled histograms, control staining of Ifngr1−/− populations; black lines, uninfected hematopoietic progenitors; red lines, hematopoietic subsets from mice infected with P. chabaudi. Data in , are from two independent experiments (n = 6 mice). () Flow cytometry of bone marrow chimeras reconstituted with Ifngr1−/− and B6.Rosa26eYFP (B6eYFP) cells (ratio, above plots) and infected with P. chabaudi. Numbers above and below outlined areas indicate percent cells in each. () Frequency and absolute number of infection-induced Lin−IL-7Rα+c-Kithi progenitors per femur in radiation chimeras analyzed at day 11 after infection! with P. chabaudi. Horizontal axis, ratio of Ifngr1−/− to wild-type bone marrow cells. Data in , are from two independent experiments with at least five mice per group (mean and s.e.m.). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to the work. * Nikolai N Belyaev & * Douglas E Brown Affiliations * Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, London, UK. * Nikolai N Belyaev, * Douglas E Brown, * Ana-Isabel Garcia Diaz & * Alexandre J Potocnik * Division of Parasitology, Medical Research Council National Institute for Medical Research, London, UK. * William Jarra & * Jean Langhorne * Flow Cytometry Core Facility, Medical Research Council National Institute for Medical Research, London, UK. * Aaron Rae * Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK. * Joanne Thompson Contributions N.N.B. and D.E.B. did experiments, analyzed data and contributed to the writing of the manuscript; A.-I.G.D. and W.J. did experiments and provided advice; A.R. did the cell sorting; J.T. provided reagents; and J.L. and A.J.P. designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alexandre J Potocnik (apotocn@nimr.mrc.ac.uk) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (924K) Supplementary Figures 1–3 and Tables 1–4 Additional data
• The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a β-catenin-dependent pathway
  - Nat Immunol 11(6):487-494 (2010)
  Nature Immunology | Article The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a β-catenin-dependent pathway * Pengyuan Yang1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Huazhang An1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Xingguang Liu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mingyue Wen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuanyuan Zheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yaocheng Rui1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xuetao Cao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume:11,Pages:487–494Year published:(2010)DOI:doi:10.1038/ni.1876Received04 December 2009Accepted09 April 2010Published online09 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Intracellular nucleic acid sensors detect microbial RNA and DNA and trigger the production of type I interferon. However, the cytosolic nucleic acid–sensing system remains to be fully identified. Here we show that the cytosolic nucleic acid–binding protein LRRFIP1 contributed to the production of interferon-β (IFN-β) induced by vesicular stomatitis virus (VSV) and Listeria monocytogenes in macrophages. LRRFIP1 bound exogenous nucleic acids and increased the expression of IFN-β induced by both double-stranded RNA and double-stranded DNA. LRRFIP1 interacted with β-catenin and promoted the activation of β-catenin, which increased IFN-β expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-β enhanceosome via IRF3. Therefore, LRRFIP1 and its downstream partner β-catenin constitute another coactivator pathway for IRF3-mediated production of type I interferon. View full text Figures at a glance * Figure 1: LRRFIP1 increases L. monocytogenes– and VSV–induced production of IFN-β in macrophages. () Enzyme-linked immunosorbent assay (ELISA) of IFN-β and TNF in supernatants of mouse primary peritoneal macrophages transfected for 48 h with LRRFIP1-specific or control (Ctrl) siRNA, then left uninfected (Medium) or infected for 18 h with L. monocytogenes (LM) at a multiplicity of infection (MOI) of 100. () Real-time PCR analysis of IFN-β mRNA expression in RAW264.7 cells transfected for 24 h with LRRFIP1-specific or control siRNA, then left untreated (Medium) or transfected for 4 h with L. monocytogenes genomic DNA (5 μg/ml); results are normalized to the expression of HPRT (hypoxanthine guanine phosphoribosyl transferase) and are presented relative to expression in cells transfected with control siRNA and not transfected with genomic DNA, set as 1. () ELISA of IFN-β and TNF in supernatants of mouse primary peritoneal macrophages transfected for 48 h with LRRFIP1-specific or control siRNA, then infected for 18 h with VSV at an MOI of 10. () ELISA of IFN-β in RAW264.! 7 cells with stable knockdown of LRRFIP1 and control RAW264.7 cells left uninfected or infected for 8 h with L. monocytogenes (MOI, 100). *P < 0.01 (Student's t-test). Data are from one experiment of three with similar results (mean ± s.d. of three samples per experiment). * Figure 2: LRRFIP1 increases dsRNA- and dsDNA-induced expression of IFN-β in macrophages. () Real-time PCR analysis of IFN-β mRNA expression in RAW264.7 cells transfected for 24 h with LRRFIP1-specific or control siRNA, then left untreated or transfected for 4 h with poly(dG:dC) (5.0 μg/ml), poly(I:C) (5.0 μg/ml) or poly(dA:dT) (5.0 μg/ml), presented as described in Figure 1b. *P < 0.01 (Student's t-test). Data are from one experiment of three with similar results (mean ± s.d.). (,) Immunoblot analysis (IB) of Flag-tagged full-length LRRFIP1 and mutant LRRFIP1ΔNBD precipitated (precip) from HEK293 cells with biotin-conjugated poly(dA:dT) or poly(dG:dC) in the absence () or presence () of unconjugated poly(dA:dT) competitor. SA, streptavidin. Data are representative of three experiments. () Luciferase activity of HEK293T cells transfected with 40 ng IFN-β–luciferase reporter plasmid and 10 ng renilla luciferase plasmid, together with 0 ng, 10 ng, 25 ng or 50 ng (wedges) of plasmid expressing LRRFIP1 or LRRFIP1ΔNBD (equalized with empty control vector (Ct! rl)), cultured for 24 h, then left unstimulated (left) or stimulated for 16 h transfection with poly(dA:dT) (2 μg/ml; right); results are presented relative to renilla luciferase activity. *P < 0.01 (Student's t-test). Data are from one experiment of three with similar results (mean ± s.d. of four samples). * Figure 3: LRRFIP1 mediates L. monocytogenes– and VSV-induced activation of β-catenin. () Immunoblot analysis of phosphorylated (p-) NF-κB p65, IRF3, p38, Jnk and β-catenin (β-cat) in mouse primary peritoneal macrophages infected for various times (above lanes) with L. monocytogenes at an MOI of 100. Bottom, total β-catenin (loading control). () Immunoblot analysis of nuclear (Nucl) and cytoplasmic (Cyt) proteins from RAW264.7 cells infected for various times (above lanes) with L. monocytogenes at an MOI of 100; equal amount of proteins were assessed with antibody to β-catenin (anti-β-catenin) and anti–histone H3 (nuclear) or with anti-β-catenin and anti-β-actin (cytoplasmic). () Immunoblot analysis of RAW264.7 cells infected for various times (above lanes) with L. monocytogenes at an MOI of 100; proteins immunoprecipitated (IP) from lysates with an LRRFIP1-specific antibody or immunoglobulin G (IgG) were detected with anti-β-catenin or anti-LRRFIP1. () Immunoblot analysis of phosphorylated β-catenin in mouse peritoneal macrophages infected for var! ious times (above lanes) with VSV at an MOI of 50 and treated with control or LRRFIP1-specific siRNA. Bottom, total β-catenin (loading control). Data are from one experiment of three with similar results. * Figure 4: Positive regulation by β-catenin of pathogen-induced production of IFN-β in macrophages. (,) ELISA of IFN-β in supernatants of mouse primary peritoneal macrophages, with (β-cat siRNA) or without (Ctrl siRNA) knockdown of β-catenin, either left untreated (Medium) or infected for 18 h with L. monocytogenes (MOI, 100; ) or stimulated for 12 h with LPS (100 ng/ml; ). () ELISA of IFN-β in supernatants of primary peritoneal macrophages isolated from β-catenin-deficient mice (β-cat-KO) or control mice (β-cat-loxP) and infected for 12 h with VSV (MOI, 10) left untreated (VSV) or treated with ultraviolet irradiation (UV-VSV), or left unstimulated (PBS) or stimulated for 6 h with LPS (100 ng/ml) or poly(I:C) (20 μg/ml). () ELISA of IFN-β in the serum of β-catenin-deficient or control mice injected intravenously for 6 h with VSV (1 × 106 plaque-forming units/g) with or without ultraviolet irradiation. () Survival of β-catenin-deficient and control mice (n = 10 mice total) injected intravenously with VSV (1 × 107 plaque-forming units/g). *P < 0.01 (Student's t-! test). Data are from one experiment of three with similar results (mean ± s.d. in –). * Figure 5: Interaction between β-catenin and IRF3. () Immunoblot analysis of β-catenin and IRF3 in HEK293T cells cotransfected for 24 h with plasmids for Myc-tagged β-catenin (Myc-β-cat) and hemagglutinin-tagged IRF3 (HA-IRF3); proteins immunoprecipitated from lysates with anti-Myc were detected with anti-hemagglutinin (HA). TCE, immunoblot analysis of total cell extracts. () IRF3 truncation mutants. Numbers in parentheses indicate amino acids included in construct. DBD (gray box), DNA-binding domain; white box, residues 141–201; IAD (gray box), interaction domain; black box, C terminus. () Immunoprecipitation and immunoblot analysis of proteins in HEK293T cells transiently transfected with Myc-tagged wild-type β-catenin together with hemagglutinin-tagged wild-type IRF3 (IRF3(1–472)) or the IRF3 truncation mutants in . () Immunoprecipitation and immunoblot analysis of proteins in HEK293T cells transiently transfected with Myc-tagged wild-type β-catenin together with Flag-tagged wild-type IRF3 or IRF3 truncation muta! nts. () β-catenin truncation mutants: β-catΔN lacks the first 140 residues of the N terminus; β-catΔC lacks the final 147 residues of the C terminus; β-catΔArm contains both the N-terminal 140 residues and the C-terminal 147 residues but lacks residues 141–633 in the armadillo domain. () Immunoprecipitation and immunoblot analysis of proteins in HEK293T cells transiently transfected hemagglutinin-tagged wild-type IRF3 together with Myc-tagged wild-type β-catenin or the truncation mutants of β-catenin in . Data are from one experiment of three with similar results. * Figure 6: Binding of β-catenin to the Ifnb1 promoter locus though interaction with IRF3, which promotes p300 recruitment and acetylation of histones H3 and H4 at the Ifnb1 promoter locus. (,) ChIP analysis of the recruitment of β-catenin to the Ifnb1 promoter in RAW264.7 cells left unstimulated or stimulated for 1 h with LPS (100 ng/ml) or poly(I:C) (20 μg/ml); Ifnb1 promoter sequences in input DNA and DNA recovered from antibody-bound chromatin segments were detected by semiquantitive PCR () or real-time PCR (). () ChIP analysis of the recruitment of β-catenin to the Ifnb1 promoter in RAW264.7 cells transfected with control or IRF3-specific siRNA and stimulated as in ,; Ifnb1 promoter sequences were detected by real-time PCR. (,) ChIP analysis of the recruitment of p300 to the Ifnb1 promoter and acetylation of histones H3 (H3Ac) and H4 (H4Ac) at the Ifnb1 promoter in β-catenin-deficient (β-cat-KO) and control (β-cat-loxP) mouse primary peritoneal macrophages stimulated as in ,; Ifnb1 promoter sequences were detected by semiquantitive PCR () or real-time PCR (). () Association of p300 and IRF3 in β-catenin-deficient or control mouse primary peritoneal ! macrophages stimulated for 1 h with poly(I:C) (20 μg/ml); p300 was immunoprecipitated from nuclear extracts and associated IRF3 was detected by immunoblot. Results in were normalized to the corresponding input controls and are presented relative to results obtained with the PBS-treated control. *P < 0.01 (Student's t-test). Data are representative of three independent experiments (–; mean and s.e.m. in ,,) or are from one experiment of three with similar results (). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Pengyuan Yang & * Huazhang An Affiliations * National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China. * Pengyuan Yang, * Huazhang An, * Xingguang Liu, * Mingyue Wen, * Yuanyuan Zheng, * Yaocheng Rui & * Xuetao Cao Contributions P.Y., H.A., X.L., M.W., Y.Z. and Y.R. did the experiments; and X.C. and H.A. designed the study and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Xuetao Cao (caoxt@immunol.org) or * Huazhang An (anhz@immunol.org) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (508K) Supplementary Figures 1–11 and Supplementary Table 1 Additional data
• Synaptotagmin-mediated vesicle fusion regulates cell migration
  - Nat Immunol 11(6):495-502 (2010)
  Nature Immunology | Article Synaptotagmin-mediated vesicle fusion regulates cell migration * Richard A Colvin1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Terry K Means1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas J Diefenbach2 Search for this author in: * NPG journals * PubMed * Google Scholar * Luis F Moita1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert P Friday1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sanja Sever4 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriele S V Campanella1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tabitha Abrazinski1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lindsay A Manice1 Search for this author in: * NPG journals * PubMed * Google Scholar * Catarina Moita1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Norma W Andrews5 Search for this author in: * NPG journals * PubMed * Google Scholar * Dianqing Wu6 Search for this author in: * NPG journals * PubMed * Google Scholar * Nir Hacohen1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew D Luster1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:495–502Year published:(2010)DOI:doi:10.1038/ni.1878Received02 December 2009Accepted12 April 2010Published online16 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release. View full text Figures at a glance * Figure 1: Screen of chemotaxis by shRNA identifies synaptotagmins SYT2, SYTL5 and SYT7. () Primary chemotaxis screen showing the chemotactic indices of SupT1 cells infected with 1,500 arrayed lentiviruses encoding shRNA targeting 300 genes. Horizontal axis, individual samples; vertical axis, natural logarithm (Ln) of the chemotactic index; boxes outline results for cells infected with viruses targeting genes selected as putative positive (green) or negative (red) regulators of chemotaxis. () Transwell chemotaxis of SupT1 cells infected with viruses containing shRNA targeting genes encoding eGFP (sheGFP), SYTL5 (shSYTL5), SYT2 (shSYT2) or SYT7 (shSYT7). () Transwell chemotaxis of THP-1 cells infected with viruses targeting genes encoding eGFP, SYTL5 or SYT2. () Quantitative PCR analysis of SYT2 mRNA abundance in SupT1 cells infected with viruses targeting genes encoding eGFP or SYT2, presented relative to the abundance of GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase). () Immmunoblot analysis of extracts from SupT1 cells infected with lentiviruses ! targeting genes encoding eGFP or SYT2, probed with antibody to SYT2 (anti-SYT2; top) or anti-β-actin (bottom). () Quantification of SYT2 protein. () Transwell chemotaxis of wild-type (WT) and Syt7−/− primary splenocytes. P values, two-way analysis of variance (ANOVA). Data are representative of an experiment done once () or experiments done three times (,,; mean and s.e.m. of two samples), at least four times in duplicate (; error bars, s.e.m.) or twice (,). * Figure 2: Knockdown of RAB27A and RAB3A inhibits T cell chemotaxis. () Transwell chemotaxis of SupT1 cells infected with viruses targeting genes encoding eGFP, Rab27a or Rab3a. () Quantitative PCR analysis of RAB27A mRNA abundance in SupT1 cells infected with viruses targeting genes encoding eGFP or Rab27a, presented relative to GAPDH mRNA abundance. () Immunoblot analysis of extracts of SupT1 cells infected with lentiviruses targeting genes encoding eGFP or Rab27a, probed with anti-Rab27a (top) or anti-β-actin (bottom). () Quantification of Rab27a protein, presented as the ratio of the Rab27a to β-actin in lysates of cells infected with lentivirus targeting RAB27A, normalized to the ratio in lysates of cells infected with lentivirus targeting the gene encoding eGFP. P values, two-way ANOVA () or Student's t-test (). Data are representative of at least three similar experiments (; mean and s.e.m. of two samples) or experiments done at least four times (; error bar, s.e.m.) or twice (,). * Figure 3: Migration of wild-type and SYT7-deficient neutrophils ex vivo. (,) Tracks of neutrophil migration, derived from stacks of images taken every 15 s for 30 min during migration in a Zigmond chamber in a gradient of fMLP. Data are representative of four similar experiments. () Migration velocity of wild-type and Syt7−/− BMDNs. Each symbol represents an individual cell; small horizontal lines indicate the mean. Data are compiled from four separate experiments. (,) Migration vectors, presented as Rose plots of data obtained in ,. P = 0.00002 () or 0.7 (; Rayleigh test for both). Data are representative of four similar experiments. () Frequency of polarized cells 25 min after exposure to an fMLP gradient (n = 100 cells examined at 25 min and categorized as polarized or unpolarized). P values, Student's t-test. Data are compiled from four similar experiments (error bars, s.e.m.). * Figure 4: Migration of wild-type and SYT7-deficient neutrophils in vivo in a model of gout. () Flow cytometry of cells recruited into the air pouch of a wild-type mouse and a Syt7−/− mouse injected with MSU crystals; cells were stained with anti-Ly6G and anti-CD11b. Numbers in quadrants indicate percent cells in each. () Total cells, neutrophils (PMN; CD11b+Ly6C+) and monocytes (Mono; CD11b+Ly6C−) recruited into the air pouches of wild-type mice (n = 11) and Syt7−/− mice (n = 11) injected with MSU crystals or wild-type mice (n = 6) and Syt7−/− mice (n =6) injected with PBS (control). P values, Student's t-test. () Enzyme-linked immunosorbent assay of IL-1β and CXCL2 (MIP-2) in supernatants of wild-type and Syt7−/− thioglycolate-recruited peritoneal macrophages primed with lipopolysaccharide, then stimulated with PBS or MSU crystals. No significant difference, wild-type versus Syt7−/− (Student's t-test). Data are representative of three (,) or two () experiments (error bars, s.e.m.). * Figure 5: CXCR4 cell surface expression and chemokine-induced F-actin polymerization and calcium release. () Cell surface expression of CXCR4 on shRNA-expressing SupT1 cells (key) stimulated with CXCL12 (concentration, horizontal axis). Differences not significant (Student's t-test). () Cell surface expression of CXCR4 on wild-type and Syt7−/− splenocytes stimulated with CXCL12 (concentration, horizontal axis), analyzed by flow cytometry. Differences not significant (Student's t-test). () F-actin polymerization in shRNA-expressing SupT1 cells (key) stimulated for various time (horizontal axis) with CXCL12, analyzed by flow cytometry. *P < 0.02, versus cells expressing shRNA targeting the gene encoding eGFP at the same time point (Student's t-test). () Flow cytometry of F-actin content in wild-type splenocytes (n = 3 mice) and Syt7−/− splenocytes (n = 3 mice) after stimulation with CXCL12 for various times (horizontal axis). () Calcium flux in SupT1 cells infected with shRNA-expressing lentivirus (right margin), loaded with the fluorescent calcium indicator Fluo-8 and act! ivated with 10 nM CXCL12, presented as emission at 520 nM after activation at 480 nM; curves were displaced on the vertical axis for clarity. Baselines of original curves were similar (data not shown). () GTP-bound RhoA, Cdc42, and Rac in cytoplasmic extracts of wild-type and Syt7−/− splenocytes stimulated for various times (horizontal axis) with 10 nM CXCL12. No significant difference, wild type versus Syt7−/− (Student's t-test). Data represent one of three similar experiments (,; average and s.e.m. of two samples), at least three experiments (; average and s.e.m. of two or three replicates of one to four per gene), an experiment done three times (; error bars, s.e.m.) or experiments done in triplicate and repeated three times (), or are representative of three experiments () or an experiment done in triplicate two times (; error bars, s.e.m.). * Figure 6: Effect of calcium on chemotaxis, F-actin polymerization, and chemokine-induced expression of LAMP-1. () Transwell chemotaxis of SupT1 cells toward CXCL12 with and without (0) treatment with 10 μM BAPTA. P value, two-way ANOVA. Data are representative of three similar experiments (mean and s.e.m. of three samples). () F-actin polymerization with and without treatment with 10 μM BAPTA. Results not significant (Student's t-test). Data are from one of three similar experiments (average and s.e.m. of two samples). () Flow cytometry analysis of the cell surface expression of LAMP-1 on wild-type BMDNs the presence (right) or absence (left) of C5a stimulation (10 nM), with or without BAPTA treatment (left margin). Numbers in outlined areas indicate percent LAMP-1+ cells. Data are representative of three experiments done in triplicate. () Flow cytometry analysis of the cell surface expression of LAMP-1 on wild-type and Syt7−/− BMDNs at baseline (without chemoattractant) and after C5a stimulation (+ C5a). Data are representative of three experiments done in triplicate. () Laser! -scanning confocal microscopy of the localization of LAMP-2 and cathepsin-L (CatL) in the podocyte cell membrane before (0 CK; top row) and after (bottom row) stimulation with 10 nM CXCL11. Left, staining with anti-LAMP-2; middle, staining with anti-cathepsin-L; right, merged images of LAMP-2 (red), cathepsin-L (green) and overlay (yellow). Original magnification, ×40. Results are representative of three experiments. * Figure 7: Localization of LysoTracker Red–stained vesicles during cell migration. () Confocal microscopy of LysoTracker Red localization during the migration of activated CD4+ lymphocytes from wild-type mice and Syt7−/− mice in a gradient (wedge) of CXCL10 (Dunn chemotaxis chamber). Original magnification, ×20. Results are representative of four experiments. () Cell body and uropod surface area of wild-type and SYT7-deficient activated CD4+ T cells migrating in a CXCL10 gradient. *P = 0.01 (Student's t-test). Data are representative of four experiments (n = 20 cells per group; error bars, s.e.m.). () LysoTracker Red localization in wild-type and Syt7−/− activated CD4+ T cells migrating in a CXCL10 gradient. *P = 0.000001 (Student's t-test). Data are representative of four experiments (n = 20 cells per group; error bars, s.e.m.). () LysoTracker Red fluorescence intensity in polarized wild-type and Syt7−/− cells (uropod/cell body). *P = 0.00025 (Student's t-test). Data are representative of four experiments (error bars, s.e.m.). () Laser-scanni! ng confocal microscopy of polarized, activated wild-type and Syt7−/− CD4+ lymphocytes (time series). Outlines (far left) indicate initial locations of polarized cells followed over time in the subsequent images. Original magnification, ×20. Results are representative of four experiments. () Adherence of polarized wild-type cells (n = 114) and Syt7−/− cells (n = 108) over a 30-minute period. *P = 8.3 × 10−30 (χ2 test). Data are representative of four experiments (error bars, s.e.m.). () Duration of the adhesion of polarized adherent wild-type cells (n = 6) and Syt7−/− cells (n = 88). *P = 0.00000045 (Student's t-test). Data are representative of four experiments (error bars, s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital (MGH) and Harvard Medical School, Boston, Massachusetts, USA. * Richard A Colvin, * Terry K Means, * Luis F Moita, * Robert P Friday, * Gabriele S V Campanella, * Tabitha Abrazinski, * Lindsay A Manice, * Catarina Moita, * Nir Hacohen & * Andrew D Luster * Ragon Institute of MGH, Massachusetts Institute of Technology (MIT) and Harvard, Boston, Massachusetts, USA. * Thomas J Diefenbach * Cell Biology of the Immune System Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. * Luis F Moita & * Catarina Moita * Division of Nephrology, Massachusetts General Hospital, Boston, Massachusetts, USA. * Sanja Sever * Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA. * Norma W Andrews * Yale University School of Medicine, Yale University, New Haven, Connecticut, USA. * Dianqing Wu * Broad Institute, Cambridge, Massachusetts, USA. * Nir Hacohen * Present address: Novartis Institute for Biomedical Research, Cambridge, Massachusetts, USA. * Richard A Colvin Contributions R.A.C. designed the experiments, did the screening, collected and analyzed data and wrote the manuscript; T.K.M. did air-pouch experiments, macrophage stimulation and GTPase assays and helped edit the manuscript; T.J.D. did confocal microscopy and analyzed lymphocytes; L.F.M. and C.M. helped with initial screens; R.P.F. assisted with air-pouch experiments; S.S. did confocal microscopy of podocytes; G.S.V.C. helped with experiments; T.A. and L.A.M. collected data; N.W.A. provided mouse strains; D.W. assisted with video migration assays of neutrophils; N.H. helped design experiments, analyzed data and helped edit the manuscript; A.D.L. helped design experiments, analyzed data and edited the manuscript; and all authors discussed results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Andrew D Luster (aluster@mgh.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (300K) Bone marrow derived neutrophils from wild-type C57BL/6 mice migrating in response to fMLP. * Supplementary Video 2 (150K) Bone marrow derived neutrophils from Syt7−/− mice migrating in response to fMLP. * Supplementary Video 3 (4M) CD4+ lymphocytes obtained from wild-type C57BL/6 mice, loaded with lysotracker red, migrating in response to CXCL10. * Supplementary Video 4 (1M) CD4+ lymphocytes obtained from wild-type C57BL/6 mice, loaded with lysotracker red, migrating in response to CXCL10. * Supplementary Video 5 (7M) CD4+ lymphocytes obtained from Syt7−/− mice, loaded with lysotracker red, migrating in response to CXCL10. * Supplementary Video 6 (640K) CD4+ lymphocytes obtained from Syt7−/− mice, loaded with lysotracker red, migrating in response to CXCL10. PDF files * Supplementary Text and Figures (840K) Supplementary Figures 1–4, Supplementary Tables 1–2 and Supplementary Methods Additional data
• Regulation of thymocyte positive selection and motility by GIT2
  Phee H Dzhagalov I Mollenauer M Wang Y Irvine DJ Robey E Weiss A - Nat Immunol 11(6):503-511 (2010)
  Nature Immunology | Article Regulation of thymocyte positive selection and motility by GIT2 * Hyewon Phee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Dzhagalov2 Search for this author in: * NPG journals * PubMed * Google Scholar * Marianne Mollenauer1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yana Wang4 Search for this author in: * NPG journals * PubMed * Google Scholar * Darrell J Irvine5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Ellen Robey2 Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur Weiss1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:503–511Year published:(2010)DOI:doi:10.1038/ni.1868Received01 February 2010Accepted22 March 2010Published online02 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein–coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2−/− double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro. By two-photon laser-scanning microscopy, we found that the scanning activity of Git2−/− thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes. View full text Figures at a glance * Figure 1: Impaired generation of CD4SP thymocytes in DO11.10+Git2−/− mice. () Flow cytometry analysis of the expression of CD4 and CD8 on thymocytes from DO11.10+ wild-type (Git2+/+) mice and DO11.10+Git2−/− mice to assess stages of thymocyte development. Numbers in quadrants indicate percent cells in each. () Cell numbers of thymic subsets from DO11.10+ wild-type and DO11.10+Git2−/− mice (n = 10–11 mice per genotype). () Expression of the TCR transgene in total thymocytes from DO11.10+ wild-type or DO11.10+Git2−/− mice, assessed with the transgene-specific antibody KJ1-26. Numbers above bracketed lines indicate percent KJ1-26+ cells. () Frequency of KJ1-26hiCD69hi cells among DP thymocytes (left) and number of KJ1-26hiCD69hi DP cells (right) from DO11.10+ wild-type or DO11.10+Git2−/− mice (n = 3 mice per genotype). () Expression of KJ1-26 in CD4+ T cells from DO11.10+ wild-type spleen (n = 3) or DO11.10+Git2−/− spleen (n = 5). Numbers above bracketed lines indicate percent KJ1-26+ cells. () Frequency of CD4+KJ1-26+ cells in t! he periphery of DO11.10+ wild-type mice (n = 3) and DO11.10+Git2−/− mice (n = 5). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t-test). Data are representative of nine (–) or three (–) experiments (error bars (,,), s.e.m.). * Figure 2: Fewer CD4SP thymocytes in TCR-transgenic Git2−/− mice because of impaired positive selection, not more apoptosis or negative selection. () Intracellular staining of BrdU+ cells after 48 h of BrdU incorporation by DP or TCR-transgenic CD4SP thymocytes from TCR-transgenic wild-type or Git2−/− mice, presented as a percentage of total live thymocytes. Vα2+ indicates cells positive for the OT-II transgene; KJ1-26+ indicates cells positive for the DO11.10 transgene. *0.01 < P < 0.05; **P < 0.001 (unpaired two-tailed Student's t-test). Data are pooled from two independent experiments (OT-II+ mice: wild-type, n = 8; Git2−/−, n = 6) or are from a single experiment (DO11.10+ mice: n = 3 per genotype; error bars, s.e.m.). () Apoptosis of DO11.10+ wild-type or DO11.10+Git2−/− thymocytes, analyzed by staining with 7-amino-actinomycin D and annexin V at 0, 4 and 24 h of incubation in medium containing 10% FBS. Data are representative of two independent experiments with two to three mice per genotype each (error bars, s.e.m.). () Intracellular staining of active caspase-3 in DO11.10+ wild-type and DO11.10+Git2! −/− thymocytes. Data are pooled from two independent experiments with five mice per genotype (error bars, s.e.m.). () Frequency of Vβ3+, Vβ5+ and Vβ11+ cells among CD4SP thymocytes or CD4+ T cells from lymph nodes from wild-type and Git2−/− mice on a BALB/c background (n = 3 per genotype). Data are representative of three experiments (error bars, s.e.m.). * Figure 3: The defect in the positive selection of TCR-transgenic Git2−/− thymocytes is intrinsic to hematopoietic cells. () Flow cytometry analysis of the generation of CD4SP thymocytes by wild-type BALB/c hosts reconstituted with DO11.10+ wild-type donor bone marrow (DO11.10+Git2+/+→Git2+/+) or DO11.10+Git2−/− donor bone marrow (DO11.10+Git2−/−→Git2+/+). Data are representative of three independent experiments with five chimeras. () Flow cytometry analysis of the generation of CD4SP thymocytes in wild-type and Git2−/− hosts reconstituted with DO11.10+ wild-type bone marrow. Data are representative of a single experiment with five chimeras per condition. In ,, numbers in outlined areas indicate percent cells in each; numbers above bracketed lines indicate percent KJ1-26+ cells. () Thymic architecture of chimeras reconstituted with DO11.10+ wild-type or DO11.10+Git2−/− bone marrow cells. Original magnification, ×4. () DP-to-CD4SP transition of thymocytes generated from OT-II+ wild-type or OT-II+Git2−/− bone marrow hematopoietic stem cells in a mixed–bone marrow chimer! a reconstituted with a 1:1 mixture of bone marrow from two donor groups with different congenic markers (OT-II+ wild type (CD45.1+CD45.2+) and OT-II+Git2−/− CD45.2+)). NS, not significant; *0.01 < P < 0.05; **P < 0.001 (unpaired two-tailed Student's t-test); P values below graph compare wild-type with Git2−/−. Data are pooled from seven independent experiments (mean and s.e.m.). * Figure 4: Greater migratory activity of Git2−/− DP thymocytes in response to SDF-1 or CCL25. () Transwell migration of wild-type and Git2−/− thymocytes in the presence or absence (Media) of SDF-1 or CCL25, quantified by flow cytometry after staining with anti-CD4 and anti-CD8 and presented as the percentage of input cells that migrated to the lower chamber. Data are from five independent experiments (means and s.e.m. of duplicate assays). () Flow cytometry analysis of the expression of CD69 and TCRβ (top) or CD69 (bottom) in DP populations of input or migrated wild-type or Git2−/− thymocytes in the presence of CCL25 or SDF-1; thymocytes were stained with anti-CD4, anti-CD8, anti-CD69 and anti-TCRβ. Numbers in quadrants (top) indicate percent TCRβ−CD69− cells (left) or TCRβ+CD69+ cells (right); numbers above bracketed lines (bottom) indicate percent cells. Data are representative of five experiments. () Migration of preselection CD69loTCRβlo (top) and post-selection CD69hiTCRβhi DP thymocytes (bottom). Data are from three independent experiments (er! ror bars, s.e.m. of duplicate assays). *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t-test). * Figure 5: Greater directional and random motility of Git2−/− thymocytes. () Transwell migration of wild-type and Git2−/− thymocytes from the upper well in the absence of SDF-1 (Media), or with SDF-1 in the top well (Top), the bottom well (Bottom) or both wells (Both). Data are representative of two independent experiments (error bars, s.e.m.). () Directional migration of wild-type and Git2−/− thymocytes on two-dimensional surfaces, monitored by time-lapse fluorescence microscopy with alginate beads; thymocytes were labeled with different fluorescent dyes, a 1:1 mixture of thymocytes in a solution of collagen (1.5 mg/ml) was placed on chambered coverglass coated with ICAM-1 (1 μg/ml) in the presence of unloaded control beads (left) or SDF-1-loaded beads (right) and cells were imaged for 40 min to 1 h at intervals of 30 s. Each symbol represents the track of one thymocyte; small horizontal lines indicate the mean. *P = 0.03 (unpaired two-tailed Student's t-test). Data are representative of two (control beads) or three (SDF-1-releasing bead! s) independent experiments (mean and s.e.m. in plots). () Average migration speed of wild-type and Git2−/− thymocytes in the presence of control beads or SDF-1-loaded beads as in , binned into intervals of 1 μm/min; the frequency of total cells migrating in each range is normalized as a percentage of the maximum (% of max). Data are representative of two (control beads) or three (SDF-1-releasing beads) independent experiments. () Directionality index of wild-type and Git2−/− thymocytes that migrated more than 20 μm toward SDF-1-releasing beads (left) and average migration speeds of wild-type and Git2−/− thymocytes that moved toward SDF-1-releasing beads by more than 20 μm, binned into intervals of 1 μm/min and presented as in (right; cells that did not move toward SDF-1-releasing beads by more than 20 μm are excluded). Each symbol (left) represents one thymocyte; small horizontal lines indicate the mean. NS, unpaired two-tailed Student's t-test. Data are re! presentative of three independent experiments. * Figure 6: More Rac1 activation and actin polymerization in Git2−/− thymocytes in response to SDF-1. () Immunoblot analysis (IB) of Rac1 activation in wild-type or Git2−/− thymocytes after 0, 0.5, 2, 5 and 15 min of SDF-1 stimulation (left), and increase in Rac1 activation normalized to GTP-Rac1 in the resting state (right). Precip, precipitation (with PAK1-PBD agarose); WCL, whole-cell lysates (control). () F-actin polymerization in Git2−/− DP thymocytes (left) and CD4SP thymocytes (right) in response to SDF-1, assessed by staining with phalloidin–Alexa Fluor 488, anti-CD4 and anti-CD8; the mean fluorescence intensity of phalloidin–Alexa Fluor 488 staining is normalized to that of wild-type cells at rest. Data are from three independent experiments per condition (error bars, s.e.m.). * Figure 7: More Git2−/− DP thymocytes overcome TCR-mediated stop signals in the presence of SDF-1. () Transwell migration of wild-type and Git2−/− DP thymocytes across filters coated with BSA or ICAM-1 (3 μg/ml), in response to media or SDF-1 in the bottom well; migration was quantified by flow cytometry after staining with anti-CD4 and anti-CD8 and is presented as migrating cell number/input cell number. () Transwell migration of wild-type and Git2−/− DP thymocytes across filters coated with ICAM-1 (3 μg/ml) plus either of two concentrations of anti-CD3 (5 μg/ml or 20 μg/ml), in response to media or SDF-1 in the bottom well. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (unpaired two-tailed Student's t-test). Data are from two independent experiments (error bars, s.e.m. of duplicate assays). * Figure 8: Altered motility of Git2−/− thymocytes in the cortex. () Wild-type (blue; CFP) and Git2−/− (green; GFP) thymocytes from the thymus of a partial mixed–bone marrow chimera at a single time point, superimposed on cell tracks (top); red, blood vessels. Below, trajectories of individual cells, presented as tracks: yellow, CFP+ wild-type thymocytes; purple, GFP+Git2−/− thymocytes. Scale bars, 20 μm. Data are representative of one experiment (wild type) or three experiments (Git2−/−). () Average migration speed of wild-type and Git2−/− thymocytes. Each symbol represents an individual tracked thymocyte; green horizontal lines indicate the mean (small black horizontal lines, s.e.m.). *P < 0.0001 (unpaired two-tailed Student's t-test). Data are compiled from one experiment (wild type) or three experiments on 2 different days (Git2−/−). () Directionality index of wild-type and Git2−/− thymocytes, plotted as a function of average speed (0–10 μm/min), binned as intervals of 2–3 μm/min, 3–4 μm/min or 4–5! μm/min (gray vertical lines); each symbol represents an individual tracked thymocyte; numbers above plots indicate average directionality index of each bin. *P = 0.0004 for 2–3 μm/min; **P < 0.0001 for 3–4 μm/min and for 4–5 μm/min (unpaired two-tailed Student's t-test). Data are compiled from one experiment (wild type) or three experiments on 2 different days (Git2−/−). () Fluorescence microcopy of the migration of Git2−/− thymocytes near blood vessels, presented as a projection of single time point with cell tracks superimposed. Scale bar, 10 μm. Data are representative of three experiments. () Immunofluorescence of thymic sections from wild-type mice injected with lectin–fluorescein isothiocyanate (to label blood vessels) and stained with anti-SDF-1, showing SDF-1+ blood vessels. Original magnification, ×20 (top) or ×40 (bottom). Data are representative of two experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Medicine, Division of Rheumatology, Rosalind Russell Medical Research Center for Arthritis, University of California San Francisco, San Francisco, California, USA. * Hyewon Phee, * Marianne Mollenauer & * Arthur Weiss * Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California Berkeley, Berkeley, California, USA. * Ivan Dzhagalov & * Ellen Robey * Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, USA. * Marianne Mollenauer & * Arthur Weiss * Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Yana Wang * Department of Biological Engineering and Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Darrell J Irvine * Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. * Darrell J Irvine Contributions H.P. designed the study, did experiments, analyzed data and wrote the manuscript; M.M. did experiments; I.D. did the two-photon experiments and analyzed data; Y.W. and D.J.I. supplied alginate beads; E.R. supervised the two-photon experiments and discussed data; and A.W. designed the study, supervised the research and revised the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Arthur Weiss (aweiss@medicine.ucsf.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (3M) Directional migration of wild-type and Git2−/− thymocytes to SDF-1. * Supplementary Video 2 (948K) Migration of wild-type and Git2−/− thymocytes in intact thymic lobes using two-photon microscopy. * Supplementary Video 3 (1M) Accumulation and movement of Git2−/− thymocyte on small blood vessels. * Supplementary Video 4 (288K) Migratory behavior of wild-type thymocytes that move away from small blood vessels. PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–8, Table 1 and Methods Additional data
• Autonomous role of medullary thymic epithelial cells in central CD4+ T cell tolerance
  Hinterberger M Aichinger M da Costa OP Voehringer D Hoffmann R Klein L - Nat Immunol 11(6):512-519 (2010)
  Nature Immunology | Article Autonomous role of medullary thymic epithelial cells in central CD4+ T cell tolerance * Maria Hinterberger1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Aichinger1 Search for this author in: * NPG journals * PubMed * Google Scholar * Olivia Prazeres da Costa2 Search for this author in: * NPG journals * PubMed * Google Scholar * David Voehringer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Reinhard Hoffmann2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ludger Klein1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:512–519Year published:(2010)DOI:doi:10.1038/ni.1874Received16 February 2010Accepted30 March 2010Published online02 May 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Medullary thymic epithelial cells (mTECs) serve an essential function in central tolerance by expressing peripheral-tissue antigens. These antigens may be transferred to and presented by dendritic cells (DCs). Therefore, it is unclear whether mTECs, in addition to being an antigen reservoir, also serve a mandatory function as antigen-presenting cells. Here we diminished major histocompatibility complex (MHC) class II on mTECs through transgenic expression of a 'designer' microRNA specific for the MHC class II transactivator CIITA (called 'C2TA' here). This resulted in an enlarged polyclonal CD4+ single-positive compartment and, among thymocytes specific for model antigens expressed in mTECs, enhanced selection of regulatory T cells (Treg cells) at the expense of deletion. Our data document an autonomous contribution of mTECs to both dominant and recessive mechanisms of CD4+ T cell tolerance and support an avidity model of Treg cell development versus deletion. View full text Figures at a glance * Figure 1: Lower MHC class II expression through Ciita silencing in vitro. () The miR-30 backbone in the retroviral vector LMP modified to encode synthetic hairpin sequences complementary to the coding region of C2TA mRNA. Below, sequence details of one such construct, C2TAsh(6); underlining indicates sequences of the predicted mature 'designer' miRNA. LTR, long terminal repeat; ψψ, packaging signal sequence; Pgk1, phosphoglycerate kinase; Puro, sequence encoding puromcyin resistance; IRES, internal ribosomal entry site. () Alignment of the C2TAsh(6) leading strand (antisense) with the three C2TA mRNA species arising through initiation of transcription from three alternative promoters (pI, pIII and pIV). UTR, untranslated region; ORF, open reading frame. () Expression of MHC class II (MHCII) in WEHI 279.1 cells infected with LMP containing C2TA-specific hairpin sequences or empty vector control; GFP+ cells were stained with antibody to MHC class II or an isotype-matched control antibody. () Immunoblot analysis of protein extracts from GFP+ WEHI 2! 79.1 cells infected with LMP-C2TAsh(6) or empty vector. β-actin (below) serves as a loading control. Data are representative of at least three independent experiments. * Figure 2: Transgenic expression of the C2TA-specific synthetic miRNA in mTECs. () The C2TAkd BAC transgene. The modified miR-30 backbone from the vector LMP-C2TAsh(6) was cloned into a BAC transgenic construct containing 152 kilobases (kb) of the 5′ region and 58 kilobases of the 3′ region flanking Aire. In this construct, the Aire start codon in exon 1 was replaced by a β-globin (βg) intron followed by the miR-30-embedded C2TA-specific sequences. pA, poly(A). () Small RNA TaqMan assay of the abundance of the processed synthetic miRNA in purified thymic stromal cells, presented in arbitrary units (AU) relative to the expression of small nucleolar RNA (snoRNA202). Values for mTEClo cells ('immature' CD80lo mTECs) were at the lower border of the linear detection range, whereas those for cTECs and DCs were below that threshold and are therefore classified as undetectable (UD). mTEChi, mature CD80hi mTECs; DC, CD11c+ DC. () Quantitative RT-PCR analysis of the abundance of Aire mRNA in the mTEC subsets defined as in ; results are presented relative to! the expression of Actb (encoding β-actin). Data are representative of three experiments (mean ± s.d.). * Figure 3: Silencing of Ciita and MHC class II genes in C2TAkd mTECs. () Quantitative RT-PCR analysis of the abundance of Ciita mRNA (left) and a representative C2TA target (the α-chain of H-2A (H2-Aa); right) in wild-type (WT) and C2TAkd mTEC subsets (defined as in Fig. 2b); results are presented relative to Actb expression. Data are representative of one experiment (mean ± s.d. of three biological replicates). () Microarray expression analysis of genes encoding MHC class II chains (H2-Aa, H2-Ab1, H2-Ea and H2-Eb1) and MHC class II–associated C2TA target genes (H2-Oa, H2-Ob, H2-DMa, H2-DMb2 and invariant chain (Cd74)) in mature (CD80hi) mTECs from wild-type, C2TAkd and Ciita−/− mice. P < 0.00001, wild-type versus C2TAkd (except H2-Eb: P = 0.003; two-tailed Student's t-test with unequal variance). () Microarray expression analysis of MHC class II–unrelated putative C2TA target genes in mature (CD80hi) mTECs from wild-type, C2TAkd and Ciita−/− mice. P > 0.05 (two-tailed Student's t-test with unequal variance). Data are representat! ive of three experiments (mean ± s.d.). * Figure 4: Phenotype, APC-function and promiscuous PTA expression of C2TAkd mTECs. () Expression of MHC class II on thymic stromal cell subsets of C2TAkd, wild-type and Ciita−/− mice, assessed by flow cytometry. cDC, conventional DCs defined by a CD11chiCD45RA− surface phenotype and subcategorized as intrathymically derived ('autochthonous'; Sirpα−) or migratory, peripherally derived (Sirpα+); pDC, plasmacytoid DCs defined by the phenotype CD11cintCD45RA+. () Immunoblot analysis of MHC class II in mTECs from C2TAkd mice. Below lanes, densitometry; ND, not defined (H-2Ea in mTEClo cells, below the threshold of sensitivity). () Flow cytometry analysis (dose-response curve) of the frequency of GFP+ cells among HA-specific GFP reporter hybridoma cells (A5 cells) cultured together for 17 h with mTECs from wild-type or C2TAkd mice in the presence of titrated amounts of HA peptide (amino acids 107–119: HA(107–119)). () Frequency of GFP+ cells among total (CD45−EpCAM+Ly51−) mTECs from Aire-GFP reporter (Adig) mice in the presence (C2TAkd; n = 11 ! mice) or absence (wild-type; n = 25 mice) of the C2TAkd transgene. Numbers above bracketed lines indicate percent GFP+ cells (the most mature Aire+ mTECs; average ± s.d.). () Distribution of Aire+ cells in medullary regions of wild-type or C2TAkd thymi, assessed in cryosections stained for medullary areas (Keratin 5; green) and Aire (red). C, cortical areas; M, medullary areas; dashed line, cortico-medullary junction. Scale bar, 100 μm. () Microarray expression analysis of genes encoding Aire-dependent and Aire-independent PTAs in mature (CD80hi) mTECs from wild-type and C2TAkd mice. P > 0.05 (two-tailed Student's t-test with unequal variance). Data representative of at least two independent experiments (,), two independent experiments (), three experiments (), three experiments with at least three organs per genotype () or three experiments (; mean ± s.d.). * Figure 5: Nonredundant contribution of mTECs and hematopoietic APCs to the deletional tolerance of CD4+ T cells. () Thymic profiles of 5- to 6-month-old C2TAkd mice (n = 9) and wild-type mice (n = 10). Numbers in plots indicate percent CD4SP cells (average ± s.d.). Total thymic cellularity was not different between the two genotypes (data not shown). P = 0.001 (two-tailed Student's t-test with unequal variance). () Frequency of CD4SP, CD8+ single-positive (CD8SP), double-positive (DP) and double-negative (DN) thymocyte subsets in C2TAkd and wild-type mice. () Thymic profiles of wild-type and C2TAkd recipients after reconstitution with wild-type or MHC class II–deficient (MHCII-KO) bone marrow: WT→WT, n = 9; WT→C2TAkd, n = 10; MHCII-KO→WT, n = 7; MHCII-KO→C2TAkd, n = 9. Numbers in plots indicate percent CD4SP cells (average ± s.e.m.). () Summary of the data in . *P < 0.01 and **P < 0.001 (two-tailed Student's t-test with unequal variance). Data are representative of two (,) or four (,) experiments (error bars, s.d.). * Figure 6: Rescue from clonal deletion and enhanced generation of OVA-specific Treg cells in DO11.10 × Aire-OVA × C2TAkd thymi. () Quantitative RT-PCR analysis of expression of the Aire-OVA transgene in the presence (C2TAkd) or absence (WT) of the C2TAkd transgene in purified stromal cells (subsets defined in Fig. 2b); results are presented relative to Actb expression. Data are from one experiment (mean ± s.d. of three biological replicates). () Thymocyte subsets in DO11.10 and DO11.10 × Aire-OVA mice in the presence (C2TAkd) or absence (WT) of mTEC-specific silencing of Ciita. Numbers above outlined areas (top) indicate percent CD4SP cells; numbers above bracketed lines (bottom) indicate frequency of DO11.10+ cells among gated CD4SP thymocytes (average ± s.d.). Data are representative of eight experiments. () Frequency of DO11.10+ cells among gated CD4SP thymocytes in (summary). Each symbol represents an individual mouse; small horizontal lines indicate the mean (DO11.10, n = 10 mice; DO11.10 × Aire-OVA, n = 11 mice; DO11.10 × Aire-OVA × C2TAkd, n = 14 mice). *P < 0.001 (two-tailed Student's t! -test with unequal variance). Data are representative of eight experiments. () Foxp3 expression (intracellular staining) in CD4SP cells from DO11.10, DO11.10 × Aire-OVA and DO11.10 × Aire-OVA × C2TAkd mice. Numbers adjacent to outlined areas indicate frequency of DO11.10+Foxp3+ cells among CD4SP thymocytes (average ± s.d.). Data are representative of eight experiments. () Total DO11.10+Foxp3+ CD4SP thymocytes in DO11.10, DO11.10 × Aire-OVA and DO11.10 × Aire-OVA × C2TAkd mice. *P < 0.001 (two-tailed Student's t-test with unequal variance). Data are representative of eight experiments (mean ± s.e.m.). () Total DO11.10+Foxp3− (apparently naive) CD4SP thymocytes in the presence of cognate antigen. *P < 0.001 (two-tailed Student's t-test with unequal variance). Data are representative of eight experiments (mean ± s.e.m.). * Figure 7: Rescue from clonal deletion and enhanced generation of HA-specific Treg cells in TCR-HA × Aire-HA × C2TAkd thymi. () Thymocyte subsets in TCR-HA or TCR-HA × Aire-HA mice in the presence or absence of mTEC-specific C2TA silencing. Numbers above outlined areas (top) indicate percent CD4SP cells; numbers above bracketed lines (bottom) indicate frequency of TCR-HA+ cells among gated CD4SP thymocytes (average ± s.d.). () Frequency of TCR-HA+ cells among gated CD4SP thymocytes in (summary). Each symbol represents an individual mouse; small horizontal lines indicate the mean (TCR-HA, n = 7 mice; TCR-HA × Aire-HA, n = 6 mice; TCR-HA × Aire-HA × C2TAkd, n = 14 mice). *P < 0.001. () Foxp3 expression in CD4SP cells, assessed by intracellular staining. Numbers adjacent to outlined areas indicate frequency of TCR-HA+Foxp3+ cells among CD4SP thymocytes (average ± s.d.). () Total TCR-HA+Foxp3+ CD4SP thymocytes. () Total TCR-HA+Foxp3−CD4SP thymocytes in the presence of cognate antigen. P values (,,), two-tailed Student's t-test with unequal variance. Data are representative of five experiments. * Figure 8: The C2TAkd-mediated cell-fate conversion of DO11.10 × Aire-OVA thymocytes is independent of cross-presentation by DCs. () Thymocyte subsets in wild-type, Aire-OVA or Aire-OVA × C2TAkd recipients 6–8 weeks after reconstitution with DO11.10 or DO11.10 × ΔDC bone marrow cells. Numbers above outlined areas (top) indicate percent CD4SP cells; numbers above bracketed lines (bottom) indicate frequency of DO11.10+ cells among gated CD4SP thymocytes (average ± s.d.). P = 0.00001, DO11.10 × ΔDC→Aire-OVA × C2TAkd versus DO11.10 × ΔDC→Aire-OVA (DO11.10+CD4+ T cells). (,) Frequency () and absolute number () of Foxp3+DO11.10+ CD4SP cells resulting from C2TA silencing in Aire × OVA and Aire-OVA × C2TAkd recipients of DO11.10 × ΔDC bone marrow. P = 0.005 (in ). () Total DO11.10+Foxp3−CD4SP thymocytes in the presence of cognate antigen. *P < 0.001. P values (,,), two-tailed Student's t-test with unequal variance. Data are representative of two experiments with at least three chimeras per group. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE20276 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Institute for Immunology, Ludwig-Maximilians-Universität, Munich, Germany. * Maria Hinterberger, * Martin Aichinger, * David Voehringer & * Ludger Klein * Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany. * Olivia Prazeres da Costa & * Reinhard Hoffmann Contributions M.H. generated and analyzed the C2TAkd model as well as the compound transgenic mice and bone marrow chimeras and was also essentially involved in all other experiments; M.A. did expression analyses by quantitative PCR; O.P.d.C. and R.H. did microarray experiments; D.V. provided ΔDC mice; and L.K. and M.H. designed experimental strategies and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ludger Klein (ludger.klein@med.uni-muenchen.de) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–5 Additional data
• Sustained signaling by canonical helper T cell cytokines throughout the reactive lymph node
  Perona-Wright G Mohrs K Mohrs M - Nat Immunol 11(6):520-526 (2010)
  Nature Immunology | Article Sustained signaling by canonical helper T cell cytokines throughout the reactive lymph node * Georgia Perona-Wright1 Search for this author in: * NPG journals * PubMed * Google Scholar * Katja Mohrs1 Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Mohrs1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:520–526Year published:(2010)DOI:doi:10.1038/ni.1866Received20 January 2010Accepted18 March 2010Published online25 April 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cytokines are soluble proteins that regulate immune responses. The present paradigm is that cytokine production in lymphoid tissues is tightly localized and signaling occurs between conjugate cells. Here we assess cytokine signaling during infection by measuring in vivo phosphorylation of intracellular signal transducer and activator of transcription (STAT) proteins. We show that interferon-γ (IFN-γ) and interleukin 4 (IL-4) signaled to the majority of lymphocytes throughout the reactive lymph node and that IL-4 conditioning of naive, bystander cells was sufficient to override opposing T helper type 1 (TH1) polarization. Our results demonstrate that despite localized production, cytokines can permeate a lymph node and modify the majority of cells therein. Cytokine conditioning of bystander cells could provide a mechanism by which chronic worm infections subvert the host response to subsequent infections or vaccination attempts. View full text Figures at a glance * Figure 1: IL-4 signals elicit STAT6 phosphorylation and IL-4R upregulation throughout a TH2-reactive lymph node. () Immunoblot analysis of total STAT6 and phosphorylated STAT6 (p-STAT6) in mesLNs cells from naive or H. polygyrus–infected (HP) wild-type (WT), Il4ra−/− and Stat6−/− mice, assessed ex vivo immediately after isolation. () STAT6 phosphorylation in lymphocyte subsets of mesLNs, in the presence (HP) or absence (Naive) of H. polygyrus infection. Below, geometric mean fluorescence intensity (MFI) of cells gated on GFP− cells to exclude activated TH2 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Max, maximum. () STAT6 phosphorylation in wild-type, Il4ra−/− and Il4−/− mesLNs 2 weeks after H. polygyrus infection, for cells gated on total lymphocytes. () STAT6 phosphorylation in CD4+ lymphocytes in mesLNs of mixed–bone marrow chimeras (1:1 ratio of wild-type (CD90.2−) cells and Il4ra−/− (CD90.2+) cells) infected with H. polygyrus. Horizontal lines indicate the mode fluorescence intensity of staining for STAT6! phosphorylation in cells from naive mice. () IL-4Rα expression in lymphocyte subsets of wild-type mesLNs in the presence or absence of H. polygyrus infection; Il4ra−/− cells serve as a negative staining control. Below, geometric mean fluorescence intensity, presented as described in . Data are representative of two or more independent experiments with three to four mice per group. * Figure 2: IFN-γ signals permeate a TH1-reactive lymph node. () STAT1 phosphorylation assessed ex vivo in the mesLNs of wild-type mice 1 week after T. gondii infection (TG) or sham infection, for cells gated on CD19+, CD4+ and CD8+ lymphocyte subsets. () STAT1 phosphorylation in wild-type and Ifng−/− mesLNs 1 week after T. gondii or sham infection, for cells gated on CD19+, CD4+ and CD8+ lymphocyte subsets. () IFN-γ receptor occupancy on CD19+, CD4+ and CD8+ lymphocyte subsets from mesLNs of T. gondii– or sham-infected wild-type or Ifngr1−/− mice, assessed by staining with the monoclonal antibody GR-20. () MHC class I (H-2Kb; MHCI) expression and isotype-matched control antibody staining in lymphocyte subsets of wild-type mesLNs, with (TG) or without (Sham) T. gondii infection. () STAT4 phosphorylation ex vivo in mesLNs from T. gondii– or sham-infected wild-type mice, for cells gated on CD4+ cells. Bottom row (,,,), geometric mean fluorescence intensity, presented as described in Figure 1b. Data are representative of at l! east three separate experiments with three to five mice per group. * Figure 3: STAT6 phosphorylation occurs in draining but not nondraining lymphoid tissues. () Expression of human CD2 (huCD2) in 4get-KN2 IL-4 dual-reporter mice infected with H. polygyrus. Numbers in top right quadrants indicate the frequency of human CD2–positive cells in the total lymphocyte population. Spl, spleen; ndLN, nondraining lymph node. Right, combined data for all mice. () STAT6 phosphorylation in 4get mice 2 weeks after H. polygyrus infection. Horizontal lines indicate mode fluorescence intensity of STAT6 phosphorylation staining in the negative control cells (mesLN cells of Il4ra−/− mice). Right, geometric mean fluorescence intensity of STAT6 phosphorylation in CD4+ GFP− cells. () Expression of human CD2 (for cells gated on CD4+ cells) in wild-type or Il4ra−/− 4get-KN2 mice infected with H. polygyrus and injected intravenously with PBS or antibody to CD3ε (α-CD3) 2 weeks later. Right, frequency of human CD2–positive cells in the GFP+ CD4+ population. (,) STAT6 phosphorylation in the mice in () or in 4get mice infected with H. polygyr! us and injected intravenously with recombinant mouse IL-4 on day 14 (), for cells gated on CD4+ GFP− cells; Il4ra−/− mice are included as a baseline staining control. Right, geometric mean fluorescence intensity of STAT6 phosphorylation in CD4+ GFP− cells. Right (–), each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are representative of two or more independent experiments with three to four mice per group. * Figure 4: STAT6 phosphorylation in the TH2-reactive lymph node is sustained and reflects continuous IL-4R triggering. () STAT6 phosphorylation in mesLN cells from 4get-KN2 IL-4 dual-reporter mice at various times after H. polygyrus infection, for cells gated on CD4+ GFP− cells. () Expression of human CD2 in mesLNs cells from 4get-Kn2 mice at various times after H. polygyrus infection, for cells gated on total lymphocytes. () STAT6 phosphorylation in cells isolated from the mesLNs of 4get mice 2 weeks after H. polygyrus infection and incubated for various times with an IL-4-neutralizing antibody (11B11). Cells are gated on CD4+ GFP− cells; dotted line indicates geometric mean fluorescence of H. polygyrus–infected Il4ra−/− controls; error bars indicate s.d. of four individual mice per group. () Binding of IL-4 to the cell surface in mesLNs cells from naive or H. polygyrus–infected wild-type 4get or Il4−/− mice, detected with antibody to IL-4 (BVD6); cells are gated on CD4+ GFP− cells. () Surface IL-4 expression in mesLN cells collected from naive or H. polygyrus–infected (! 2 weeks) wild-type 4get mice, stained immediately (Ex vivo) or incubated for 60 min with the IL-4-neutralizing antibody 11B11 (Stripped); cells were gated on CD4+ GFP− cells. In ,, (bottom) and , each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are representative of two to four independent experiments with four mice per group. * Figure 5: Estimation of the effective interstitial IL-4 concentration in the TH2-reactive lymph node. () IL-4Rα expression in naive (CD44lo GFP−) CD4+ cells sorted from wild-type 4get (CD90.1+) and Stat6−/− 4get (CD90.1−) lymph nodes and cultured together for 24 h in the presence of graded concentrations of IL-4 without additional stimuli or antibodies. Vertical lines indicate the mode fluorescence intensity of IL-4Rα staining on cells cultured in medium alone. Below, geometric mean fluorescence intensity of IL-4Rα staining on each gated population. () IL-4Rα expression in CD4+ GFP− cells from the mesLNs of mixed–bone marrow chimeras (1:1 ratio of wild-type (CD90.1+) 4get cells and Stat6−/− (CD90.1−) 4get cells) infected with H. polygyrus and analyzed 2 weeks later (n = 4 mice per group). Data are representative of two to four independent experiments. * Figure 6: IL-4 conditioning of naive lymphocytes alters subsequent helper T cell polarization. () STAT6 phosphorylation in 4get DO11.10 TCR-transgenic CD4+ cells from draining and contralateral popliteal lymph nodes 18 h after transfer into 4get mice that had been immunized with H. polygyrus larvae in the footpad 6 d before adoptive transfer. () Frequency of DO11.10 cells in draining popliteal lymph nodes of 4get mice injected with H. polygyrus and adoptively transferred with DO11.10 cells, as in , and immunized in the same footpad 7 d later with Y. pestis–pulsed DCs loaded with OVA peptide (DC-pOVA) or an irrelevant peptide control (DC-pHA). () Cytokine polarization of transferred DO11.10 cells assessed 7 d after DC immunization, measured by intracellular staining after stimulation of lymph node cells for 18 h in the presence or absence of OVA peptide (IFN-γ; left), or quantified with the 4get reporter and presented as the frequency of GFP+ cells in the activated (CD44hi) DO11.10 population (IL-4; right). In ,, each symbol represents an individual mouse; small hor! izontal lines indicate the mean. Data are representative of two to four independent experiments with four mice per group. Author information * Abstract * Author information * Supplementary information Affiliations * Trudeau Institute, Saranac Lake, New York, USA. * Georgia Perona-Wright, * Katja Mohrs & * Markus Mohrs Contributions G.P.-W. and M.M. designed the research and prepared the manuscript; G.P.-W. and K.M. did the experiments; G.P.-W. analyzed and interpreted the results; and M.M. guided the study. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Markus Mohrs (mmohrs@trudeauinstitute.org) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–9 Additional data
• The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation
  Chang HC Sehra S Goswami R Yao W Yu Q Stritesky GL Jabeen R McKinley C Ahyi AN Han L Nguyen ET Robertson MJ Perumal NB Tepper RS Nutt SL Kaplan MH - Nat Immunol 11(6):527-534 (2010)
  Nature Immunology | Article The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation * Hua-Chen Chang1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarita Sehra1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Ritobrata Goswami1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Weiguo Yao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Qing Yu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gretta L Stritesky1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Rukhsana Jabeen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Carl McKinley1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ayele-Nati Ahyi1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ling Han1 Search for this author in: * NPG journals * PubMed * Google Scholar * Evelyn T Nguyen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael J Robertson3 Search for this author in: * NPG journals * PubMed * Google Scholar * Narayanan B Perumal4 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert S Tepper1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen L Nutt5 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark H Kaplan1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:527–534Year published:(2010)DOI:doi:10.1038/ni.1867Received28 October 2009Accepted22 March 2010Published online02 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg CD4+ helper T cells acquire effector phenotypes that promote specialized inflammatory responses. We show that the ETS-family transcription factor PU.1 was required for the development of an interleukin 9 (IL-9)-secreting subset of helper T cells. Decreasing PU.1 expression either by conditional deletion in mouse T cells or the use of small interfering RNA in human T cells impaired IL-9 production, whereas ectopic PU.1 expression promoted IL-9 production. Mice with PU.1-deficient T cells developed normal T helper type 2 (TH2) responses in vivo but showed attenuated allergic pulmonary inflammation that corresponded to lower expression of Il9 and chemokines in peripheral T cells and in lungs than that of wild-type mice. Together our data suggest a critical role for PU.1 in generating the IL-9-producing (TH9) phenotype and in the development of allergic inflammation. View full text Figures at a glance * Figure 1: PU.1 is required for optimal IL-9 production in mouse T cells. () ELISA of IL-9 in supernatants of naive wild-type (WT) and Sfpi1fl/flLck-Cre CD4+ T cells cultured for 5 d under TH9 conditions and stimulated for 1, 2 or 3 d with anti-CD3. () Quantitative PCR analysis of Il9 mRNA among RNA isolated from TH2 or TH9 cultures stimulated for 24 h with anti-CD3. () Intracellular staining for IL-4 and IL-9 in wild-type and Sfpi1fl/flLck-Cre CD4+ T cells cultured for 5 d under TH9 conditions and stimulated for 5 h with the phorbol ester PMA and ionomycin. Numbers in top left quadrants indicate percent IL-9+IL-4− cells. () Quantitative PCR analysis of Sfpi1 expression in naive CD4+ T cells cultured under TH1, TH2, TH9 or TH17 conditions. () Intracellular staining for IL-9 and Foxp3 in naive CD4+ T cells cultured for 5 d under TH9 or iTreg conditions and stimulated for 6 h with anti-CD3. Numbers in outlined areas indicate percent Foxp3+IL-9− cells (top left) or Foxp3−IL-9+ cells (bottom right). () Intracellular staining for IL-9 and IL-17 i! n naive CD4+ T cells cultured for 5 d under TH9 or TH17 conditions and stimulated for 6 h with PMA and ionomycin. Numbers in outlined areas indicate percent IL-9+IL-17− cells (top left) or IL-9−IL-17+ cells (bottom right). () ELISA of IL-9 and IL-17 in supernatants of naive CD4+ T cells cultured for 5 d under TH9 or TH17 conditions and stimulated for 24 h with anti-CD3. () Expression of Il4, Il9 and Sfpi1 assessed in RNA from TH2 cultures separated into IL-4lo and IL-4hi populations by magnetic selection. Quantitative PCR results are presented relative to the expression in IL-4lo cells (normalized to β2-microglobulin expression), calculated by the change-in-threshold method. *P < 0.05 (two-tailed Student's t-test). Data are representative of more than four experiments (–; average and s.d. of three mice), are an average of three to five () or three () experiments (error bars, s.d.), or are representative of at least three experiments (–; mean and s.d.). * Figure 2: PU.1 promotes IL-9 production. (,) IL-9 secretion (top) and Il9 expression (bottom) by eGFP+ cells sorted from TH9 cultures () or TH2 cultures () transduced with control retrovirus (MIEG) or 1 (Sfpi1)-expressing retrovirus (RV) and stimulated for 24 h with anti-CD3. Expression results (bottom) were normalized to β2-microglobulin and are presented relative to expression in cells transduced with control virus. () Intracellular staining of IL-4 and IL-9 in eGFP+ populations from TH9 or TH2 cultures transduced with control or 1-expressing retrovirus. Numbers in quadrants indicate percent cells in each (average ± s.e.m. of two to three mice). Data are representative of two to three experiments (error bars (,), s.e.m.). * Figure 3: IL-9 and IL-10 are not coordinately regulated in TH9 cells. () Quantitative PCR analysis of Il9 and Il10 among RNA isolated from naive CD4+ T cells cultured for 5 d under TH2 or TH9 conditions, then left unstimulated (U) or stimulated for 6 h or 24 h with plate-bound anti-CD3; results were normalized to β2-microglobulin and are presented relative to expression in unstimulated cells. () Intracellular staining of IL-10 and IL-9 in TH2 and TH9 cultures derived as in and left unstimulated (Control) or stimulated for 5 h with anti-CD3. Numbers in outlined areas indicate percent IL-10+IL-9− cells (top left) or IL-10−IL-9+ cells (bottom right). () ELISA of IL-9 and IL-10 in supernatants of naive CD4+ T cells cultured for 5 d under TH9 conditions in the presence or absence of anti-IL-10, then restimulated for 24 h with anti-CD3. () ELISA of IL-9 and IL-10 in supernatants of naive CD4+ T cells cultured for 5 d under TH9 conditions, then restimulated for 24 h with anti-CD3 in the presence or absence of TGF-β1. () ELISA of IL-10 in supern! atants of wild-type and Sfpi1fl/flLck-Cre naive CD4+ T cells cultured for 5 d under TH9 conditions, then stimulated for 24 h with anti-CD3. Data are representative of at least three experiments (error bars (–), s.e.m.). * Figure 4: Histone modifications at the Il9 locus. () VISTA plot of CNSs adjacent to the Il9 locus. (,) ChIP analysis of histone modifications in naive CD4+ T cells isolated and analyzed immediately (Naive) or differentiated for 5 d under TH1, TH2, TH9, TH17 or iTreg culture conditions. H3K27me3, trimethylation of H3K27; AcH3K9-18, acetylation of H3K9 and H3K18; AcH4, acetylation of histone H4; AcH3, acetylation of histone H3. (,) ChIP analysis of histone modifications in naive wild-type and Sfpi1fl/flLck-Cre CD4+ T cells cultured under TH9 conditions. () Consensus PU.1-binding sites in Il9 CNS1 (above) and DNA-affinity–precipitation assay of the binding of PU.1 to site 2 in TH2 or TH9 extracts (below), with (right) or without (left) competition of extracts incubated with a fivefold excess of unlabeled PU.1 consensus double-stranded oligonucleotide. Input (far right), expression of PU.1 in cell extracts. () ChIP analysis of PU.1 binding in naive CD4+ T cells differentiated for 5 d under TH1, TH2 or TH9 culture conditions. ! Data are representative of two to four experiments (average and s.d. of percent input with subtraction of control IgG (–) or with anti-PU.1 and IgG control ChIP values shown separately ()) or are representative of three experiments (). * Figure 5: PU.1 promotes IL-9 production in human T cells. () Quantitative PCR analysis of gene expression by naive human CD4+ T cells cultured for 5 d under TH2 or TH9 conditions and then restimulated for 24 h with anti-CD3; results were normalized to β2-microglobulin and are presented relative to expression in TH2 cells. () ELISA of IL-9 in supernatants of TH2 and TH9 cultures derived and restimulated as in . () Intracellular staining for IL-9 and IL-13 in cells from TH2 and TH9 cultures derived as in and stimulated for 6 h with anti-CD3. (,) Quantitative PCR analysis of SPI1 expression () and ELISA of IL-9 production () by TH9 cultures derived as in and transfected for 24 h with control or SPI1-specific siRNA. () Multiplex analysis of IL-9 in supernatants of PBMCs from infants (18–30 months of age) classified as atopic (n = 49) or nonatopic (n = 33) on the basis of positive allergen-specific serum IgE; cells were stimulated for 48 h with anti-CD3. *P < 0.04 (unpaired Student's t-test). Data are representative of three to five ! experiments with different donors (–) or one study (). * Figure 6: PU.1 expression in T cells is required for the development of allergic inflammation. () ELISA of TH2 cytokines in supernatants of splenocytes from wild-type and Sfpi1fl/flLck-Cre mice sensitized and challenged intranasally with OVA; cells collected 48 h after the final intranasal challenge were stimulated for 72 h with anti-CD3 (left) or OVA (right). () Hematoxylin and eosin staining of paraffin-embedded sections of lungs from mice challenged as in (left) or left unchallenged (right). Original magnification, ×100 (top row) or ×400 (bottom row). () Whole-body plethysmography of airway hyper-reactivity in wild-type and Sfpi1fl/flLck-Cre mice at 24 h after the final intranasal challenge as in , tested at baseline (B), after saline inhalation (S) and after the inhalation of various doses (horizontal axis) of methacholine. Penh, enhanced pause. () Cells recovered by BAL in mice challenged as in (C) or left unchallenged (UC; left), and specific cell types in BAL from mice challenged as in , assessed by flow cytometry (right). T, T cell; B, B cell; Neu, neutrophi! l; Eos, eosinophil; Mφ, macrophage; DC, dendritic cell. () ELISA of IL-9 and TH2 cytokines present in BAL fluid of mice challenged as in . (,) Histological analysis of lungs, assessed as in (), and BAL fluid cells, analyzed as in (), from wild-type mice sensitized and challenged intranasally daily for 5 d with ovalbumin and injected intravenously with control immunoglobulin (control Ab) or anti-IL-9 30 min before the first, third and fifth intranasal challenge; lungs and BAL fluid were obtained 48 h after the final intranasal challenge. Original magnification (), ×100 (top row) or ×400 (bottom row). *P < 0.05 (two-tailed Student's t-test). Data are representative of three experiments (average and s.e.m. of five to seven mice in ,–,). * Figure 7: PU.1 is required for the expression of IL-9 and chemokines in allergen-sensitized mice. () Quantitative PCR analysis of the expression of Il9 and chemokines associated with allergic inflammation among RNA isolated from splenocytes stimulated with OVA as described in Figure 6a, presented relative to expression in 1-deficient cultures. () Quantitative PCR analysis of the expression of Ccl17 and Ccl22 in eGFP+ cells sorted from TH9 cultures transduced with control (MIEG) retrovirus or 1-expressing retrovirus and stimulated for 6 h with anti-CD3, presented relative to expression in 1-deficient cultures. () Quantitative PCR analysis of Ccl17 and Ccl22 mRNA among total lung RNA isolated from sensitized and challenged wild-type and Sfpi1fl/flLck-Cre mice or unchallenged wild-type mice (UC), presented relative to expression in unchallenged wild-type cultures. *P < 0.05 (two-tailed Student's t-test). Data are representative of three (,) or two () experiments. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Hua-Chen Chang & * Sarita Sehra Affiliations * Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Hua-Chen Chang, * Sarita Sehra, * Ritobrata Goswami, * Weiguo Yao, * Qing Yu, * Gretta L Stritesky, * Rukhsana Jabeen, * Carl McKinley, * Ayele-Nati Ahyi, * Ling Han, * Evelyn T Nguyen, * Robert S Tepper & * Mark H Kaplan * Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Ritobrata Goswami, * Gretta L Stritesky, * Ayele-Nati Ahyi & * Mark H Kaplan * Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Michael J Robertson * School of Informatics, Indiana University-Purdue University, Indianapolis, Indianapolis, Indiana, USA. * Narayanan B Perumal * The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. * Stephen L Nutt Contributions M.H.K. designed and supervised the study and wrote the manuscript; H.-C.C., R.G., R.J. and L.H. did experiments in Figures 1,2,3 and Supplementary Figure 1; Q.Y., R.G. and G.L.S. did experiments in Figure 4 and Supplementary Figure 2; W.Y., M.J.R. and R.S.T. obtained human samples; W.Y. did all experiments in Figure 5; S.S., E.T.N., C.M. and A.-N.A. did experiments in Figures 6 and 7 and Supplementary Figure 3; N.B.P. provided bioinformatics analysis; and S.L.N. provided mice. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mark H Kaplan (mkaplan2@iupui.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (104KK) Supplementary Figures 1–3 Additional data
• PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells
  Good-Jacobson KL Szumilas CG Chen L Sharpe AH Tomayko MM Shlomchik MJ - Nat Immunol 11(6):535-542 (2010)
  Nature Immunology | Article PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells * Kim L Good-Jacobson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Courtney G Szumilas1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lieping Chen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Arlene H Sharpe3 Search for this author in: * NPG journals * PubMed * Google Scholar * Mary M Tomayko4 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark J Shlomchik1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:535–542Year published:(2010)DOI:doi:10.1038/ni.1877Received03 March 2010Accepted12 April 2010Published online09 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Memory B and plasma cells (PCs) are generated in the germinal center (GC). Because follicular helper T cells (TFH cells) have high expression of the immunoinhibitory receptor PD-1, we investigated the role of PD-1 signaling in the humoral response. We found that the PD-1 ligands PD-L1 and PD-L2 were upregulated on GC B cells. Mice deficient in PD-L2 (Pdcd1lg2−/−), PD-L1 and PD-L2 (Cd274−/−Pdcd1lg2−/−) or PD-1 (Pdcd1−/−) had fewer long-lived PCs. The mechanism involved more GC cell death and less TFH cell cytokine production in the absence of PD-1; the effect was selective, as remaining PCs had greater affinity for antigen. PD-1 expression on T cells and PD-L2 expression on B cells controlled TFH cell and PC numbers. Thus, PD-1 regulates selection and survival in the GC, affecting the quantity and quality of long-lived PCs. View full text Figures at a glance * Figure 1: Differences in the expression of PD-1 and its ligands by B cell subsets. () Flow cytometry analysis of NIP binding and Fas expression by splenic CD19+EMA− cells obtained from B6 mice (n = 3–5) 12 d after immunization with NP-CGG in alum; numbers adjacent to outlined areas indicated percent cells in each gate. EMA, ethidium monoazide. (–) Flow cytometry analysis of the expression of PD-L2 (), PD-L1 () and PD-1 () by B cell populations from the mice in , gated (as outlined in ) as follows: NIP−Fas− (naive; thin solid lines), NIPintFashi (GC; dashed lines) and NIP+Fas+ (emerging memory; thick solid lines). (–) Frequency (–) and kinetics (–) of the expression of B7 family members and PD-1 on B cell subsets at 12 d and 4 and 14 weeks after immunization as described in . The CD19+NIP+IgG1+CD38+κlo phenotype (κ, immunoglobulin κ-chain) was used to identify memory B cells 14 weeks after immunization because of the very low frequency of detectable cells. Data are representative of at least three independent experiments (mean ± s.e.m. i! n –). * Figure 2: Long-lived PCs are lower in abundance in the absence of PD-1 signaling. (–) Enzyme-linked immunospot (ELISPOT) analysis of long-lived PC and memory responses in spleen and bone marrow from wild-type control mice (B6), Pdcd1lg2−/− mice (; n ≥ 16), Cd274−/−Pdcd1lg2−/− mice (; n ≥ 10) and Pdcd1−/− mice (; n ≥ 14) at least 12 weeks after immunization with NP-CGG or alum (n ≥ 5 mice per genotype). Data are combined from two (Cd274−/−Pdcd1lg2−/−) or at least seven (Pdcd1lg2−/− or Pdcd1−/−) independent experiments (error bars, s.e.m.). () Memory B cell frequency after immunization as in , assessed in live splenocytes gated as CD19+NIP+IgG1+CD38+κlo. The CD38 marker was included to gate out any residual GC (CD38−) B cells; because naive B cells are also CD38+, IgG1 was included to separate memory B cells from naive B cells, although this precludes analysis of any IgM+ memory B cells present. Number above bracketed line (left) indicates percent CD19+ cells; numbers adjacent to outlined areas indicate percent I! gG1+NIP+ cells (middle) or κloCD38+ cells (right). Data are representative of at least 15 experiments. (–) Frequency of memory B cells in wild-type control mice (B6 or BALB/c), Pdcd1lg2−/− mice (), Cd274−/−Pdcd1lg2−/− mice () and Pdcd1−/− mice (). Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are combined from two (Cd274−/−Pdcd1lg2−/−) four (Pdcd1lg2−/−) or six (Pdcd1−/−) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney nonparametric, two-tailed test). * Figure 3: The decrease in PC numbers occurs during the late GC response and affects both IgG1 and IgM. (–) ELISPOT analysis of NP+IgG1+ AFCs in spleen and bone marrow from wild-type control mice (B6 or BALB/c; n ≥ 6), Pdcd1lg2−/− mice (; n ≥ 6), Cd274−/−Pdcd1lg2−/− mice (; n ≥ 10) and Pdcd1−/− mice (; n ≥ 12) ~4 weeks after immunization with NP-CGG or alum (n ≥ 3 mice per genotype). Data are combined from at least two independent experiments (error bars, s.e.m.). (–) NP+IgG1+ AFCs in spleen and bone marrow of wild-type control mice, Pdcd1lg2−/− mice (), Cd274−/−Pdcd1lg2−/− mice () and Pdcd1−/− mice () at various times after immunization with NP-CGG (day 28 includes days 27–31, as also presented in –; day 12 is also in Supplementary Fig. 1; data beyond day 84, Fig. 2). Data are combined from at least two independent experiments, except data for days 18 and 21 for Cd274−/−Pdcd1lg2−/− mice represent one independent experiment per time point. (,) Circulating IgG1 and IgM in wild-type mice, Pdcd1lg2−/− mice (; n = 6–8! ) and Pdcd1−/− mice (; n = 6) at week 4 () and week 3 () after immunization with NP-CGG. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are representative of at least two independent experiments (error bars, s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney nonparametric, two-tailed test). * Figure 4: Greater cell death but normal proliferation in GCs of Pdcd1−/− mice. () Flow cytometry analysis of CaspGLOW and BrdU staining of splenic CD19+NIPintFashi cells from a B6 mouse at day 12 after immunization with NP-CGG, pulsed with BrdU (3 mg) 2 h before analysis. Numbers above bracketed lines indicate percent CaspGLOW+ cells (left) or BrdU+ cells (right). (–) Frequency over time of splenic GC B cells (; day 12 also in Supplementary Fig. 1) that are CaspGLOW+ () or BrdU+ () among splenic CD19+NIPintFashi cells from wild-type and Pdcd1−/− B6 mice immunized with NP-CGG and pulsed with BrdU 2 h before analysis (3 mg/mouse); results are flow cytometry of cells visualized by detection with CaspGLOW or BrdU immediately after collection at days 10, 12, 14, 16, 18 and 21 after immunization. () Flow cytometry analysis of CaspGLOW+ emerging memory (mem) cells (left) and plasmablasts-PCs (intermediate–positive for NIP and positive for syndecan) from mice treated as described in – (n ≥ 4 per time point). () ELISPOT analysis of NP2-specific vers! us NP17-specific IgG1+ bone marrow AFCs from knockout mice (open bars; n ≥ 6) and wild-type mice (filled bars); one molecule of bovine serum albumin conjugated to two (NP2-BSA) or seventeen (NP17-BSA) NP molecules was used as capture antigen. *P < 0.05, **P < 0.01 (Mann-Whitney nonparametric, two-tailed test). Data are combined from or representative of at least two independent experiments (error bars, s.e.m.). * Figure 5: More cells of a TFH phenotype correlates with lower cytokine production in the absence of PD-1 signaling. (,) Flow cytometry analysis of TFH cell frequency among CD4+ cells () and number of TFH cells () from wild-type (BALB/c) and Cd274−/−Pdcd1lg2−/− mice on days 12, 15 and 18 after immunization with NP-CGG; CD4+CD90.2+EMA− cells were gated for the TFH cell markers of PD-1 expression and CCR7 downregulation. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are representative of two independent experiments at all time points (Cd274−/−Pdcd1lg2−/−), days 15 or 16 (Pdcd1lg2−/−) or days 15 and 18 (Pdcd1−/−). () TFH cell frequency among cells from wild-type (BALB/c or B6), Pdcd1lg2−/− and Pdcd1−/− mice (n = 3–5) at day 15 after immunization with NP-CGG, for cells gated as in ,. Data are representative of at least two independent experiments during the late GC response. () Quantitative PCR analysis of the expression of Il4, Il21 and Ifng by cells from wild-type and Pdcd1−/− mice immunized with NP-CGG in a! lum, sorted for ICOS expression and CCR7 downregulation (n = 3, day 12), in conjunction with CXCR5 expression (n = 2, day 15; n = 3, day 18; n indicates mice per genotype). Results were calculated by the comparative threshold method relative to actin expression as follows: 2(actin CT − cytokine CT). Data are from three independent experiments (one per time point). NS, not significant; *P < 0.05, **P < 0.01 (Mann-Whitney nonparametric, two-tailed test). * Figure 6: The lower abundance of AFCs is due to impaired interactions between PD-ligands on B cells and PD-1 on T cells. () B cell transfer system. KO, knockout; BCR, B cell antigen receptor; TG, transgenic. () T cell transfer system. TCR, T cell antigen receptor. (,) ELISPOT analysis of NP+IgG1+ AFCs in spleen and bone marrow from AM14 Vκ8R recipients of transferred control B cells (filled bars) or B cells from Pdcd1lg2−/− B1-8 heterozygous (HET) mice (; beyond day 63; n = 16; open bars) or Pdcd1lg2−/− mice (; days 26–28; n = 14–15; open bars) immunized with alum or NP-CGG. () ELISPOT analysis of splenic NP+IgG1+ AFCs in AM14 Vκ8R recipients of transferred B cells (n = 8) or OT-II recipients of transferred T cells (n = 14) from B6 wild-type or Pdcd1−/− mice, assessed at 4 weeks after immunization. (,) Frequency and number of NIP+IgG1+CD38+κlo B cells after transfer of wild-type (filled bars) or Pdcd1−/− (open bars) T cells () or B cells (), assessed at 4 weeks after immunization. *P < 0.05, **P < 0.001 (Mann-Whitney nonparametric, two-tailed test). Data are combined fro! m three () or four () independent experiments or are combined from two (B cell transfer) or three (T cell transfer) independent experiments (–; error bars, s.e.m.). * Figure 7: AFC production, memory B cell formation and TFH cells are altered in mixed–bone marrow chimeras. (,) ELISPOT analysis of spleen () and bone marrow () from mixed–bone marrow chimeras generated by injection of a mixture (80:20) of Igh-J−/− bone marrow plus BALB/c, Cd274−/−Pdcd1lg2−/−, Pdcd1lg2−/− or Pdcd1−/− bone marrow (open bars) or wild-type or knockout bone marrow only (without Igh-J−/− bone marrow; filled bars) into lethally irradiated BALB/c recipients, which were allowed to rest for 6 weeks before immunization with NP-CGG, then were assessed 25–26 days later. Igh-J−/− + BALB/c, n = 19; Igh-J−/− + Pdcd1lg2−/−, n = 12; Igh-J−/− + Cd274−/−Pdcd1lg2−/−, n = 8; Igh-J−/− + Pdcd1−/−, n = 5. () Flow cytometry analysis of memory B cell frequency among CD19+ cells in the mixed–bone marrow chimeras in ,. () ELISPOT analysis of spleen from the mixed–bone marrow chimeras in ,, as in Figure 4f. (,) Flow cytometry analysis of TFH cell frequency among CD4+CD90.2+ cells in the mixed–bone marrow chimeras in ,. Igh-J−! /− + BALB/c, n = 15; Igh-J−/− + Pdcd1lg2−/−, n = 9; Igh-J−/− + Cd274−/−Pdcd1lg2−/−, n = 4; Igh-J−/− + Pdcd1−/−, n = 5. *P < 0.05, **P < 0.001 (Mann-Whitney nonparametric, two-tailed test). Data are combined from or are representative of two independent experiments (error bars, s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Laboratory Medicine and Department of Immunobiology, Yale University, New Haven, Connecticut, USA. * Kim L Good-Jacobson, * Courtney G Szumilas & * Mark J Shlomchik * Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Lieping Chen * Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. * Arlene H Sharpe * Department of Dermatology, Yale University, New Haven, Connecticut, USA. * Mary M Tomayko Contributions K.L.G.-J., M.M.T and M.J.S. designed research; K.L.G.-J. and C.G.S did research; L.C. and A.H.S. generated and contributed knockout mice; and K.L.G.-J. and M.J.S. analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mark J Shlomchik (mark.shlomchik@yale.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–3 Additional data
• Erratum: The basic leucine zipper transcription factor E4BP4 is essential for natural killer cell development
  - Nat Immunol 11(6):543 (2010)
  Nature Immunology | Erratum Erratum: The basic leucine zipper transcription factor E4BP4 is essential for natural killer cell development * Duncan M Gascoyne Search for this author in: * NPG journals * PubMed * Google Scholar * Elaine Long Search for this author in: * NPG journals * PubMed * Google Scholar * Henrique Veiga-Fernandes Search for this author in: * NPG journals * PubMed * Google Scholar * Jasper de Boer Search for this author in: * NPG journals * PubMed * Google Scholar * Owen Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Benedict Seddon Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Coles Search for this author in: * NPG journals * PubMed * Google Scholar * Dimitris Kioussis Search for this author in: * NPG journals * PubMed * Google Scholar * Hugh JM Brady Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume:11,Page:543Year published:(2010)DOI:doi:10.1038/ni0610-543a Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.10, 1118–1124 (2009); published online 13 September 2009; corrected after print 5 October 2009 In the version of this article initially published, the equal contribution of Duncan M. Gascoyne and Elaine Long is not noted. The error has been corrected in the HTML and PDF versions of the article. Additional data
• Erratum: TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation
  - Nat Immunol 11(6):543 (2010)
  Nature Immunology | Erratum Erratum: TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation * Björn F Lillemeier Search for this author in: * NPG journals * PubMed * Google Scholar * Manuel A Mörtelmaier Search for this author in: * NPG journals * PubMed * Google Scholar * Martin B Forstner Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes B Huppa Search for this author in: * NPG journals * PubMed * Google Scholar * Jay T Groves Search for this author in: * NPG journals * PubMed * Google Scholar * Mark M Davis Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume:11,Page:543Year published:(2010)DOI:doi:10.1038/ni0610-543b Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.11, 90–96 (2010); published online 13 December 2009; corrected after print 29 January 2010 In the version of this article initially published, some rows in Table 1 were misaligned. The error has been corrected in the HTML and PDF versions of the article. Additional data
• Corrigendum: Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands
  - Nat Immunol 11(6):543 (2010)
  Nature Immunology | Corrigendum Corrigendum: Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands * Roman Barbalat Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Lau Search for this author in: * NPG journals * PubMed * Google Scholar * Richard M Locksley Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory M Barton Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume:11,Page:543Year published:(2010)DOI:doi:10.1038/ni0610-543c Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.10, 1200–1207 (2009); published online: 4 October; corrected after print 11 November 2009 In the version of this article initially published, the label along the vertical axis of the top row in Figure 6a is incorrect. The correct label should be Ly6G, and the related text in the legend should read "Numbers adjacent to outlined areas indicate Ly6G+CD11b+ cells (top row) or Ly6C+CD11b+ cells (bottom row)." The error has been corrected in the HTML and PDF versions of the article. Additional data
• Corrigendum: MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis
  - Nat Immunol 11(6):543 (2010)
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• Corrigendum: Immunological synapse formation inhibits, via NF-κB and FOXO1, the apoptosis of dendritic cells
  - Nat Immunol 11(6):543 (2010)
  Nature Immunology | Corrigendum Corrigendum: Immunological synapse formation inhibits, via NF-κB and FOXO1, the apoptosis of dendritic cells * Lorena Riol-Blanco Search for this author in: * NPG journals * PubMed * Google Scholar * Cristina Delgado-Martín Search for this author in: * NPG journals * PubMed * Google Scholar * Noelia Sánchez-Sánchez Search for this author in: * NPG journals * PubMed * Google Scholar * Luis M Alonso-C Search for this author in: * NPG journals * PubMed * Google Scholar * María Dolores Gutiérrez-López Search for this author in: * NPG journals * PubMed * Google Scholar * Gloria Martínez del Hoyo Search for this author in: * NPG journals * PubMed * Google Scholar * Joaquín Navarro Search for this author in: * NPG journals * PubMed * Google Scholar * Francisco Sánchez-Madrid Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Cabañas Search for this author in: * NPG journals * PubMed * Google Scholar * Paloma Sánchez-Mateos Search for this author in: * NPG journals * PubMed * Google Scholar * José Luis Rodríguez-Fernández Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume:11,Page:543Year published:(2010)DOI:doi:10.1038/ni0610-543e Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.10, 753–760 (2009); published online 7 June 2009; corrected after print 11 February 2010 In the version of this article initially published, a citation was omitted. It should be cited in the third paragraph of the Discussion section after the second sentence as follows: In this context, it has been suggested that Notch1, another receptor located at the IS(DC), may inhibit the apoptosis of DCs by inducing activation of Akt and STAT3, a transcription factor that promotes cell survival51. The bibliographic information is as follows: 51. Luty, W.H., Rodeberg, D., Parness, J. & Vyas, YM.Antiparallel segregation of notch components in the immunological synapse directs reciprocal signaling in allogeneic Th:DC conjugates. J. Immunol., 819–829 (2007). The error has been corrected in the HTML and PDF versions of the article. Additional data