Latest Articles Include:
- Gene-environment interactions in chronic inflammatory disease
- Nat Immunol 12(4):273-277 (2011)
Nature Immunology | Commentary Gene-environment interactions in chronic inflammatory disease * Harald Renz1 * Erika von Mutius2 * Per Brandtzaeg3 * William O Cookson4 * Ingo B Autenrieth5 * Dirk Haller6 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:273–277Year published:(2011)DOI:doi:10.1038/ni0411-273Published online21 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Chronic inflammatory diseases represent a major challenge for both clinical research and patient care, and evidence indicates that these disorders develop as a result of complex gene-environment interactions. Better understanding of their cause-and-effect relationship is the basis for emerging proposals for therapy and prevention. View full text Figures at a glance * Figure 1: A new paradigm for the development of chronic inflammation. The traditional simplistic paradigm of chronic inflammatory disease development () has been revised to a more complex understanding of the cornerstones of these conditions (). In , epigenetics (blue) represents a key mechanism linking genetic components (yellow) and the environment. Disease development depends on intimate interaction between intrinsic mechanisms (red) and extrinsic mechanisms (green) in a temporal and spatial way. * Figure 2: Metabolic and immune-mediated pathologies with inflammatory processes in various target organs as the key driver of disease initiation and/or perpetuation. Extrinsic and intrinsic events (red) lead to the manifestation of disease (black). Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Harald Renz is with the Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps University, Marburg, Germany. * Erika von Mutius is with Dr. von Hauner Children's Hospital, Ludwig-Maximilians University Munich, Germany. * Per Brandtzaeg is with the Laboratory for Immunohistochemistry and Immunopathology, Centre for Immune Regulation, University of Oslo, and the Department of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway. * William O. Cookson is with the National Heart & Lung Institute Imperial College London, United Kingdom. * Ingo B. Autenrieth is with the Institute of Medical Microbiology and Hygiene, University Hospital Tübingen, Tübingen, Germany. * Dirk Haller is with the Section of Biofunctionality, Research Center for Nutrition and Food Science, and Center for Diet and Disease, Technical University Munich, Munich, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Harald Renz Author Details * Harald Renz Contact Harald Renz Search for this author in: * NPG journals * PubMed * Google Scholar * Erika von Mutius Search for this author in: * NPG journals * PubMed * Google Scholar * Per Brandtzaeg Search for this author in: * NPG journals * PubMed * Google Scholar * William O Cookson Search for this author in: * NPG journals * PubMed * Google Scholar * Ingo B Autenrieth Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Haller Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - To kill, you have to duck an HDAC
- Nat Immunol 12(4):279-281 (2011)
Nature Immunology | News and Views To kill, you have to duck an HDAC * Amnon Altman1 * Kok-Fai Kong1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:279–281Year published:(2011)DOI:doi:10.1038/ni0411-279Published online21 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Analysis of the serine-threonine phosphoproteome leads to the intriguing find that phosphorylation of the histone deacetylase HDAC7 is key to cytotoxic T lymphocyte function. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Amnon Altman and Kok-Fai Kong are in the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Amnon Altman Author Details * Amnon Altman Contact Amnon Altman Search for this author in: * NPG journals * PubMed * Google Scholar * Kok-Fai Kong Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Developing intestinal fortitude
- Nat Immunol 12(4):281-282 (2011)
Nature Immunology | News and Views Developing intestinal fortitude * Dietmar J Kappes1Journal name:Nature ImmunologyVolume: 12,Pages:281–282Year published:(2011)DOI:doi:10.1038/ni0411-281Published online21 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The development and function of TCRαβ+CD8αα+ intraepithelial lymphocytes remain poorly understood. These cells are now shown to require transforming growth factor-β for development and proper expression of characteristic surface homodimers of CD8α. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Dietmar J. Kappes is with Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Dietmar J Kappes Author Details * Dietmar J Kappes Contact Dietmar J Kappes Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Maturation of effector regulatory T cells
- Nat Immunol 12(4):283-284 (2011)
Nature Immunology | News and Views Maturation of effector regulatory T cells * Naganari Ohkura1, 2 * Shimon Sakaguchi1, 2 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:283–284Year published:(2011)DOI:doi:10.1038/ni0411-283Published online21 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Regulatory T cells adopt specialized differentiation programs controlled by transcription factors. The transcription factors Blimp-1 and IRF4 are now shown to be pivotal in the maturation of effector regulatory T cells. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Naganari Ohkura and Shimon Sakaguchi are in the Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan * World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan. * Naganari Ohkura & * Shimon Sakaguchi Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shimon Sakaguchi Author Details * Naganari Ohkura Search for this author in: * NPG journals * PubMed * Google Scholar * Shimon Sakaguchi Contact Shimon Sakaguchi Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Defeating sepsis by misleading MyD88
- Nat Immunol 12(4):284-286 (2011)
Nature Immunology | News and Views Defeating sepsis by misleading MyD88 * Katherine A Smith1 * Rick M Maizels1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:284–286Year published:(2011)DOI:doi:10.1038/ni0411-284Published online21 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Helminth parasites are adept at dampening immunity. New data showing that intracellular degradative pathways are manipulated have important implications for therapy. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Katherine A. Smith and Rick M. Maizels are with the Institute of Immunology and Infection Research, University of Edinburgh, Ashworth Laboratories, Edinburgh, UK. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rick M Maizels Author Details * Katherine A Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Rick M Maizels Contact Rick M Maizels Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research Highlights
- Nat Immunol 12(4):287 (2011)
Nature Immunology | Research Highlights Research Highlights Journal name:Nature ImmunologyVolume: 12,Page:287Year published:(2011)DOI:doi:10.1038/ni0411-287Published online21 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Time and memory IgM+ memory B cells exist in mice; however, their contribution to the antigen-specific memory pool has remained unclear because of technical difficulties in detecting rare endogenous antigen-specific cells. In Science, Jenkins and colleagues use an antigen-based technique for the enrichment of rare cells to show that immunization with antigen generates both immunoglobulin-switched and IgM+ memory B cells. IgM+ cells, which show little evidence of selection in the germinal center or affinity maturation, are more numerous and have a longer lifespan than immunoglobulin-switched cells but expand their populations poorly during a secondary response. In contrast, immunoglobulin-switched populations expand greatly and dominate the secondary response because of their ability to be activated in the presence of high-affinity serum immunoglobulin. Thus, early memory relies on shorter-lived immunoglobulin-switched cells, whereas more durable IgM+ reserves account for late memory. IV Science (10 February 2011) doi:10.1126/science.1201730 View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Quantitative events determine the differentiation and function of helper T cells
- Nat Immunol 12(4):288-294 (2011)
Nature Immunology | Perspective Quantitative events determine the differentiation and function of helper T cells * Anne O'Garra1 * Leona Gabryšová1 * Hergen Spits2 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:288–294Year published:(2011)DOI:doi:10.1038/ni.2003Published online21 March 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In recent years, numerous qualitative discoveries have been made in immunology research. However, the effect of quantitative events, long recognized as the driving factors for determinism in developmental biology, that dictate the quality of the immune response elicited to an antigen in concert with microbial products still requires serious attention. Here we discuss how the often-neglected issue of quantification affects the specification, differentiation and commitment of helper T cells. As reductionist in vitro approaches have been instrumental in the elucidation of the factors determining the development of helper T cells, in this perspective we highlight the need for the standardization of protocols, also fundamental for the comparison of immune responses in mice and humans. Improving understanding of how these in vitro quantitative events translate to immune responses in vivo, which can be studied in mouse models, is of importance in obtaining information on immune res! ponses in humans, thus empowering translational research. View full text Figures at a glance * Figure 1: Plasticity and commitment of helper T cells. () Heterogeneity and reversal of helper T cell phenotypes, based on data obtained from flow cytometry analysis of the synthesis of intracellular cytokines in polarized TH1 and TH2 populations obtained from TCRαβ-transgenic CD4+ T cells stimulated in vitro with APCs plus ovalbumin in the presence of IL-12 plus monoclonal antibody to IL-4 (TH1) or IL-4 (TH2) for 1 week (top) or 3 weeks (middle); bottom, hypothetical helper T cell pool in vivo (from ref. 26). The number of superscripted plus symbols corresponds to the amount of cytokine. () Enzyme-linked immunosorbent assay of cytokines in supernatants of TH1 and TH2 cells stimulated for 48 h by the phorbol ester PMA and ionomycin, assessed after 1 week or 3 weeks of polarization in vitro (from ref. 26). () Flow cytometry of intracellular IFN-γ production (top) and enzyme-linked immunosorbent assay of secreted soluble IFN-γ production (bottom) by cells generated from a resting TH1 clone obtained from TCRαβ-transgenic CD4+! T cells stimulated for 3 weeks in vitro with APCs plus ovalbumin in the presence of IL-12 plus monoclonal antibody to IL-4, then restimulated for 4–6 h PMA, with brefeldin A added for final 2 h (top) or 48 h (bottom) with PMA and ionomycin. Modified with permission from ref. 26. () Graded expression of transcription factors. Modified from ref. 20. * Figure 2: Several factors determine the absolute amount of helper T cell cytokines produced in response to infection. The greater abundance of CD4+IFN-γ+ T cells in the lungs of Il10−/− mice infected with aerogenic M. tuberculosis strain H37Rv (–) leads to more IFN-γ protein in supernatants of lung cell suspensions restimulated ex vivo with purified protein derivative () and more IFN-γ protein in the serum (). WT, wild-type. *P < 0.05 and **P < 0.01 (unpaired Student's t-test). Modified with permission from ref. 90. Author information * Abstract * Author information Affiliations * Division of Immunoregulation, The Medical Research Council National Institute for Medical Research, London, UK. * Anne O'Garra & * Leona Gabryšová * Academic Medical Center, University of Amsterdam, The Netherlands. * Hergen Spits Competing financial interests H.S. works one day per week for AIMM Therapeutics. Corresponding author Correspondence to: * Hergen Spits Author Details * Anne O'Garra Search for this author in: * NPG journals * PubMed * Google Scholar * Leona Gabryšová Search for this author in: * NPG journals * PubMed * Google Scholar * Hergen Spits Contact Hergen Spits Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2
- Nat Immunol 12(4):295-303 (2011)
Nature Immunology | Article The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2 * Greg M Delgoffe1 * Kristen N Pollizzi1 * Adam T Waickman1 * Emily Heikamp1 * David J Meyers2 * Maureen R Horton3 * Bo Xiao4 * Paul F Worley4 * Jonathan D Powell1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:295–303Year published:(2011)DOI:doi:10.1038/ni.2005Received04 November 2010Accepted04 February 2011Published online27 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The kinase mTOR has emerged as an important regulator of the differentiation of helper T cells. Here we demonstrate that differentiation into the TH1 and TH17 subsets of helper T cells was selectively regulated by signaling from mTOR complex 1 (mTORC1) that was dependent on the small GTPase Rheb. Rheb-deficient T cells failed to generate TH1 and TH17 responses in vitro and in vivo and did not induce classical experimental autoimmune encephalomyelitis (EAE). However, they retained their ability to become TH2 cells. Alternatively, when mTORC2 signaling was deleted from T cells, they failed to generate TH2 cells in vitro and in vivo but preserved their ability to become TH1 and TH17 cells. Our data identify mechanisms by which two distinct signaling pathways downstream of mTOR regulate helper cell fate in different ways. These findings define a previously unknown paradigm that links T cell differentiation with selective metabolic signaling pathways. View full text Figures at a glance * Figure 1: Rheb controls mTORC1 activity in T cells. () Immunoblot analysis of mTOR activation, assessed as phosphorylation (p-) of S6K1 and Akt, in lysates of wild-type (WT) and T-Rheb−/− CD4+ T cells stimulated for 1 h or 3 h with anti-CD3 and anti-CD28 (TCR + costim) in serum-free medium. () Densitometry analysis of the activity of mTORC1 (p-S6K1), mTORC2 (p-Akt(Ser473)) and PI(3)K (p-Akt(Thr308)) in the cells in (n = 4 mice per group). NS, not significant. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test). () Flow cytometry of splenocytes from wild-type and T-Rheb−/− mice. Plots of CD3 and B220 (top) are gated on lymphocyte forward and side scatter; plots of CD4 and CD8 (middle) are gated on CD3+ cells; plots of Foxp3 and CD25 Treg cells (bottom) are gated CD3+CD4+ cells. () CFSE dilution in wild-type and T-Rheb−/− CD4+ T cells stimulated for 48 h (Day 2) or 96 h (Day 4) with anti-CD3 and irradiated autologous antigen-presenting cells (APCs). () IL-2 production in wild-type and T-Rheb−/− CD4+ T cell! s stimulated overnight with anti-CD3 and anti-CD28. Numbers in quadrants or adjacent to outlined areas (,) indicate percent cells in each. Data are representative of at least three independent experiments (–) or four experiments (,; error bars (,), s.e.m.). * Figure 2: TH1 and TH17 differentiation require mTORC1 in vitro, but mTORC1 is dispensable for TH2 differentiation. () IFN-γ production in wild-type and T-Rheb−/− CD4+ T cells stimulated with anti-CD3 and APCs in TH1- or TH2-skewing conditions (horizontal axis), followed by 5 d of population expansion with IL-2 and restimulation in vitro with anti-CD3 and anti-CD28. () IL-17 production in wild-type and T-Rheb−/− CD4+ T cells stimulated with anti-CD3 and APCs in TH1- or TH17-skewing conditions (left margin), followed by 5 d of population expansion and restimulation with anti-CD3 and APCs in the presence of a protein-transport inhibitor. Numbers in outlined areas indicate percent IL-17+CD4+ cells. () IL-4 production in wild-type and T-Rheb−/− CD4+ T cells treated as in . () CFSE dilution and cytokine production of wild-type and T-Rheb−/− CD4+ T cells labeled and skewed as in and restimulated with anti-CD3 and anti-CD28 in the presence of a protein-transport inhibitor. Numbers in quadrants indicate percent cells in each. Data are representative of at least three independent ! experiments (error bars (,), s.e.m.). * Figure 3: T cells deficient in mTORC1 cannot skew toward TH1 or TH17 in vivo. () Cytokine production in C57BL/6 (Thy-1.2+) mice immunized with vaccinia virus expressing ovalbumin and given adoptive transfer of 1 × 106 wild-type or T-Rheb−/− CD4+ OT-II (Thy-1.1+) T cells; splenocytes obtained 4 d later were rechallenged overnight in vitro with OVA peptide in the presence of a protein-transport inhibitor. Plots are gated on Thy-1.1+ cells. TNF, tumor necrosis factor. () Cytokine production by cells from mice treated as in . Each symbol represents an individual mouse (n = 3); small horizontal lines indicate the mean. P < 0.01 for IFN-γ or 0.05 for IL-4 (Student's t-test). () Cytokine production in C57BL/6 (Thy-1.2+) mice given mock immunization or immunized as in and given adoptive transfer as in , except with CFSE-labeled cells. () Cytokine production from Peyer's patch lymphocytes (PPLs) isolated from wild-type and T-Rheb−/− mice (n = 6) and stimulated with PMA and ionomycin. *P < 0.01 (Student's t-test). Numbers in quadrants (,) or outlined ! areas () indicate percent cells in each. Data are representative of at least three independent experiments. * Figure 4: T-Rheb−/− mice do not develop EAE but instead develop an alternative autoimmune disease. () Disease progression in wild-type and T-Rheb−/− mice (n = 7) immunized with MOG and complete Freund's adjuvant to induce EAE. *P < 0.01 (one-way analysis of variance). () Immunohistochemistry of CD45 and CD4 in sections of the central nervous system from wild-type mice, frozen at the height of disease. () Cytokine production in samples from the central nervous system, allowed to 'rest' for 16 h in unsupplemented medium and stimulated with PMA and ionomycin in the presence of a protein-transport inhibitor (n = 4 mice). Plots (left) are gated by CD4 expression and side scatter and numbers in quadrants indicate percent cells in each; each symbol (right) represents an individual mouse. P < 0.05 for IFN-γ or 0.01 for IL-17 (Student's t-test). () Incidence of classical or nonclassical EAE in mice immunized with MOG and complete Freund's adjuvant. () Immunohistochemistry of CD45 and CD4 in the cerebellum, assessed as in . Original magnification (,), ×20. Data are representa! tive of three independent experiments (error bars (,), s.e.m.). * Figure 5: T cells deficient in mTORC2 cannot skew toward TH2 but retain TH1 and TH17 skewing. () Immunoblot analysis of mTOR activation (assessed as in Fig.1a) in lysates of wild-type and T-Rictor−/− CD4+ T cells stimulated for 1 h or 3 h with anti-CD3 and anti-CD28 in serum-free medium. () Densitometry analysis of the activity of mTORC1, mTORC2 and PI(3)K (assessed as in 1b; n = 4 mice). *P < 0.05 (Student's t-test). () IFN-γ production in wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells stimulated overnight with anti-CD3 and APCs in TH1- or TH2-skewing conditions, followed by population expansion for 5 d with IL-2 and restimulation in vitro with anti-CD3 and anti-CD28. () IL-17 production by wild-type and T-Rictor−/− CD4+ T cells stimulated with anti-CD3 and APCs in TH1- or TH17-skewing conditions, followed by population expansion for 5 d and restimulation with anti-CD3 and APCs in the presence of a protein-transport inhibitor. Numbers in outlined areas indicate percent IL-17+CD4+ cells. () IL-4 production in cells treated as in . () Cytokine prod! uction by splenocytes (treated as in Fig. 3a) from C57BL/6 (Thy-1.2+) mice immunized with vaccinia virus expressing ovalbumin and given adoptive transfer of 1 × 106 wild-type, T-Rheb−/− or T-Rictor−/− CD4+ OT-II (Thy-1.1+) T cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.01 (Student's t-test). () IL-4 production in CD4+ T cells from wild-type, T-Rheb−/− and T-Rictor−/− mice immunized with alum (Naive) or ovalbumin plus alum (OVA primed) and boosted after 14 d; splenocytes were stimulated 48 h with OVA protein (100 μg/ml) with protein-transport inhibitor present for the final 8 h. Numbers in outlined areas indicate percent IL-4+CD4+ cells. () OVA-specific serum immunoglobulin G1 (IgG1) antibody titers in the mice in . Data are representative of four (,) or two () experiments or at least three independent experiments (–,,; error bars (,,,), s.e.m.). * Figure 6: Both mTORC1 and mTORC2 influence cytokine signaling by inhibiting SOCS proteins differently. () Immunoblot analysis of mTOR activation (assessed as in Fig. 1a) in lysates of wild-type, naive T cells stimulated for 8 h with anti-CD3 and anti-CD28 plus various doses of rapamycin (above lanes) in serum-containing medium. () Cytokine production by wild-type 5C.C7 T cells stimulated for 48 h with pigeon cytochrome c peptide in TH1- or TH2-skewing conditions in the presence (VLD rapamycin) or absence (Untreated) of a very low dose of rapamycin (100 pM) and washed, followed by population expansion for 5 d in fresh drug and IL-2, then restimulation with anti-CD3 and anti-CD28. Numbers in quadrants indicate percent IL-4+IFN-γ− cells (top left) or IL-4−IFN-γ+ cells (bottom right). () Immunoblot analysis of STAT phosphorylation in lysates of freshly isolated wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells resuspended for 1 h in serum-free medium and stimulated for 30 min with IL-12, IL-4 or IL-6. () SOCS3 and SOCS5 mRNA in wild-type, T-Rheb−/− and T-Rictor�! ��/− CD4+ T cells left unstimulated (Unstim) or activated overnight with anti-CD3 and anti-CD28 (Activated); results are normalized to those of 18s rRNA and are presented relative to those of wild-type unstimulated cells (for SOCS3) or stimulated cells (for SOCS5). () Immunoblot analysis of SOCS3 and SOCS5 in cells treated as in . () Kinetic analysis of SOCS3 and SOCS5 in lysates of wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells stimulated for 48, 72 or 96 h with anti-CD3 and anti-CD28; actin serves as a loading control. () IFN-γ production in wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells transfected by nucleofection with control siRNA (siCtrl) or siRNA specific for SOCS3 (siSOCS3) or SOCS5 (siSOCS5; data not shown), allowed to 'rest' for 16 h and then stimulated for 48 h in TH1- or TH2-skewing conditions, followed by population expansion for 48 h in IL-2 and restimulation with anti-CD3 and anti-CD28. Numbers in outlined areas indicate percent IFN-! γ+CD4+ cells. () IL-4 production by the cells in . TH1, wild-! type TH1 cells (negative control). Data are representative of at least three independent experiments (–) or two experiments (,; error bars (,), s.e.m.). * Figure 7: Control of the induction of transcription factors by mTORC1 and mTORC2. () Expression of mRNA for lineage-specific transcription factors in wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells skewed toward the TH1, TH2 or TH17 subset; results are presented relative to those for 18S rRNA (T-bet and GATA-3) or as arbitrary units for the change in cycling threshold (RORγt). ND, not detected. () Flow cytometry of wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells skewed toward the TH1, TH2 or TH17 subset and stained for T-bet, GATA-3 or RORγt. Numbers above outlined areas indicate percent cells in each. () Flow cytometry of cells transfected with siRNA (as in Fig. 6g) and skewed toward the TH1 or TH2 subset. Shaded histograms indicate wild-type TH2 cells (T-bet) or wild-type TH1 cells (GATA-3). () Flow cytometry of naive 5C.C7 T cells skewed toward the TH1 or TH2 subset in the presence or absence of a very low dose of rapamycin. Numbers in quadrants indicate percent GATA-3+T-bet− cells (top left) or GATA-3− T-bet+ cells (bottom ri! ght). Data are representative of at least three independent experiments (,,; error bars (), s.e.m.) or two experiments (). * Figure 8: Inhibition of both mTORC1 and mTORC2 is required for spontaneous induction of Treg cells. () Expression of Foxp3 and CD25 in wild-type, T-Rheb−/−, T-Rictor−/− and T-Mtor−/− CD4+ T cells stimulated overnight with anti-CD3 and APCs and allowed to 'rest' for 5 d in IL-7-supplemented medium. () CFSE dilution in wild-type T cells given no stimulation (No stim) or stimulated in vitro with anti-CD3 APCs alone (No supp) or in combination with Foxp3+ Treg cells (suppressor/responder (Supp/resp) ratio, above plots) generated from wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells (with TGF-β and IL-2); Treg cells were enriched by CD25 magnetic separation, and Foxp3 expression was confirmed by intracellular staining. () Expression of Foxp3 and CD25 in wild-type, T-Rheb−/− and T-Rictor−/− CD4+ T cells stimulated with anti-CD3 and APCs in the presence of a very low dose or conventional (500 nM) dose of rapamycin and allowed to 'rest' for 5 d in IL-7-supplemented medium (with drug). () Expression of Foxp3 and CD25 (left) by wild-type T cells stimula! ted with anti-CD3 and APCs in the presence (DMK1) or absence (Untreated) of DMK1, followed by population expansion for 5 d in the presence of IL-2 and drug, and immunoblot analysis (right) of mTOR activation in lysates of wild-type T cells stimulated for 1 or 3 h with anti-CD3 and anti-CD28 in serum-free medium with or without DMK1. Numbers adjacent to (,) or in () outlined areas indicate percent CD25+Foxp3+ cells. Data are representative of at least three independent experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Sidney-Kimmel Comprehensive Cancer Research Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Greg M Delgoffe, * Kristen N Pollizzi, * Adam T Waickman, * Emily Heikamp & * Jonathan D Powell * Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * David J Meyers * Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Maureen R Horton * Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Bo Xiao & * Paul F Worley Contributions G.M.D. did research, helped design experiments and wrote the paper; K.N.P. assisted with in vivo experiments and biochemistry; A.T.W. assisted with EAE induction and central nervous system isolation and did immunohistochemistry; E.H. assisted with very-low-dose rapamycin experiments; D.J.M. synthesized the mTOR kinase inhibitor; M.R.H. helped design experiments and contributed reagents; B.X. and P.F.W. generated the original mouse line with loxP-flanked Rheb alleles; and J.D.P. designed experiments, oversaw research and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jonathan D Powell Author Details * Greg M Delgoffe Search for this author in: * NPG journals * PubMed * Google Scholar * Kristen N Pollizzi Search for this author in: * NPG journals * PubMed * Google Scholar * Adam T Waickman Search for this author in: * NPG journals * PubMed * Google Scholar * Emily Heikamp Search for this author in: * NPG journals * PubMed * Google Scholar * David J Meyers Search for this author in: * NPG journals * PubMed * Google Scholar * Maureen R Horton Search for this author in: * NPG journals * PubMed * Google Scholar * Bo Xiao Search for this author in: * NPG journals * PubMed * Google Scholar * Paul F Worley Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan D Powell Contact Jonathan D Powell Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (5M) Rheb-deficient T cells induce nonclassical EAE. Video of a representative T-Rheb−/− mouse 14 days after immunization with MOG peptide + CFA to induce EAE. The mouse displays symptoms of ataxia without paralysis. PDF files * Supplementary Text and Figures (9M) Supplementary Figures 1–13 Additional data - The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells
- Nat Immunol 12(4):304-311 (2011)
Nature Immunology | Article The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells * Erika Cretney1, 2, 6 * Annie Xin1, 2, 6 * Wei Shi1, 3 * Martina Minnich4 * Frederick Masson1, 2 * Maria Miasari1, 2 * Gabrielle T Belz1, 2 * Gordon K Smyth1, 5 * Meinrad Busslinger4 * Stephen L Nutt1, 2 * Axel Kallies1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:304–311Year published:(2011)DOI:doi:10.1038/ni.2006Received11 November 2009Accepted04 February 2011Published online06 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Regulatory T cells (Treg cells) are required for peripheral tolerance. Evidence indicates that Treg cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of Treg cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector Treg cells. Thus, our study defines a differentiation pathway that leads to the acquisition of Treg cell effector functions and requires both IRF4 and Blimp-1. View full text Figures at a glance * Figure 1: Expression of Blimp-1 in a subset of Treg cells. () Blimp-1–GFP expression (right) in CD4+CD25+ splenic T cells isolated from a 51-week-old Blimp1+/GFP mouse. Number in outlined area (left) indicates percent CD25+CD4+ cells. Data are representative of seven independent experiments with similar results, each with one to three mice. () Quantitative RT-PCR analysis of Blimp1 mRNA expression in sorted CD4+ T cells pooled from the spleens and lymph nodes of Blimp1+/GFP mice (n = 10); results are presented relative to the expression of Hprt1 (hypoxanthine guanine phosphoribosyl transferase). *P = 0.04 (t-test). Data are representative of three independent experiments (mean and s.e.m. of triplicate wells). () Blimp-1–GFP expression on CD4+ T cells from the spleen, mesenteric lymph nodes (mesLN) and thymus of an 8-week-old Blimp1+/GFP mouse. Numbers in quadrants indicate percent positive cells in each. Data are representative of three to eight experiments. () Foxp3 expression in splenic CD4+CD25+ T cells (filled histograms) an! d CD4+CD25− T cells (open histograms) pooled from Blimp1+/GFP mice (n = 3) and isolated according to Blimp-1–GFP expression. Numbers in plots indicate percent Foxp3+ cells. Data are representative of two independent experiments. () Proliferation (assessed as [3H]thymidine incorporation) of CD4+CD25− responder T cells (CD25−) cultured in vitro at various ratios (horizontal axis) with GFP− or GFP+ CD4+CD25+ T cells (CD25+) pooled from the spleens and lymph nodes of Blimp1+/GFP mice. R (far right), responder cells cultured without CD4+CD25+ cells. Data are representative of two independent experiments with similar results (mean ± s.e.m. of triplicate wells). * Figure 2: Blimp-1-expressing Treg cells have an effector phenotype, produce IL-10 and localize to mucosal sites. () Flow cytometry of CD4+CD25+ T cells from the mesenteric lymph nodes of Blimp1+/GFP mice. Numbers in quadrants indicate percent cells in each. () Cytokine production by pooled Blimp1+/GFP splenic and lymph node CD4+ T cells sorted according to various markers (horizontal axis) and stimulated for 24 h with monoclonal anti-CD3 and anti-CD28. *P = 0.005 and **P = 0.0005 (t-test). () Blimp-1–GFP expression in CD4+CD25+ T cells from Peyer's patches (PP) and among intraepithelial lymphocytes (IEL) of Blimp1+/GFP mice. Numbers in plots indicate percent Blimp-1–GFP+ cells. () Quantitative RT-PCR analysis of the expression of Irf4 and Tbx21 in Blimp1+/GFP Treg cells and conventional CD4+ T cell populations sorted as in ; results are presented relative to Hprt1 expression. Data are representative of two to three experiments with two to three mice each (,) or three independent experiments (,; mean and s.d. () or s.e.m. ()). * Figure 3: Blimp-1 is dispensable for the generation of effector Treg cells but is required for their IL-10 production and tissue homeostasis. (,) Flow cytometry of splenic CD4+ T cells from wild-type (Ly5.1+)Blimp1GFP/GFP (Ly5.2+) mixed–bone marrow chimeras (WTBlimp1GFP/GFP), for gated cells. () Bead assay of cytokines in the supernatants of GFP− and GFP+ CD4+CD25+ T cells sorted from Blimp1+/GFP mice (+/GFP) or from the Blimp1GFP/GFP compartment of mixed–bone marrow chimeras (GFP/GFP) and stimulated for 24 h with monoclonal anti-CD3 and anti-CD28. *P = 0.0009 (t-test). (,) Flow cytometry analysis of Foxp3 expression () and frequency of Foxp3+ cells among total CD4+ T cells () for cells from or among the peripheral lymph nodes (pLN), spleen, Peyer's patches, intraepithelial lymphocytes and lungs of chimeras generated with a mixture of wild-type (WT; Ly5.1+) and Blimp1GFP/GFP (GFP/GFP; Ly5.2+) bone marrow. *P = 0.03 and **P = 0.0002 (t-test). () ICOS expression on Treg cells from the lungs and lung-draining mediastinal lymph nodes (mLN) of wild-typeBlimp1GFP/GFP mixed chimeras before (Naive) 10 d after (HKx31! ) infection with influenza virus (strain HKx31). () ICOS expression on wild-type and Blimp1GFP/GFP Foxp3+CD4+ cells in the lungs of naive mice (open histograms) and influenza-infected mice (filled histograms). Numbers in quadrants (,,,) indicate percent cells in each. Data are representative of two to three independent experiments with similar results, each with three to five mice per group (mean and s.e.m. of triplicate wells in ; mean and s.d. in ). * Figure 4: Blimp-1 limits numbers of Treg cells and is induced by IL-2 and inflammatory signals. (,) GFP expression in CD4+ T cells () and number of Ly5.2+CD4+ T cells () in the spleens of Rag1−/− Ly5.1+ mice 8 weeks after transfer of CD4+CD25−CD62L+ T cells, isolated from wild-type (Ly5.1+) mice, plus either GFP− or GFP+ CD4+CD25+ T cells, sorted from Blimp1+/GFP(Ly5.2+) mice. *P = 0.002 (t-test). () Blimp-1–GFP expression in CD4+CD25+ T cells (left) and number of GFP+ and GFP−CD4+CD25+ T cells (right) in the spleen 7 d after injection of PBS or IL-2–anti-IL-2 complexes (IL-2c) into Blimp1+/GFP mice. Numbers in plots indicate percent Blimp-1–GFP+ cells. () Flow cytometry of CD4+ T cells on day 5 after injection of PBS or monoclonal anti-CD40 or PBS into Blimp1+/GFP mice on days 0, 2 and 4. Numbers in quadrants (,) indicate percent cells in each. Data are representative of three independent experiments with similar results with three mice per group (,; mean and s.e.m. in ) or two independent experiments with similar results, with one to two mice per grou! p (PBS) or two to three mice per group (IL-2–anti-IL-2 or anti-CD40; ,; mean ± s.d. in ). * Figure 5: IRF4 is required for the generation of Blimp-1-expressing effector Treg cells, but T-bet is not. (,) Flow cytometry of CD4+ T cells from the spleens and mesenteric lymph nodes of Tbx21−/−Blimp1+/GFP mice () or Irf4−/−Blimp1+/GFP mice () and their respective littermate controls. () ChIP analysis of purified Treg cells with an IRF4-specific antibody and PCR primers specific for the following regulatory regions of Blimp1: promoter (Prom), conserved noncoding sequence (CNS) and region 3′ to Blimp1 (3′). Numbers above bars indicate enrichment relative to results obtained with the Irf4−/− control. (–) Flow cytometry of Treg cells and CD4+ T cells from the mesenteric lymph nodes (,) and mediastinal lymph nodes and lungs () of wild-type (Ly5.1+)–Irf4−/− (Ly5.2+) mixed–bone marrow chimeras (WT–Irf4−/−). () Contribution of IRF4-deficient Treg cells to various tissues in wild-type–Irf4−/− chimeric mice, presented as the ratio of Ly5.2+CD4+Foxp3+ T cells to Ly5.2−CD4+Foxp3+ T cells. *P < 0.01, **P < 0.001 and ***P < 0.0001 (t-test). () Flow c! ytometry of CD4+Foxp3+ T cells from mediastinal lymph nodes and lungs before and 10 d after infection of wild-type–Irf4−/− chimeric mice with influenza virus (strain HKx31). Numbers in outlined areas or quadrants (,,,,,) indicate percent cells in each. Data are representative of two to three experiments with two to three mice in each (,,,), three independent experiments () or two independent experiments with three to five mice per group (–; mean and s.e.m. in ). * Figure 6: Blimp-1 and IRF4 jointly regulate the effector Treg cell differentiation program. () Microarray analysis of Blimp-1–GFP+ Treg cells isolated from the Blimp1GFP/GFP or Blimp1+/GFP compartment of bone–marrow chimeric mice, presented as a heat map of the 30 genes with the greatest differences in expression (presented as normalized intensity (log2)). Data are from three experiments (mean). () Quantitative RT-PCR analysis of gene expression in Treg cell populations from Blimp1+/GFP and Blimp1GFP/GFP mice, presented as expression in Blimp-1–GFP+ cells relative to that in Blimp-1–GFP− Treg cells. NS, not significant (P = 0.13); *P = 0.03, **P = 0.02 and ***P = 0.01 (t-test). Data are representative of three experiments (mean ± s.d. of three biological replicates). (,) Flow cytometry of Treg cells from wild-type (Ly5.1+)–Blimp1GFP/GFP (Ly5.2+) or wild-type (Ly5.1+)–Blimp1+/GFP (Ly5.2+) mixed–bone marrow chimeras before or after infection with influenza virus (strain HKx31); cell gating, above plots; genotype or organ, left margins. Numbers in qua! drants indicate percent cells in each. Data are representative of three experiments with two to three mice each. () Comparative analysis of genes with different expression (DE) in the Blimp-1 data set (Blimp1GFP/GFP and Blimp1+/GFP mice) and the IRF4 data set (mice expressing Cre recombinase from the Foxp3 promoter, with loxP-flanked (fl) alleles: Irf4−/fl and Irf4+/fl); results are presented as normalized intensity (log2) relative to those of wild-type mice. Data are from three (Blimp1 DE) or two (Irf4 DE) experiments (mean). * Figure 7: Binding of IRF4 and Blimp-1 to regulatory regions in the Il10 and Ccr6 loci. () ChIP analysis of purified Irf4−/− and Irf4+/+ Treg cells, assessed with an IRF4-specific antibody and PCR primers specific for regulatory regions of Il10 and Ccr6. Numbers above bars indicate enrichment relative to that in Irf4−/− T cells. () ChIP analysis of the Il10 locus with antiserum specific for acetylation of histone H3 at Lys9 (H3K9ac) or trimethylation of histone H3 at Lys4 (H3K4me3) or Lys27 (H3K27me3). () Binding of Blimp-1 to intron 1 of Il10, analyzed by streptavidin-mediated ChIP analysis of in vitro–activated Treg cells from Blimp1Bio/BioRosa26BirA/BirA mice. Input DNA and precipitated DNA were quantified by real-time PCR with primer pairs amplifying a conserved sequence in Il10 intron 1 or a control sequence in the 3′ region of Cd19; the same chromatin was used for control ChIP experiments with immunoglobulin G–coupled Dynabeads. Precipitated DNA is presented relative to input DNA. Data are representative of three to four independent experime! nts (,) or two independent experiments (; mean and s.d.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE27143 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this study. * Erika Cretney & * Annie Xin Affiliations * The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. * Erika Cretney, * Annie Xin, * Wei Shi, * Frederick Masson, * Maria Miasari, * Gabrielle T Belz, * Gordon K Smyth, * Stephen L Nutt & * Axel Kallies * Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia. * Erika Cretney, * Annie Xin, * Frederick Masson, * Maria Miasari, * Gabrielle T Belz, * Stephen L Nutt & * Axel Kallies * Department of Computer Science and Software Engineering, The University of Melbourne, Parkville, Victoria, Australia. * Wei Shi * Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria. * Martina Minnich & * Meinrad Busslinger * Department of Mathematics and Statistics, The University of Melbourne, Parkville, Victoria, Australia. * Gordon K Smyth Contributions E.C., A.X., M.M., F.M., M.M. and A.K. designed and did experiments; W.S. and G.K.S. analyzed the microarray data; G.T.B. and M.B. designed experiments; and S.L.N. and A.K. designed experiments and wrote the paper and contributed equally to this work. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Axel Kallies or * Stephen L Nutt Author Details * Erika Cretney Search for this author in: * NPG journals * PubMed * Google Scholar * Annie Xin Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Shi Search for this author in: * NPG journals * PubMed * Google Scholar * Martina Minnich Search for this author in: * NPG journals * PubMed * Google Scholar * Frederick Masson Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Miasari Search for this author in: * NPG journals * PubMed * Google Scholar * Gabrielle T Belz Search for this author in: * NPG journals * PubMed * Google Scholar * Gordon K Smyth Search for this author in: * NPG journals * PubMed * Google Scholar * Meinrad Busslinger Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen L Nutt Contact Stephen L Nutt Search for this author in: * NPG journals * PubMed * Google Scholar * Axel Kallies Contact Axel Kallies Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–12, Supplementary Tables 1–2 and Supplementary Methods Additional data - Control of the development of CD8αα+ intestinal intraepithelial lymphocytes by TGF-β
- Nat Immunol 12(4):312-319 (2011)
Nature Immunology | Article Control of the development of CD8αα+ intestinal intraepithelial lymphocytes by TGF-β * Joanne E Konkel1 * Takashi Maruyama1 * Andrea C Carpenter2 * Yumei Xiong2 * Brian F Zamarron1 * Bradford E Hall3 * Ashok B Kulkarni3 * Pin Zhang1 * Remy Bosselut2 * WanJun Chen1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:312–319Year published:(2011)DOI:doi:10.1038/ni.1997Received30 November 2010Accepted11 January 2011Published online06 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell–specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4−CD8−TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells. View full text Figures at a glance * Figure 1: Mice that lack TGF-β-signaling have fewer TCRαβ+CD8αα+ IELs. () Staining of CD8α versus CD8β on TCRβ+ cells in 2- to 3-week-old Tgfb1−/− mice and age-matched wild-type (WT) control littermates. (,) Frequency () and total number () of CD8αβ+ or CD8αα+ TCRαβ+ IELs in the mice in . () Staining of CD8α versus CD8β on TCRγδ+ cells from the mice in . (,) Frequency () and total number () of CD8αα+ TCRαβ+ IELs in the mice in (n ≥ 8 per genotype). () Staining of CD8α versus CD8β on TCRβ+ cells from 3- to 4-week-old Tgfbr1f/fCd4-Cre+ mice and age-matched control (Tgfbr1f/+CD4-Cre+) littermates. (,) Frequency () and total number () of CD8αβ+ or CD8αα+ TCRαβ+ IELs in the mice in . () Staining of CD8α versus CD8β on TCRγδ+ cells from the mice in . (,) Frequency () and total number () of CD8αα+ TCRαβ+ IELs in the mice in (n = 18 (Tgfbr1f/fCd4-Cre+) or 19 (Tgfbr1f/+Cd4-Cre+)). Numbers in quadrants (,,,) indicate percent cells in each. *P < 0.05 and **P < 0.005 (unpaired two-tailed Student's t-test). Data are r! epresentative of at least five independent experiments (mean and s.e.m. in ,,,,,,,). * Figure 2: Smad3−/− mice have fewer TCRαβ+CD8αα+ IELs. (a) Staining of CD8α versus CD8β on TCRβ+ cells from wild-type and Smad3−/− mice. Numbers in quadrants indicate percent cells in each. () Frequency of TCRβ+CD8αβ+, TCRβ+CD8αα+ and TCRβ+CD8− IELs in wild-type mice (n = 14) and Smad3−/− mice (n = 16). *P < 0.0007 (unpaired two-tailed Student's t-test). Data are representative of at least three independent experiments (mean and s.e.m. in ). * Figure 3: Overexpression of TGF-β1 from T cells leads to a larger population of TCRαβ+CD8αα+ IELs. () Staining of CD8α versus CD8β on TCRβ+ cells from 8-week-old β1glo+Cd4-Cre+ mice and age-matched control littermates (β1glo−Cd4-Cre+, β1glo+Cd4-Cre− or β1glo−Cd4-Cre−). Numbers in quadrants indicate percent cells in each. (,) Frequency () and total number () of CD8αβ+ or CD8αα+ TCRαβ+ IELs in the β1glo+Cd4-Cre+ mice (n = 7) and control littermates (n = 8) in . *P = 0.0331 and **P = 0.0003 (unpaired two-tailed Student's t-test). Data are representative of at least three independent experiments (mean and s.e.m. in ,). * Figure 4: TGF-β-deficient mice have a smaller population of DN TCRαβ+CD5+ thymocytes. () Gating of DN TCRαβ+CD5+ thymocytes. Numbers adjacent to outlined areas indicate percent cells in each. SSC, side scatter; FSC, forward scatter. () Frequency (left) and total number (middle) of DN TCRαβ+CD5+ thymocytes, and frequency of all DN thymocytes (right), in Tgfb1−/− mice (n = 14) and their wild-type control littermates (n = 12). *P < 0.01 (unpaired two-tailed Student's t-test). () Staining of annexin-V (AnnV) versus 7-amino-actinomycin D (7-AAD) on DN TCRαβ+CD5+ thymocytes from wild-type and Tgfb1−/− mice. Numbers in quadrants indicate percent cells in each. (,) Staining of Bcl-2 () and Bim () on DN TCRαβ+CD5+ thymocytes from wild-type and Tgfb1−/− mice. (–) Staining of Ki67 (), CD127 (), α4β7 () and CD103 () on DN TCRαβ+CD5+ thymocytes from wild-type and Tgfb1−/− mice. Data are representative of two to five experiments with at least two mice per group. * Figure 5: TGF-β induces CD8α expression on DN TCRαβ+CD5+ thymocytes. Quantitative PCR analysis of the expression of CD8α (), CD8β () and Th-POK () in flow cytometry–sorted DN TCRαβ+CD5+ thymocytes from wild-type thymi stimulated overnight with anti-CD3 (α-CD3; 1 μg/ml) in the presence or absence of TGF-β1 (2 ng/ml); results are presented relative to those of control cultures without TGF-β1 treatment. *P ≤ 0.0244 (paired two-tailed Student's t-test). Data are representative of two independent experiments. * Figure 6: TGF-β is needed to maintain expression of CD8 on peripheral T cells. () Staining of CD4 versus CD8α (top) or CD8β versus CD8α (bottom) on TCRβ+ splenocytes from wild-type and Tgfb1−/− mice. () Staining of CD8α, CD8β and CD4 on wild-type control cells and Tgfb1−/− cells gated in . () Staining as in on TCRβ+ splenocytes from Tgfbr1f/+Cd4-Cre+ and Tgfbr1f/fCd4-Cre+ mice. () Staining as in on Tgfbr1f/+Cd4-Cre+ and Tgfbr1f/fCd4-Cre+ cells gated in . Outlined areas (gates) on top row (,) indicate CD4+CD8α− cells (top left) or CD4−CD8α+ cells (bottom right); outlined areas (gates) on bottom row (,) indicate CD8αβ+ cells. Data represent at least three independent experiments with at least two mice per group. * Figure 7: TGF-β induces expression of CD8α on peripheral CD4+ T cells in a Smad3-dependent manner. () Staining of CD4 versus CD8α (left) on CD4+CD25− cells cultured for 4 d with anti-CD3 in the presence or absence of TGF-β1 (2 ng/ml). Numbers adjacent to outlined areas indicate percent CD4+ cells in the starting population (left) or CD4+CD8α+ TCRαβ+ cells (middle and right). Far right, frequency of CD8β− (CD8αα+) cells or CD8β+ (CD8αβ+) cells in the CD4+CD8α+ TCRβ+ population. Data are representative of five independent experiments. () Quantitative PCR analysis of the expression of CD8α and CD8β in CD4+CD25− cultures at 6 h and 48 h, stimulated with anti-CD3 alone (Control) or anti-CD3 plus TGF-β1 (TGF-β1); results are presented relative to those of control cultures. *P < 0.005 (paired two-tailed Student's t-test). Data are representative of four independent experiments (mean and s.e.m.). () Staining of CD4 versus CD8α (left) on CD4+CD25− cells from Smad3−/− mice or wild-type control littermates, cultured for 4 d with anti-CD3 with or without! TGF-β1. Right, frequency of CD8α+CD4+TCRβ+ cells after culture with anti-CD3 and various concentrations (horizontal axis) of TGF-β1. *P < 0.004 (unpaired two-tailed Student's t-test). Data represent three independent experiments (mean and s.e.m.). () Quantitative PCR analysis of CD8α expression in wild-type and Smad3−/− CD4+CD25− T cell cultures stimulated for 6 h with anti-CD3 alone or anti-CD3 plus TGF-β1, presented relative to expression in control cultures without TGF-β1 treatment. *P < 0.005 (paired two-tailed Student's t-test). Data are representative of two independent experiments (mean and s.e.m.). * Figure 8: Expression of Th-POK and Runx3 in CD4+CD8α− and CD4+CD8α+ cells. () Expression of Runx3-RFP in CD4+CD8α− or CD4+CD8α+ CD4+CD25− cells cultured for 4 d without stimulation (media only) or with anti-CD3 in the presence (α-CD3+TGF-β1) or absence (α-CD3+media) of TGF-β1 (2 ng/ml). () Expression of Th-POK–GFP in CD4+CD8α− cells cultured for 4 d without stimulation (media only) or with anti-CD3 in the presence (+α-CD3+TGF-β1) or absence (+α-CD3+media) of TGF-β1 (2 ng/ml). () Expression of CD4 and CD8α (left) by cells cultured as in . Numbers adjacent to outlined areas indicate percent CD4+CD8α− cells (left) and CD4+CD8α+ cells (right). Right, Th-POK–GFP expression in CD4+CD8α− and CD4+CD8α+ cells. (,) Frequency of CD8α+CD4+TCRβ+ cells in cultures of CD4+CD25− cells sorted from wild-type mice (Zbtb7b+/+) and mice heterozygous for Th-POK deficiency (Zbtb7b+/−; ) and from Th-POK-transgenic mice and control littermates (), cultured for 4 d with anti-CD3 (left) or anti-CD3 plus anti-CD28 (right) in the presence or! absence of TGF-β1 (1 ng/ml). *P < 0.0202 (unpaired two-tailed Student's t-test). Data are representative of three (–) or two (,) independent experiments (mean and s.e.m. in ,). Author information * Abstract * Author information * Supplementary information Affiliations * Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA. * Joanne E Konkel, * Takashi Maruyama, * Brian F Zamarron, * Pin Zhang & * WanJun Chen * Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. * Andrea C Carpenter, * Yumei Xiong & * Remy Bosselut * Functional Genomics Section, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA. * Bradford E Hall & * Ashok B Kulkarni Contributions J.E.K. designed and did experiments, analyzed data and wrote the manuscript; T.M., B.F.Z. and P.Z. did experiments; B.E.H. and A.B.K. provided critical materials; A.C.C. and Y.X. did experiments and provided critical materials, R.B. provided materials and read the manuscript; and W.C. initiated and directed the research, designed experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * WanJun Chen Author Details * Joanne E Konkel Search for this author in: * NPG journals * PubMed * Google Scholar * Takashi Maruyama Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea C Carpenter Search for this author in: * NPG journals * PubMed * Google Scholar * Yumei Xiong Search for this author in: * NPG journals * PubMed * Google Scholar * Brian F Zamarron Search for this author in: * NPG journals * PubMed * Google Scholar * Bradford E Hall Search for this author in: * NPG journals * PubMed * Google Scholar * Ashok B Kulkarni Search for this author in: * NPG journals * PubMed * Google Scholar * Pin Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Remy Bosselut Search for this author in: * NPG journals * PubMed * Google Scholar * WanJun Chen Contact WanJun Chen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–10 Additional data - RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota
- Nat Immunol 12(4):320-326 (2011)
Nature Immunology | Article RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota * Shinichiro Sawa1, 2 * Matthias Lochner1, 2, 3 * Naoko Satoh-Takayama4, 5 * Sophie Dulauroy1, 2 * Marion Bérard6 * Melanie Kleinschek7 * Daniel Cua7 * James P Di Santo4, 5 * Gérard Eberl1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:320–326Year published:(2011)DOI:doi:10.1038/ni.2002Received27 August 2010Accepted01 February 2011Published online20 February 2011Corrected online27 February 2011 Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Lymphoid cells that express the nuclear hormone receptor RORγt are involved in containment of the large intestinal microbiota and defense against pathogens through the production of interleukin 17 (IL-17) and IL-22. They include adaptive IL-17-producing helper T cells (TH17 cells), as well as innate lymphoid cells (ILCs) such as lymphoid tissue–inducer (LTi) cells and IL-22-producing NKp46+ cells. Here we show that in contrast to TH17 cells, both types of RORγt+ ILCs constitutively produced most of the intestinal IL-22 and that the symbiotic microbiota repressed this function through epithelial expression of IL-25. This function was greater in the absence of adaptive immunity and was fully restored and required after epithelial damage, which demonstrates a central role for RORγt+ ILCs in intestinal homeostasis. Our data identify a finely tuned equilibrium among intestinal symbionts, adaptive immunity and RORγt+ ILCs. View full text Figures at a glance * Figure 1: RORγt+ ILCs are the main producers of IL-22 in the intestine. () Flow cytometry analysis of intracellular staining of IL-22 in small intestine lamina propria leukocytes (SI-LPLs) from E15, 2-week-old and 8-week-old Rorc-GFP mice, assessed after 3 h of stimulation with IL-23 ex vivo (left), and of the expression of CD3ε and CD4 by IL-22+RORγt+ cells (right; outlined in red at left). Numbers in quadrants indicate percent cells in each. () Quantitative PCR analysis of Il22 expression in the terminal ileum of 8-week-old wild-type mice (WT), RAG-2-deficient mice (RAG-2-KO), RORγt-deficient mice (RORγt-KO) and mice deficient in both RAG-2 and RORγt (RAG-2–RORγt–DKO); results are presented relative to those for Hsp90ab1 (heat-shock protein 90) and Gapdh (glyceraldehyde phosphate dehydrogenase). ND, not detected. NS, not significant (unpaired t-test). () Quantitative PCR analysis of the expression of Reg3b and Reg3g by epithelial cells from 8-week-old wild-type, RAG-2-deficient and RORγt-deficient mice; results are presented relativ! e to those for Hsp90ab1 and Gapdh. *P < 0.05 (unpaired t-test). () Surface expression of CD4 and NKp46 (right) on RORγt+ ILCs (GFP+CD3ε− cells) sorted at left. Numbers adjacent to outlined areas and in quadrants indicate percent cells in each. Data are representative of three independent experiments () or two (,) or four () experiments with two mice per group (mean and s.e.m. of triplicates in ,). * Figure 2: The production of proinflammatory cytokines by RORγt+ ILCs. (,) Real-time quantitative PCR analysis of Il22 () and Il17a () transcript expression (left) and flow cytometry analysis IL-22 () and IL-17 () protein expression (right) by SI-LPLs obtained from E15, 2-week-old and 8-week-old Rorc-GFP mice and stimulated for 3 h ex vivo with recombinant IL-23 or the phorbol ester PMA plus ionomycin, respectively. PCR results (left) are presented relative to those for Hsp90ab1 and Gapdh; numbers above bracketed lines (right) indicate percent IL-22+ cells () or IL-17+ cells (; mean ± s.d. of ten mice). Blue (, right), isotype-matched control antibody. LTi4, CD4+ LTi. () Real-time quantitative PCR analysis of the expression of transcripts encoding lymphotoxin-α (Lta) and IFN-γ (Ifng); results are presented relative to those for Hsp90ab1 and Gapdh. *P < 0.05 (unpaired t-test). Data are representative of five (flow cytometry) or three (PCR) independent experiments (error bars, s.e.m.). * Figure 3: The microbiota represses IL-22 production by RORγt+ ILCs. () Absolute number of RORγt+ ILCs and CD4+ T cells among SI-LPLs from 10-week-old germ-free (GF) and SPF mice (left) and in 6-week-old antibiotic-treated (+Abx) and untreated (control) mice (right). *P < 0.05 (unpaired t-test). () Quantitative PCR analysis of Il22 expression by RORγt+ ILCs and CD4+ T cells from 8-week-old SPF or germ-free Rorc-GFP mice (n = 3 per group) and flow cytometry analysis of IL-22 expression by RORγt+ ILCs and CD4+ T cells obtained from the preceding mice or from untreated or antibiotic-treated Rorc-GFP mice and stimulated for 3 h ex vivo with IL-23 (n = 4 mice per group). PCR results are presented relative to those for Hsp90ab1 and Gapdh; numbers above bracketed lines indicate percent IL-22+ cells (mean ± s.d.). *P < 0.05 (Student's t-test). () Expression of Il17a by RORγt+ ILCs and CD4+ T cells from SPF or germ-free Rorc-GFP mice; results are presented relative to those for Hsp90ab1 and Gapdh. Data are compiled from two experiments with four ! mice per group (; error bars, s.e.m.) or are representative of three experiments ( (mean and s.e.m. of triplicates) and (mean and s.e.m.)). * Figure 4: The microbiota represses the activity of RORγt+ ILCs via IL-25. () Quantitative PCR analysis of Il25 expression by epithelial cells isolated by laser-capture microdissection from the terminal ileum of 6-week-old SPF or germ-free C57BL/6 mice (left) or 2-week-old or 8-week-old SPF C57BL/6 mice (right; n = 2 per group); results are presented relative to those for Hsp90ab1 and Gapdh. () Absolute number of RORγt+ ILCs among SI-LPLs from 8-week-old wild-type and IL-25-deficient (IL-25-KO) mice (n = 3 per group). () Expression of Il22 transcripts (right) and IL-22 protein (left) by RORγt+ ILCs isolated from 8-week-old wild-type and IL-25-deficient mice and analyzed without further treatment (Il22 analysis) or stimulated for 3 h ex vivo with IL-23 (IL-22 analysis). PCR results (right) are presented relative to those for Hsp90ab1 and Gapdh; numbers above bracketed lines (left) indicate percent IL-22+ cells (n = 4 mice per group; mean ± s.d.). () IL-22 production by RORγt+ ILCs from 6-week-old Rorc-GFP mice (n = 6) treated with PBS or recombi! nant IL-25. Numbers above bracketed lines indicate percent IL-22+ cells (mean ± s.e.m.). () Expression of Reg3b and Reg3g by epithelial cells from 6-week-old mice treated with PBS or recombinant IL-25; results are presented relative to those for Hsp90ab1 and Gapdh. () Expression of IL-17RB (left) by lineage-negative (CD5−B220−CD11b−Gr-1−Ter119−) cells from 6-week-old Rorc-GFP mice (top row) and by CD3ε−CD19− SI-LPLs from wild-type mice (bottom row) treated with PBS or recombinant IL-25. Numbers in quadrants indicate percent cells in each. Right, histology of cryptopatches in terminal ileum sections from 4-week-old Rorc-GFP mice. Scale bar, 50 μm. (,) Quantitative PCR analysis of Il22 expression by RORγt+ ILCs isolated from 4-week-old Rorc-GFP mice and cultured for 3 d in the presence of PBS or recombinant IL-25, with IL-17RB+CD11c+ cells in the same well (, right; , left) or separated by a Transwell (pore size, 0.4 μm; , right), followed by resorting of R! ORγt+ ILCs; results are presented relative to those for Hsp90! ab1 and Gapdh. *P < 0.05 (unpaired t-test). Data are representative of two ( (mean and s.e.m. of triplicates) and (mean and s.e.m.)) or three (, right, and , (mean and s.e.m. in )) experiments, or two (, left) or three () independent experiments, or are the compilation of two independent experiments (,; mean and s.e.m. of triplicate wells). * Figure 5: Adaptive immunity represses the activity of RORγt+ ILCs. () Intracellular IL-22 expression by SI-LPLs obtained from 6-week-old wild-type or RAG-2-deficient Rorc-GFP mice (n = 6 per group) and stimulated for 3 h ex vivo with IL-23. Numbers above bracketed lines indicate percent IL-22+ cells (mean ± s.d.). Data are representative of three independent experiments. () Absolute number of RORγt+ ILCs cells from 6-week-old wild-type or RAG-2-deficient mice (n = 4). *P < 0.05 (unpaired t-test). Data are representative of two experiments (mean and s.e.m.). () Expression of IL-22 by RORγt+ ILCs from RAG-2-deficient Rorc-GFP mice (n = 6 per group) treated with PBS or recombinant IL-25. Numbers above bracketed lines indicate percent IL-22+ cells (mean ± s.d.). Data are representative of three independent experiments. * Figure 6: Epithelial damage de-represses the activity of RORγt+ ILCs. () Quantitative PCR analysis of the expression of Il22, Il23 and Il25 in total terminal ileum (left and middle) or microdissected epithelial cells (right) from the terminal ileum of mice (n = 2 per group) treated with H2O or DSS; results are presented relative to those for Hsp90ab1 and Gapdh. () Expression of Il22 transcripts (right) and IL-22 protein (left) by RORγt+ ILCs isolated from 8-week-old Rorc-GFP mice (n = 6 per group) treated with water or DSS. PCR results (right) are presented relative to those for Hsp90ab1 and Gapdh; numbers above bracketed lines (left) indicate percent IL-22+ cells (mean ± s.d.). () Absolute number of RORγt+ ILCs among SI-LPLs from 8-week-old mice (n = 3 per group) treated with H2O or DSS.). () Expression of IL-22 by RORγt+ ILCs from 6-week-old Rorc-GFP mice (n = 6 per group) treated with DSS and injected daily with PBS or recombinant IL-25. Numbers above bracketed lines (left) indicate percent IL-22+ cells (mean ± s.e.m.). *P < 0.05 (unpa! ired t-test). Data are representative of two experiments ( (mean and s.e.m. of triplicates) and (mean and s.e.m.)), three experiments (, right (error bars, s.e.m.)), two independent experiments (, right (error bars, s.e.m.)) or three independent experiments (, (left)). * Figure 7: The activity of RORγt+ ILCs is required for protection and recovery from colitis. (,) Body weight of C57BL/6 mice (B6) injected with PBS or recombinant IL-25 () or of RAG-2-deficient mice injected with PBS or recombinant IL-25 or PBS, or mice deficient in both RAG-2 and RORγt, all treated with DSS (). †, death of an IL-25-injected RAG-2-deficient mouse day 9. *P < 0.05 (unpaired t-test). Data are representative of two experiments with five mice per group (mean ± s.e.m.). () Expression of IL-22 by RORγt+ ILCs from 9-week-old RAG-2-deficient mice (n = 3 per group) injected with PBS or recombinant IL-25 every 2 d and treated with DSS; cells were stimulated for 3 h ex vivo with PMA and ionomycin. Numbers above bracketed lines indicate percent IL-22+ cells (mean ± s.d.). Data are representative of three experiments. () Absolute number of RORγt+ ILCs from the small intestinal lamina propria of RAG-2-deficient mice injected with PBS or recombinant IL-25 and treated with DSS (n = 3 mice per group). *P < 0.05 (unpaired t-test). Data are representative of th! ree experiments (mean and s.e.m.). Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Entrez Nucleotide * NM_008302 * NM_008084 * NM_010735 * NM_011613 * NM_016971 * NM_010552 * NM_145856 * NM_031252 * NM_080729 * NM_011036 * NM_011260 * NM_011281 * NM_144548 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationErratum 27 February 2011In the version of this article initially published online, the first sentence of the abstract was incorrect. This sentence should begin "Lymphoid cells that express the nuclear hormone receptor RORγt...." The error has been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Affiliations * Institut Pasteur, Lymphoid Tissue Development Unit, Paris, France. * Shinichiro Sawa, * Matthias Lochner, * Sophie Dulauroy & * Gérard Eberl * Centre National de la Recherche Scientifique, Unités de Recherche Associées 1961, Paris, France. * Shinichiro Sawa, * Matthias Lochner, * Sophie Dulauroy & * Gérard Eberl * Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research (joint venture of the Medical School Hannover and the Helmholtz Centre for Infection Research), Hannover, Germany. * Matthias Lochner * Institut Pasteur, Innate Immunity Unit, Paris, France. * Naoko Satoh-Takayama & * James P Di Santo * Institut National de la Santé et de la Recherche Médicale U668, Paris, France. * Naoko Satoh-Takayama & * James P Di Santo * Institut Pasteur, Animalerie Centrale, Paris, France. * Marion Bérard * Merck Research Laboratories, DNAX Discovery Research, Palo Alto, California, USA. * Melanie Kleinschek & * Daniel Cua Contributions S.S. and G.E. designed the study and wrote the manuscript; S.S. did most of the experimental work; M.L. contributed to the analysis of T cells and DSS-mediated colitis; N.S.-T. contributed to the analysis of NKp46+ ILCs; S.D. did laser-capture microdissection; M.B. generated germ-free mice; M.K. and D.C. generated and provided IL-25-deficient mice; and J.P.D.S. contributed to data analysis and manuscript writing. Competing financial interests D.C. and M.K. are employees of Merck Research Laboratories, DNAX Discovery Research. Corresponding author Correspondence to: * Gérard Eberl Author Details * Shinichiro Sawa Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Lochner Search for this author in: * NPG journals * PubMed * Google Scholar * Naoko Satoh-Takayama Search for this author in: * NPG journals * PubMed * Google Scholar * Sophie Dulauroy Search for this author in: * NPG journals * PubMed * Google Scholar * Marion Bérard Search for this author in: * NPG journals * PubMed * Google Scholar * Melanie Kleinschek Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Cua Search for this author in: * NPG journals * PubMed * Google Scholar * James P Di Santo Search for this author in: * NPG journals * PubMed * Google Scholar * Gérard Eberl Contact Gérard Eberl Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5 Additional data - CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs
- Nat Immunol 12(4):327-334 (2011)
Nature Immunology | Article CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs * Jie Sun1, 2 * Haley Dodd1 * Emily K Moser1 * Rahul Sharma3 * Thomas J Braciale1, 2, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:327–334Year published:(2011)DOI:doi:10.1038/ni.1996Received12 November 2010Accepted11 January 2011Published online06 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin (IL)-10 is an important regulatory cytokine that can modulate excessive immune mediated injury. Several distinct cell types have been demonstrated to produce IL-10, including most recently CD8+ cytotoxic T lymphocytes (CTLs) responding to respiratory virus infection. Here we report that CD4+ T cell help in the form of IL-2 is required for IL-10 production by CTLs, but not for the induction of CTL effector cytokines. We show that IL-2 derived from CD4+ helper T cells cooperates with innate immune cell–derived IL-27 to amplify IL-10 production by CTLs through a Blimp-1-dependent mechanism. These findings reveal a previously unrecognized pathway that coordinates signals derived from innate and helper T cells to control the production of a regulatory cytokine by CTLs during acute viral infection. View full text Figures at a glance * Figure 1: Induction of IL-10-producing CTLs in vivo requires IL-27 and CD4+ T cells. () Production of IL-10 and IFN-γ by CTLs from MLNs or lungs of WT or Il12a−/− mice infected with influenza, measured 7 d.p.i. by intracellular staining (ICS). (,) Production of IL-10 and IFN-γ by CTLs from MLNs or lungs of WT or Ebi3−/− mice infected with influenza, measured 7 d.p.i. by intracellular staining (ICS). () Normalized percentages of IL-10+ cells in influenza-specific CTLs (IFN-γ+) from MLNs or lungs of infected WT and Ebi3−/− mice. () Production of IL-10 and IFN-γ by CTLs from MLNs or lungs of WT or Il21r−/− mice infected with influenza, measured 7 d.p.i. by ICS. (–) Production of IL-10 and IFN-γ by CTLs from MLNs or lungs of WT mice, Ebi3−/− mice or Ebi3−/− mice depleted of CD4+ T cells with anti-CD4 (α-CD4), infected with influenza and measured 7 d.p.i. by ICS. () Normalized percentages of IL-10+ cells in influenza-specific CTLs (IFN-γ+) from MLNs or lungs of WT and Ebi3−/− mice. () Mean fluorescence intensity (MFI) of IL-10! in IL-10+ cells from infected lungs. Numbers are the percentages of cells in gated population. *P ≤ 0.05; **P ≤ 0.01. Data are from one experiment but are typical of at least two. Error bars, s.e.m. * Figure 2: CD4+ T cell help is selectively required for the induction of IL-10-producing CTLs in vivo. (,) Production of IL-10, IFN-γ and TNF () by CTLs from MLNs or lungs of WT mice injected with rat immunoglobulin G (IgG) or anti-CD4 to deplete CD4+ T cells and infected with influenza, measured 8 d.p.i. by ICS. Numbers are the percentages of cells in gated population. () Normalized percentages of IL-10+ or TNF+ cells in influenza-specific CTLs (IFN-γ+) from MLNs or lungs. () Normalized percentages of IL-10+ or TNF+ cells in influenza-specific CTLs (IFN-γ+) from lungs of WT and H2-Ab1−/− mice infected with influenza, 7 d.p.i. () Percentages of cells 7 d.p.i. positive for IL-10–enhanced green fluorescent protein (eGFP) in influenza-specific PA224 or NP366 tetramer+ cells from Vert-X mice injected with rat IgG or anti-CD4 and infected with influenza. () Percentages of IL-10–eGFP+ cells among influenza-specific lung PA224 or NP366 (nucleoprotein peptide 366–374) tetramer+ cells from H2-Ab1+/− Vert-X or H2-Ab1−/− Vert-X mice infected with influenza, 7 d.p.i. (! ) Percentages of IFN-γ–eYFP+ cells among influenza-specific lung PA224 (polymerase A peptide 224–232) or NP366 tetramer+ cells from Yeti mice injected with rat IgG or anti-CD4 and infected with influenza, 7 d.p.i. *P ≤ 0.05; **P ≤ 0.01. Data in –, are from one experiment but are typical of three. Error bars, s.e.m. Data in are pooled from four experiments. * Figure 3: IL-2 provides the help from CD4+ T cell to CTLs for IL-10 production in vitro. () Production of IL-10 and IFN-γ by CL-4 cells, measured through ICS. CFSE-labeled CD8+ CL-4 cells were stimulated with influenza-infected DCs in the presence or absence of CD4+ TS-1 cells for 4 d. The cultured cells were treated with antibody to mouse IL-2 (α-mIL-2) or with antibody plus human IL-2 (hIL-2) as indicated at top. (,) Percentages of IL-10–eGFP+ cells in V-OT-I cells in CD8+ Vert-X-OT-I (V-OT-I) cells stimulated with influenza-OVA–infected DCs in the absence or presence of OT-II cells (), and release of IL-2 into medium after 2 d in culture, measured by ELISA (). () Expression of IL-10–eGFP by V-OT-I cells after 2 d in culture, measured by flow cytometry. V-OT-I or OT-II cells were activated separately by influenza-OVA–infected DCs for 4 d. The activated V-OT-I cells were unmanipulated or were cultured with the activated OT-II cells. Cells were then either left unstimulated or stimulated with plate-bound anti-CD3. The cultured cells were treated as ind! icated at top. () Release of IL-2 into medium after culture overnight of cells treated as in , measured by ELISA. () Expression of Il10, measured by quantitative RT-PCR. OT-I cells were stimulated with influenza-OVA–infected DCs. After 4 d in culture, OT-I cells were treated with human IL-2 or with human IL-2 plus plate-bound anti-CD3 for 4 h. Numbers are the percentages of cells in the gated population. Data are from one experiment but are representative of at least three replicates. Error bars, s.e.m. * Figure 4: IL-2 is required for the induction IL-10-producing CTLs in vivo. (,) IL-2 production by gated Thy-1+ cells from influenza-infected lung, measured by ICS. () IL-2 production by Thy-1+CD4+ and Thy-1+CD4− cells. () IL-2 mean fluorescence intensity. () IL-2 production by cultured MLN and lung cells from influenza-infected mice treated with antibodies, determined by ELISA 6 d.p.i. () Normalized percentages of IL-10+ cells among influenza-specific CTLs (IFN-γ+) from influenza-infected lungs after transfer of WT or Il2ra−/− T cells into Thy-1.1 mice, 8 d.p.i. (,) Production of IL-10, TNF and IFN-γ by CTLs, measured 7 d.p.i. by ICS for WT:Il2ra−/− chimeric mice infected with influenza. () Normalized percentages of IL-10+ or TNF+ cells among lung influenza-specific (IFN-γ+) CTLs. () Normalized percentages of lung IL-10+ cells among specific IFN-γ+CD8+ T cells 7 d.p.i., after infection of WT:Il2ra−/− chimeric mice. (,) Expression of IL-10–eGFP by CTLs, measured by flow cytometry. After anti-CD4 treatment and influenza infection,! Vert-X mice received IL-2 or PBS at 5 and 6 d.p.i. () Percentages of IL-10–eGFP+ cells among gated CTLs, 7 d.p.i. Numbers represent percentages of cells in gated population. *P ≤ 0.05; **P ≤ 0.01. Error bars, s.e.m. Data are representative of at least two experiments. * Figure 5: IL-2 and IL-27 synergistically induce IL-10 production by both mouse and human CTLs. (,) Production of IL-10 and IFN-γ by CFSE-labeled CD8+ CL-4 cells stimulated with influenza-infected DCs in the presence or absence of CD4+ TS-1 cells for 4 d, measured through ICS (). The cultured cells were treated as indicated at top. Numbers are the percentages of cells in gated population. () The mean fluorescence intensity of IL-10 in the IL-10+ cells. () Expression of transcripts for IL-27 subunits Ebi3 and Il27 (p28) measured through quantitative RT-PCR in various cell types, sorted as described in Online Methods, from lungs of infected WT mice. MΦ, macrophage. () IL-27 p28, measured 6 d.p.i. through ELISA, in BALF from WT mice injected with rat IgG or anti-CD4 and infected with influenza. () IL-10, measured by ELISA, in medium from purified human CD8+ T cells stimulated for 3 d with anti-CD3 plus anti-CD28 under the conditions indicated at bottom. Data in – are representative of at least three separate experiments. Data in are representative of at least two sepa! rate experiments using an additional donor. Error bars, s.e.m. * Figure 6: Induction of IL-10-producing CTLs by IL-2 and IL-27 is Blimp-1 dependent. () Expression of Il10 and Prdm1, measured by quantitative RT-PCR. Vert-X mice were infected with influenza. At 7 d.p.i., lung CD8+CD44hiIL-10–eGFP− cells and CD8+CD44hiIL-10–eGFP+ cells were sorted by flow cytometry. () Expression of Il10, Prdm1, Ifng and Tbx21 (encoding T-bet), determined 7 d.p.i. by quantitative RT-PCR in lung CD8+ cells isolated from influenza-infected H2-Ab1+/− or H2-Ab1−/− mice. () Expression of Il10, Prdm1, Ifng and Maf, determined by quantitative RT-PCR 7 d.p.i., in lung CD8+ cells isolated from WT or Ebi3−/− mice infected with influenza. () Expression of Prdm1, measured by quantitative RT-PCR, in OT-I cells cultured with influenza-OVA–infected DCs in the absence or presence of human IL-2, IL-27 or human IL-2 plus IL-27 for 4 d. () IL-10 production by OT-I cells, measured by ICS. OT-I cells were transduced with control human CD2 (hCD2)-expressing (vector) or hCD2–Blimp-1–expressing (Blimp-1) retrovirus, then cultured for 3 d. hCD! 2−, untransduced cells; hCD2+, transduced cells. () IL-10 production by CTLs, measured by ICS. CFSE-labeled CD8+ T cells from WT or Cd4-cre Prdm1fl/fl (Prdm1 cKO) mice were stimulated with DCs plus soluble anti-CD3 for 4 d under the conditions indicated at top. Numbers are the percentages of cells in the gated population. Data are representative of at least two independent experiments. *P 0.05. Error bars, s.e.m. * Figure 7: Blimp-1 deficiency in T cells results in diminished IL-10 production and enhanced pulmonary inflammation. Cd4-cre Prdm1fl/+ (control) or Prdm1 cKO mice were infected with influenza. () Production of IL-10 and IFN-γ by CTLs 7 d.p.i., measured by ICS. Numbers are the percentages of cells in gated population. () Normalized percentages of IL-10+ or TNF+ cells in influenza-specific CTLs (IFN-γ+) in control or Prdm1 cKO mice. () IL-10 and IFN-γ in BALF, measured 7 d.p.i. through ELISA. () Numbers of lung monocytes and neutrophils, measured 9 d.p.i. through flow cytometry. () Normalized percentages of IL-10+ cells in influenza-specific CTLs (IFN-γ+) from infected lungs. WT or Prdm1 cKO T cells were transferred into Thy-1.1+ WT mice and infected with influenza. At 7 d.p.i., the production of IL-10 and IFN-γ by CTLs was measured by ICS after restimulation with influenza-infected bone marrow DCs. Data in , are from three pooled experiments. Data in are pooled from two experiments. *P ≤ 0.05; **P ≤ 0.01. Error bars, s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * The Beirne B. Carter Center for Immunology Research, The University of Virginia, Charlottesville, Virginia, USA. * Jie Sun, * Haley Dodd, * Emily K Moser & * Thomas J Braciale * Department of Pathology, The University of Virginia, Charlottesville, Virginia, USA. * Jie Sun & * Thomas J Braciale * Department of Medicine, The University of Virginia, Charlottesville, Virginia, USA. * Rahul Sharma * Department of Microbiology, The University of Virginia, Charlottesville, Virginia, USA. * Thomas J Braciale Contributions J.S. designed the project, performed most of the experimental work, analyzed the data and wrote the manuscript. H.D. and E.K.M. performed some of the quantitative RT-PCR and ELISA experiments. R.S. contributed to reagents and suggestions. T.J.B. supervised the project, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas J Braciale Author Details * Jie Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Haley Dodd Search for this author in: * NPG journals * PubMed * Google Scholar * Emily K Moser Search for this author in: * NPG journals * PubMed * Google Scholar * Rahul Sharma Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas J Braciale Contact Thomas J Braciale Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–22 Additional data - IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during infection with Escherichia coli strain O157:H7
- Nat Immunol 12(4):335-343 (2011)
Nature Immunology | Article IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during infection with Escherichia coli strain O157:H7 * Fengyi Wan1, 2 * Amanda Weaver1 * Xiaofei Gao3 * Michael Bern1 * Philip R Hardwidge3 * Michael J Lenardo1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:335–343Year published:(2011)DOI:doi:10.1038/ni.2007Received18 January 2011Accepted09 February 2011Published online13 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg NF-κB is a major gene regulator in immune responses, and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how nuclear translocation of RPS3 is regulated. Here we report that phosphorylation of RPS3 Ser209 by the kinase IKKβ was crucial for nuclear localization of RPS3 in response to activating stimuli. Moreover, virulence protein NleH1 of the foodborne pathogen Escherichia coli strain O157:H7 specifically inhibited phosphorylation of RPS3 Ser209 and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but resulting in less mortality in a gnotobiotic piglet-infection model. Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promoted specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7. View full text Figures at a glance * Figure 1: RPS3 is phosphorylated and associates with IKKβ in response to NF-κB activation. () 32P-labeling assay of HEK293T cells stimulated for 0–30 min (above lanes) with TNF (20 ng/ml), followed by immunoprecipitation (IP) of whole-cell lysates with antibody to RPS3 (anti-RPS3) and autoradiography (top) or immunoblot analysis with anti-RPS3 (bottom). () Immunoassay of whole-cell lysates of Jurkat cells stimulated for 0–15 min (above lanes) with TNF and analyzed by immunoblot for total or phosphorylated (p-) proteins directly (Input) or after immunoprecipitation with anti-RPS3. () Immunoprecipitation and immunoblot analysis of the interaction between hemagglutinin (HA)-tagged RPS3 and Flag-tagged IKKβ in HEK293T cells. () Immunoassay of whole-cell lysates of Jurkat cells stimulated as in and analyzed by immunoblot for IKKα, IKKβ or RPS3 directly or after immunoprecipitation with anti-RPS3. Data are representative of at least two independent experiments. * Figure 2: IKKβ kinase activity is required for the nuclear translocation of RPS3. () Confocal microscopy (left) of RPS3 (red) and counterstained nuclei (blue) in Jurkat cells transfected for 72 h with siRNA specific for IKKα or IKKβ or scrambled nonspecific siRNA (NSp), then left untreated (Unstim) or stimulated with TNF (50 ng/ml) or with PMA (50 ng/ml) plus ionomycin (1.5 mM; PMA+I). Original magnification, ×630. Right, quantification of cells with nuclear RPS3. () Immunoblot analysis of whole-cell lysates (WCL) or nuclear subcellular fractions (Nuc) of Jurkat cells transfected with siRNA (top) and then left unstimulated (−) or stimulated for 30 min with TNF (T) or PMA plus ionomycin (P). () NF-κB luciferase assay (left) of Jurkat cells transfected with empty vector (Vec) or plasmid encoding Flag-tagged SSAA or SSEE IKKβ mutant together with a luciferase reporter driven by 5× immunoglobulin κB sites, and immunoblot analysis (right) of cytosolic (C) and nuclear (N) subcellular fractions of Jurkat cells overexpressing Flag-tagged IKKβ constructs! . Heat-shock protein 90 (hsp90) and poly(ADP-ribose) (PARP) serve as cytosolic and nuclear markers, respectively, and/or loading controls throughout. () Quantification of Jurkat cells with nuclear RPS3 with (Flag+) or without (Flag−) expression of Flag-tagged IKKβ, assessed by confocal microscopy after fixation and staining for RPS3, Flag and nuclei. Data are representative of three (,), two () or four () independent experiments with at least 200 cells each (mean and s.d. in ,; mean and s.d. of triplicates in ). * Figure 3: Importin-α-mediated nuclear translocation of RPS3 is dependent on degradation of IκBα. () Immunoassay of whole-cell lysates of Jurkat cells stimulated for 0–15 min with TNF and analyzed by immunoblot for importin-α (Imp-α), importin-β (Imp-β) or RPS3 directly or after immunoprecipitation with anti-RPS3. () Immunoprecipitation and immunoblot analysis of the association of endogenous importin-α or importin-β with RPS3 in Jurkat cells overexpressing hemagglutinin-tagged wild-type IκBα or SSAA IκBα mutant and stimulated for 0–45 min with TNF. () Immunoassay of whole-cell lysates of Jurkat cells transfected for 72 h with nonspecific or IκBα-specific siRNA, then analyzed by immunoblot for importin-α or RPS3 directly or after immunoprecipitation with anti-RPS3. () Immunoblot analysis of cytosolic (Cyt) and nuclear (Nuc) subcellular fractions of Jurkat cells transfected with nonspecific or IκBα-specific siRNA. () Immunoassay of whole-cell lysates of Jurkat cells given no pretreatment (−) or pretreated for 2 h (+) with 800 mM sodium pervanadate (Pv! ), followed by no stimulation or 30 min of TNF stimulation, then analyzed by immunoblot directly or after immunoprecipitation with anti-RPS3. Data are representative of at least two experiments. * Figure 4: IKKβ phosphorylates RPS3 at Ser209. () Autoradiography (above) and Coomassie blue staining (below) of in vitro kinase assays with recombinant GST or GST-RPS3 plus recombinant human IKKα (rIKKα) or IKKβ (rIKKβ); p-IKKα and p-IKKβ (right) indicate autophosphorylated IKK proteins, and p-RPS3 indicates phosphorylated RPS3. () In vitro kinase assay of recombinant RPS3 protein with recombinant human IKKβ; after digestion, samples were enriched for phosphorylated peptides by TiO2 and were fragmented by a mass spectrometer; intensity of ions is presented relative to the tallest peak in the spectrum, set as 100%. The spectrum of the 1+ fragment ion displays indicative of KPLPDHVpSIVEPK, based on a Mascot algorithm database search. The y6 ion (red) shows incorporation of the site of phosphorylation (the sixth amino acid from the C terminus of the fragment), further confirmed by the loss of H3PO4 from several ions. Top, RPS3 with characterized domains (NLS, nuclear localization signal; KH, K homology) and the IKK�! � Ser209 phosphorylation site in red. () Autoradiography (above) and Coomassie blue staining (below) of in vitro kinase assays with GST-tagged recombinant wild-type RPS3 or RPS3(S209A) plus recombinant human IKKβ. () Immunoprecipitation and immunoblot analysis of the serine phosphorylation of ectopically expressed RPS3 in HEK293T cells transfected with a plasmid expressing Flag-tagged wild-type RPS3 or RPS3(S209A) with or without a plasmid expressing IKKβ. () Immunoblot analysis (below) of phosphorylated and total RPS3 in whole-cell lysates or Jurkat cells stimulated for 0–30 min with TNF; above, densitometry, normalized as the intensity of each phosphorylated RPS3 band to the corresponding total RPS3 band and presented as the ratio of phosphorylated RPS3 to RPS3 relative to that of unstimulated cells. Data are representative of four (,) or two (–) independent experiments. * Figure 5: Phosphorylation of RPS3 at Ser209 is critical for its nuclear translocation and NF-κB-specifier function. () Immunoblot analysis (left) of cytosolic and nuclear subcellular fractions of Jurkat cells overexpressing Flag-tagged wild-type (WT) RPS3 or RPS3(S209A), stimulated with PMA plus ionomycin. Right, densitometry, normalized to the loading control (hsp90 or PARP) and presented relative to results of cells without PMA plus ionomycin (0 min), set as 100%. () Immunoblot analysis (left) of cytosolic and nuclear subcellular fractions of Jurkat cells overexpressing IKKβ together with Flag-tagged wild-type RPS3 or RPS3(S209A). Right, densitometry, normalized as in and presented relative to results of cells without overexpression of IKKβ (−IKKβ), set as 100%. () Immunoblot analysis of endogenous and ectopically expressed RPS3 in whole-cell lysates of Jurkat cells transfected with siRNA targeting the 3′ UTR of RPS3 mRNA (RPS3-3′ UTR) or nonspecific siRNA, plus construct encoding Flag-tagged wild-type RPS3 or RPS3(S209A). β-actin serves as a loading control throughout. () NF-! κB luciferase assay of Jurkat cells transfected as in , along with a luciferase reporter construct (as in Fig. 2c). NS, not significant; *P < 0.01 (Student's t-test). () Chromatin-immunoprecipitation analysis of the recruitment of Flag-tagged RPS3 and endogenous p65 proteins to the promoters of NFKBIA, IL8 or ACTB in extracts of Jurkat cells transfected as in , then left untreated or stimulated for 1 h with PMA plus ionomycin; quantitative real-time PCR analysis of DNA bound to Flag-RPS3 and p65 (primers, above graphs) was normalized to input DNA and is presented relative to that of cells transfected with the construct encoding Flag-tagged wild-type RPS3 and left untreated. Data are representative of at least two independent experiments (mean and s.d. of triplicates in ,). * Figure 6: NleH1 blocks the phosphorylation of RPS3 Ser209. () Immunoblot analysis of whole-cell lysates of HEK293T cells transfected with plasmids encoding hemagglutinin-tagged control protein (VN) or NleH1. () NF-κB luciferase assay of HEK293T cells transfected as in , along with a luciferase reporter construct (as in Fig. 2c). () Immunoblot analysis of whole-cell lysates of HEK293T cells overexpressing plasmids as in and left unstimulated (−) or stimulated (+) for 15 min with TNF (50 ng/ml). () Immunoblot analysis (left) of whole-cell lysates of HeLa cells left uninfected (Uninf) or infected for 3 h with wild-type, ΔescN or ΔnleH1 E. coli O157:H7, followed by treatment for 0–60 min (above lanes) with TNF. Right, densitometry, normalized as in Figure 4e and presented as the ratio of phosphorylated RPS3 to RPS3 relative to that of untreated infected cells, set as 1. () RT-PCR analysis of mRNA for IL8, TNFAIP3 and NFKBIA in HeLa cells infected for 3 h with E. coli O157:H7 strains as in , normalized to expression of GAPDH (glyc! eraldehyde phosphate dehydrogenase) and presented relative to expression in uninfected cells. () Immunohistochemistry of the phosphorylation of RPS3 Ser209 in paraffin-embedded colons of gnotobiotic piglets (n = 2) infected with wild-type or ΔnleH1 E. coli O157:H7 EDL933, assessed with anti-phosphorylated RPS3 and 3,3′-diaminobenzidine as a substrate (brown); nuclei were counterstained with hematoxylin (blue). Scale bar, 25 μm. Data are representative of two (,), four (), three (,) or six () independent experiments (mean and s.d. of triplicates in ,). * Figure 7: NleH1 alters the substrate specificity of IKKβ to block IKKβ-mediated phosphorylation of RPS3. () Autoradiography (left) and Coomassie blue staining (right) of in vitro kinase assays with recombinant histidine-tagged (His-) NleH1 or NleH1(K159A), showing autophosphorylated NleH1 (p-NleH1) and total NleH1 (right margin). () Immunoblot analysis (above) of whole-cell lysates of HeLa cells overexpressing hemagglutinin-tagged control protein (−), wild-type NleH1 or NleH1(K159A), left unstimulated (−) or stimulated for 30 min (+) with TNF (50 ng/ml). Below, densitometry, normalized and presented as in Figure 4d. () Immunoblot analysis (above) of whole-cell lysates of HeLa cells left uninfected or infected for 3 h with wild-type or ΔnleH C. rodentium or with ΔnleH C. rodentium complemented with wild-type NleH1 (ΔnleH+nleH1) or NleH1(K159A) (ΔnleH+nleH1(K159A)), followed by TNF treatment for 30 min. Below, densitometry, normalized as in Figure 4d, and presented as the ratio of phosphorylated RPS3 to RPS3 relative to that in untreated cells without infection, set as 1.! () Immunoblot analysis (above) of nuclear proteins from HeLa cells transfected for 48 h with Flag-tagged IKKβ constructs (top), then left uninfected (Mock) or infected for 3 h with wild-type, ΔescN or ΔnleH1 E. coli O157:H7. Below, densitometry, normalized as the intensity of each RPS3 band to the corresponding PARP band and presented as the change in nuclear RPS3 relative to that in mock-infected cells, set as 1. () Autoradiography (left) and Coomassie blue staining (right) of in vitro kinase assays with recombinant RPS3 or GST-tagged IκBα peptide of amino acids 1–54 (GST-IκBα (1–54)) as the substrate and recombinant NleH1 or human IKKβ as the kinase. Data are representative of at least of two experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Fengyi Wan, * Amanda Weaver, * Michael Bern & * Michael J Lenardo * Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA. * Fengyi Wan * Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA. * Xiaofei Gao & * Philip R Hardwidge Contributions F.W. and M.J.L. designed the experiments; F.W., A.W., X.G. and M.B. did the experiments; and F.W., P.R.H. and M.J.L. analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael J Lenardo Author Details * Fengyi Wan Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda Weaver Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaofei Gao Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Bern Search for this author in: * NPG journals * PubMed * Google Scholar * Philip R Hardwidge Search for this author in: * NPG journals * PubMed * Google Scholar * Michael J Lenardo Contact Michael J Lenardo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–13 Additional data - The helminth product ES-62 protects against septic shock via Toll-like receptor 4–dependent autophagosomal degradation of the adaptor MyD88
- Nat Immunol 12(4):344-351 (2011)
Nature Immunology | Article The helminth product ES-62 protects against septic shock via Toll-like receptor 4–dependent autophagosomal degradation of the adaptor MyD88 * Padmam Puneet1, 2 * Mairi A McGrath3 * Hwee Kee Tay3 * Lamyaa Al-Riyami4 * Justyna Rzepecka4 * Shabbir M Moochhala5 * Shazib Pervaiz1, 6 * Margaret M Harnett3 * William Harnett4 * Alirio J Melendez1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:344–351Year published:(2011)DOI:doi:10.1038/ni.2004Received29 November 2010Accepted03 February 2011Published online27 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition. View full text Figures at a glance * Figure 1: Effects of ES-62 on human macrophages. () Confocal microscopy of the colocalizion of ES-62 and TLR4 in primary human macrophages treated for 10 min with ES-62. Original magnification, ×40. () Immunoblot analysis of immune complexes containing TLR4 and ES-62 in primary human macrophages treated for 10 min with vehicle (–) or ES-62 (+), followed by immunoprecipitation (IP) of TLR4 (left) or ES-62 (right). () TLR4 expression in macrophages incubated for 2 h with (ES-62) or without (None) ES-62. Isotype, isotype-matched control antibody. () NF-κB activity in cells cultured with medium alone (Basal) or stimulated for 30 min with LPS after no pretreatment (LPS) or pretreatment for 2 h with ES-62 (ES-62 + LPS). () Release of proinflammatory mediators 24 h after the addition of PBS (Basal) or LPS to macrophages pretreated as in . () TLR2 expression in macrophages incubated for 6 h with or without ES-62. () NF-κB activity in cells cultured with medium alone (Basal) or stimulated for 30 min with BLP after no pretreatm! ent (BLP) or pretreatment for 6 h with ES-62 (ES-62 + BLP). () Release of cytokines 24 h after the addition of PBS (Basal) or BLP to macrophages pretreated as in . () MyD88 expression in untreated control macrophages (0) and in macrophages treated for 3 h or 6 h with ES-62 (above lanes); α-tubulin serves as a loading control. *P < 0.01, versus LPS-induced control macrophages (Student's t-test). Data are from one experiment representative of at least three independent experiments (mean ± s.d. of triplicates in ,,,). * Figure 2: Effect of ES-62 on phagocytes from patients with sepsis. (,) Expression of TLR4 () and TLR2 () in macrophages and neutrophils from healthy volunteers (Normal) and patients with sepsis, left untreated (Sepsis) or treated for 3 h with ES-62 (Sepsis + ES-62). () MyD88 in macrophages and neutrophils obtained from patients with sepsis and left untreated (0) or pretreated for 3 h or 6 h with ES-62 (above lanes). () Degranulation of macrophages and neutrophils obtained from patients with sepsis and cultured for 60 min with PBS (None; basal) or with LPS or BLP after no pretreatment (LPS or BLP) or after pretreatment for 6 h with ES-62 (ES-62 + LPS or ES-62 + BLP), assessed as the release of β-hexosaminidase (macrophages) or β-glucorinidase (neutrophils). () Release of proinflammatory mediators from macrophages 24 h after treatment of cells as in . *P < 0.01, ES-62 versus control (Student's t-test). Data are from one experiment representative of at least three independent experiments (mean and s.d. of triplicates in ,). * Figure 3: ES-62 targets TLR4 and MyD88 to early and late endosomes. Microscopy of the intracellular vesicle colocalization of TLR4 and MyD88 trafficking into EEA-1+ early endosomes (20 min; ), LAMP-1+ late endosomes and/or lysosomes (1 h; ) and LC3+ autophagosomes (3 h; ) in human macrophages exposed to LPS or ES-62. Original magnification, ×40. Data are representative of at least three independent experiments. * Figure 4: ES-62 induces autophagosome formation. () Electron microscopy of human macrophages treated with vehicle (Veh) or ES-62 (time, above images). Red arrows indicate double-membraned vacuole structures. () Immunoblot analysis of the expression of ATG5 and ATG7 in primary mouse macrophages left untreated (Basal) or treated with siRNA specific for ATG5 (siRNA-ATG5) or ATG7 (siRNA-ATG7) or control siRNA (siRNA-Ctrl). () Immunoblot analysis of MyD88 expression in primary mouse macrophages pretreated for 6 h with medium (Basal) or ES-62 (+Control) in the presence or absence of siRNA as in . () Immunoblot analysis of the expression of LC3I, LC3II and p62 in primary mouse macrophages treated for 0–18 h (above lanes) with vehicle or ES-62. () Expression of p62 (SQSTM1) mRNA in primary mouse macrophages treated with vehicle or ES-62. () Expression of p62 protein in primary mouse macrophages treated with ES-62 or vehicle in the presence or absence (0) of cyclohexamide (cyclohex). () Immunoblot analysis of the expression of LC! 3I, LC3II, p62 and MyD88 in cells treated with ES-62 (time, above lanes) in the presence of E64D and pepstatin A. () Immunoblot analysis of MyD88 and LC3II in human or mouse macrophages left untreated (Basal) or pretreated for 6 h with ES-62 in the presence or absence (+Control) of lactacystin (LAC), chloroquine or NH4Cl. β-tubulin serves as a loading control (– and –). *P < 0.01, ES-62 versus vehicle (Student's t-test). Data are from one experiment representative of at least three independent experiments (mean ± s.d. of triplicates in ). * Figure 5: ES-62 protects against endotoxic shock. () Survival of PBS-injected control mice (PBS), and mice given no pretreatment (LPS) or pretreated with PBS (PBS + LPS) or ES-62 (ES-62 + LPS) 2 h before injection with a lethal dose of LPS. () Lung and liver sections from mice treated as in , assessed 12 h after treatment. Scale bars, 50 μm. () TNF, IL-1β, IL-6, MIP-1α and HMGB1 in serum from mice treated as in (n = 10 mice per group), assessed 12 h after treatment. *P < 0.01, compared with control mice given LPS (Student's t-test). Data are from one experiment representative of at least three independent experiments (mean and s.d. in ). * Figure 6: ES-62 protects against polymicrobial sepsis. () Survival of sham-operated mice (Sham), mice given CLP without pretreatment (CLP) and mice injected with PBS (PBS + CLP) or ES-62 (ES-62 + CLP) starting 2 h before CLP. () Lung and liver sections from mice treated as in , assessed 12 h after surgery. Scale bars, 50 μm. () Infiltration of peritoneal neutrophils (left) and monocytes (right) after surgery in mice treated as in . () IL-1β, IL-3, IL-5, IL-6, TNF, chemokine CXCL1 (KC), MCP-1, MIP-1α and HMGB1 in serum of mice treated as in , assessed 12 h after surgery. (,) Bacteria in the peritoneal fluid () and peripheral blood () of mice injected with PBS or ES-62 starting 2 h before CLP, assessed 6 h or 24 h after surgery. CFU, colony-forming units. Each symbol represents an individual mouse (n = 10 mice per group); small horizontal bars indicate the mean. *P < 0.01, compared with PBS pretreatment and CLP (Student's t-test). Data are from one experiment representative of at least three independent experiments (mean ± s.d! . in ,). * Figure 7: ES-62 protects via autophagy in vivo. () Survival of wild-type (WT), Tlr2−/− or Tlr4−/− mice given CLP without pretreatment (CLP) or injected with ES-62 (ES-62 + CLP) starting 2 h before CLP. () Immunoblot analysis of the expression of ATG5 (left) and ATG7 (right) in PBMCs from mice treated in vivo with PBS (Basal), siRNA specific for ATG5 (siRNA-ATG5) or ATG7 (siRNA-ATG7) or control siRNA (siRNA-Ctrl); β-tubulin serves as a loading control. () Survival of sham-operated mice, mice given CLP without pretreatment, mice injected with ES-62 starting 2 h before CLP, and mice pretreated with siRNA specific for ATG5 or ATG7 or control siRNA and injected with ES-62 or saline starting 2 h before CLP (key, top to bottom). *P < 0.01, compared with CLP without pretreatment (Student's t-test). Data are from a single experiment representative of three independent experiments with ten mice per group. * Figure 8: Therapeutic role for ES-62 in polymicrobial sepsis. () Survival of sham-operated mice, mice given CLP without additional treatment and mice injected with ES-62 starting 1 h, 3 h or 6 h after CLP (time, key). (,) TNF, IL-1β, IL-6 and MIP-1α () and HMGB1 () in the serum of mice treated as in , assessed 12 h after surgery. () Hematoxylin and eosin staining of liver and lung sections from mice treated as in , assessed 12 h after surgery. Scale bars, 50 μm. () Survival sham-operated mice, mice given CLP without additional treatment and mice injected with ES-62 or amoxicillin–clavulanic acid (Co-Am) alone or together starting 6 h after CLP (n = 10 mice per group). *P < 0.01, compared with CLP without pretreatment (Student's t-test). Data are from one experiment representative of three independent experiments (error bars (,), s.d.). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Physiology, National University of Singapore, Singapore. * Padmam Puneet, * Shazib Pervaiz & * Alirio J Melendez * Institute of Translational Medicine, Department of Molecular & Clinical Pharmacology, Medical Research Council Centre for Drug Safety Studies, University of Liverpool, Liverpool, UK. * Padmam Puneet & * Alirio J Melendez * Institute of Infection, Immunity and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK. * Mairi A McGrath, * Hwee Kee Tay & * Margaret M Harnett * Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK. * Lamyaa Al-Riyami, * Justyna Rzepecka & * William Harnett * Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore. * Shabbir M Moochhala * NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School and Singapore-MIT Alliance, Singapore. * Shazib Pervaiz Contributions P.P., M.A.M., H.K.T., L.A.-R. and J.R. did experiments; S.M.M. supplied reagents; A.J.M. conceived of the study; A.J.M., M.M.H., S.P. and W.H. planned the experiments, supervised the study and wrote the paper; and all authors analyzed data. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alirio J Melendez Author Details * Padmam Puneet Search for this author in: * NPG journals * PubMed * Google Scholar * Mairi A McGrath Search for this author in: * NPG journals * PubMed * Google Scholar * Hwee Kee Tay Search for this author in: * NPG journals * PubMed * Google Scholar * Lamyaa Al-Riyami Search for this author in: * NPG journals * PubMed * Google Scholar * Justyna Rzepecka Search for this author in: * NPG journals * PubMed * Google Scholar * Shabbir M Moochhala Search for this author in: * NPG journals * PubMed * Google Scholar * Shazib Pervaiz Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret M Harnett Search for this author in: * NPG journals * PubMed * Google Scholar * William Harnett Search for this author in: * NPG journals * PubMed * Google Scholar * Alirio J Melendez Contact Alirio J Melendez Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (270K) Supplementary Figures 1–5 Additional data - Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes
- Nat Immunol 12(4):352-361 (2011)
Nature Immunology | Resource Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes * Maria N Navarro1 * Jurgen Goebel1 * Carmen Feijoo-Carnero1 * Nick Morrice2 * Doreen A Cantrell1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:352–361Year published:(2011)DOI:doi:10.1038/ni.2008Received28 October 2010Accepted09 February 2011Published online13 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL ! functional program. View full text Figures at a glance * Figure 1: Analysis of the basal and TCR-regulated phosphoproteome in CTLs. () SILAC ratio (TCR-stimulated/unstimulated) and signal intensity of phosphopeptides (n = 2,078) identified by mass spectrometry in lysates of P14 CTLs labeled with different isotopes (R10K8 (unstimulated) or R0K0 (stimulated)) in SILAC media and then left unstimulated (unstim) or triggered for 1 h via their TCR with cognate peptide (TCR stim), followed by cell lysis and purification of phosphopeptides by HILIC-IMAC enrichment (Supplementary Methods): black dots, phosphopeptides with phosphorylation downregulated or upregulated by TCR stimulation; gray dots, phosphopeptides not regulated by the TCR; in parentheses, selected proteins previously shown to change phosphorylation after TCR stimulation. A SILAC ratio of 1.5-fold serves as the threshold for regulation. Data are from a single experiment. () Molecular and cellular function of all 955 phosphoproteins identified (as assessed by Ingenuity Pathway Analysis): transcription (TC), developmental processes of lymphocytes (Dev! ), cell cycle (CC), lymphocyte homeostasis (LH) and phosphorylation (P). P values (vertical axis) indicate the probability that the function assigned is due to chance alone; numbers along the horizontal axis indicate total proteins in each group. Data are from a single experiment. * Figure 2: Identification of consistent phosphorylations in CTLs by Ingenuity Pathway Analysis software. () Analysis of 742 phosphorylations of 473 proteins identified by SILAC and HILIC-IMAC purification protocols, including switching of amino acid labeling, with the following isotope combinations (unstimulated and stimulated): experiment 1, R10K8 and R0K0; experiment 2, R0K0 and R6K6; experiment 3 and 4, R0K0 and R6K4; CTLs were stimulated with cognate peptide for 1 h (experiments 1, 2 and 3) or 10 min (experiment 4), and phosphorylations reproducible in three of four experiments were considered for analysis. Protein functions: RNA post-translational modification (PTM), cell death (CD), transcription (TC), actin polymerization (Pol) and protein synthesis (Prot). () TCR-regulated phosphorylation (n = 94) of 78 different proteins consistently identified; a SILAC ratio of 1.5-fold serves as the threshold for regulation. Protein functions: cell growth (CG), survival (Surv), actin polymerization (Pol) and cell cycle (CC). () Frequency of kinases predicted to be active in CTLs, by ! MaxQuant software analysis, with a sequence window ± six amino acids around the identified phosphorylation site to determine the kinase that could phosphorylate the motif. Data are representative of four experiments. * Figure 3: Phosphorylated chromatin regulators in CTLs. () Manual sequencing (below) of acquired multistage activation spectra for the peptides KTVpSEPNLK, KEpSAPPSLR and pTRSEPLPPSATASPLLAPLQPR, with Biemann-Roepstorff nomenclature; asterisks (y and b ion series) indicate loss of phosphate. Above: vertical lines indicate a fragmented bond after collision-induced dissociation; horizontal lines indicate the fragment retaining the charge. Data are from one of four experiments. () Spectral counting of HDAC7 and HDAC4, the two class IIa HDACs detected in CTLs, calculated for phosphopeptide enrichment and 14-3-3 affinity-purification screens, respectively. Data are representative of four experiments (average and s.e.m.). * Figure 4: Subcellular distribution of HDAC7 in CTLs. () Immunoblot analysis of cytosolic (Cyt) and nuclear (Nuc) extracts of P14 CTLs left untreated (–) or treated for 30 min with peptide (TCR) or phorbol-12-13-dibutyrate (PD); antibody to IκBα (anti-IκBα) and anti-SCM1 serve as controls for fraction purity, and antibody to phosphorylated Erk (Anti-p-Erk1/2) serves as an activation control. Right margin, molecular size in kilodaltons (kDa). () Immunoprecipitation (IP) and immunoblot analysis of cytosolic and nuclear extracts of P14 CTLs left untransduced (–) or retrovirally transduced with GFP-HDAC7 (+), immunoprecipitated and probed with anti-GFP (top); cytosolic extracts of GFP-HDAC7-transduced CTLs left unstimulated (–) or stimulated for 2 h with phorbol-12-13-dibutyrate or peptide (as in ), immunoprecipitated with anti-GFP and probed with antibody to phosphorylated HDAC7 (middle); and cytosolic extracts of cells treated as in middle blot, followed by affinity purification with 14-3-3–sepharose (14-3-3 precip), ! probed with anti-GFP (bottom). () Immunoblot analysis of HDAC7 expression in cytosolic and nuclear extracts of CTLs from OT-I (ovalbumin-specific) TCR–transgenic mice and polyclonal, non–TCR-transgenic wild-type (WT) mice (n = two mice, WT1 and WT2). () Immunoblot analysis of HDAC7 in cytosolic and nuclear extracts of the following populations of naive T cells: CD8+ T cells from OT-I TCR–transgenic mice (left), and polyclonal CD4+ T cells (middle) and total T cells (right) from non–TCR-transgenic wild-type mice. () Confocal microscopy of P14 CTLs retrovirally transduced with vector encoding GFP-HDAC7. Original magnification, ×100. () Microscopy (left) of GFP-HDAC7-expressing P14 CTLs left untreated (U) or treated for 3 h with leptomycin B (L) and stained with the DNA-intercalating dye DAPI. Original magnification, ×100. Middle and right, frequency of cells with nuclear GFP-HDAC7 (n ≥ 100 cells per experiment and time point) after 1, 2 or 3 h of LMB treatment (mi! ddle) or after 3 h of LMB treatment (right). Data are represen! tative of three () or two (,,) experiments, ten independent experiments () or seven experiments (; average and s.e.m.). * Figure 5: Exclusion of HDAC7 from the nucleus is required for normal CTL function. (,) Microscopy of the subcellular distribution of GFP-HDAC7-ΔP in retrovirally transduced P14 CTLs, without () or with () DAPI staining. Original magnification, ×100. Data are representative of four experiments. () Flow cytometry of CTLs transduced with GFP-HDAC7 or GFP-HDAC7-ΔP. FSC, forward scatter. Data are representative of ten experiments. () Proliferative expansion of CTLs transduced as in and cultured for 6 d in IL-2. Data are representative of three experiments (average ± s.e.m.). () Microarray analysis of the expression profiles of CTLs transduced with GFP-HDAC7-ΔP and sorted on the basis of GFP expression, presented as the distribution of the intensity ratio (log2 change, cells expressing GFP-HDAC7-ΔP (GFP+) relative to control (GFP−) cells) plotted by the average of the normalized intensity values for 23,653 probes identified as being present in at least one sample; black dots indicate probes with a change of twofold or greater (1,457 probes; 993 annotated! genes), and gray dots indicate probes with no significant change (P < 0.05) or a change below twofold (22,196 probes; 11,148 annotated genes).Data are representative of a single microarray analysis with triplicate samples. * Figure 6: Exclusion of HDAC7 from the nucleus is required for expression of the high-affinity IL-2 receptor. () Expression of CD25 mRNA in sorted P14 CTLs expressing GFP-HDAC7-ΔP, presented in arbitrary units relative to its expression in sorted GFP− cells, set as 1. () Flow cytometry of P14 CTLs transduced with GFP-HDAC7-ΔP and stained for CD25, with electronic gating of GFP+ and GFP− cells for comparison of CD25 expression in each. () Flow cytometry of P14 CTLs transduced with GFP-HDAC7 or GFP-HDAC7-ΔP and stained for CD25, with electronic gating for comparison of CD25 expression in each population. (,) Flow cytometry of P14 CTLs stimulated for 4 h with cognate peptide before analysis of surface CD25 () and intracellular IFN-γ expression (, with gating as in ). () Intracellular staining of IFN-γ in P14 CTLs expressing GFP-HDAC7-ΔP or GFP alone, stimulated for 4 h with peptide. Data are representative of three independent experiments (–; average and s.e.m. in ), four experiments (,; average and s.e.m. in ) or three experiments (). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE27092 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * The College of Life Sciences, Division of Immunology and Cell Biology, The University of Dundee, Dundee, Scotland, UK. * Maria N Navarro, * Jurgen Goebel, * Carmen Feijoo-Carnero & * Doreen A Cantrell * The Beatson Institute for Cancer Research, Glasgow, Scotland, UK. * Nick Morrice Contributions M.N.N., J.G. and C.F.-C. did the experiments and analyzed the results; N.M. supervised SILAC methodology and bioinformatic analysis; and M.N.N. and D.A.C. designed the experiments, analyzed the results and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Doreen A Cantrell Author Details * Maria N Navarro Search for this author in: * NPG journals * PubMed * Google Scholar * Jurgen Goebel Search for this author in: * NPG journals * PubMed * Google Scholar * Carmen Feijoo-Carnero Search for this author in: * NPG journals * PubMed * Google Scholar * Nick Morrice Search for this author in: * NPG journals * PubMed * Google Scholar * Doreen A Cantrell Contact Doreen A Cantrell Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (11M) Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Methods Additional data - The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding
- Nat Immunol 12(4):362-363 (2011)
Nature Immunology | Addendum The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding * Nicolas Fazilleau * Louise J McHeyzer-Williams * Hugh Rosen * Michael G McHeyzer-WilliamsJournal name:Nature ImmunologyVolume: 12,Pages:362–363Year published:(2011)DOI:doi:10.1038/ni0411-362aPublished online21 March 2011 Figures at a glance * Figure 1: Upregulation of both Bcl6 and Bcl6b in antigen-specific TFH cells analyzed immediately after isolation. Quantitative PCR analysis of Bcl6 mRNA and Bcl6b mRNA in naive helper T cells (Va11+Vb3+CD44loCD62Lhi) or PCC-specific TFH cells (PI−B220− CD11b−CD8−Va11+Vb3+CD44hiCD62LloCXCR5hi) sorted from draining lymph nodes on day 7 after subcutaneous priming with PCC in Ribi adjuvant (2 × 103 cells), followed by cDNA synthesis and amplification with Platinum SYBR Green (details as for in the original study (Nat. Immunol., 375–384 (2009))); results are presented in arbitrary units (AU) relative to the expression of β2-microglobulin mRNA, set as 1. Other antigen-specific helper T cell subsets are sorted on the basis of differences in the expression of CD62L and CXCR5 as described in detail in the original study (Nat. Immunol., 375–384 (2009)). See larger * Figure 2: Primers for Bcl6 and Bcl6b amplify the expected PCR products from naive helper T cells and TFH cells. PCR analysis of Bcl6 and Bcl6b in naive helper T cells or antigen-specific TFH cells (5 × 103) sorted (as described in Addendum Fig. 1) on day 8 of the memory response to PCC; cDNA synthesized from RNA by random-hexamer priming was analyzed by 40 cycles of PCR with both sets of primers (Addendum Fig. 1), then primers were removed and PCR products were analyzed directly with a BigDye Terminator Cycle Sequencing kit on a 3130 Genetic Analyzer. See larger * Figure 3: Equivalent upregulation of Bcl6 and Bcl6b in CXCR5+ TFH cells assessed immediately after isolation. Reverse transcription and quantitative PCR analysis of Bcl6 and Bcl6b in naive helper T cells (PI−CD8−CD11b−CD4+CD44loCD62LhiCXCR5−) or TFH cells (PI−CD8−CD11b−CD4+CD44hiCD62LloCXCR5+) from the draining lymph nodes of mice immunized with Ribi adjuvant only and sorted, results are presented in arbitrary units relative to the expression of β-actin mRNA. See larger * Figure 4: Selective upregulation of Bcl6 and Bcl6b in antigen-specific TFH cells with the use of DCs but not PCs for antigen presentation in vitro. Quantitative PCR analysis of Bcl6 and Bcl6b in cells derived from CD11c+ DCs or IgM−CD138+ plasma cells (PC) sorted ex vivo with protein antigen and cultured for 4 d in vitro together with cytosolic dye CFSE–labeled naive CD4+ PCC-specific 5C.C7 αβTCR–transgenic helper T cells, followed by sorting of CD44hi 5C.C7 αβTCR–transgenic helper T cells that diluted CFSE, for RNA extraction, cDNA synthesis and SYBR Green–based quantitative PCR amplification of Bcl6 and Bcl6b (details as for Fig. 4 in the original study (Nat. Immunol., 1110–1118 (2010))); results are presented in arbitrary units relative to β2-microglobulin mRNA expression. See larger Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.10, 375–384 (2009); published online 1 March 2009; corrected online 8 March 2009; addendum published after print 8 March 2011 It has been called to our attention that in a series of studies on the development of follicular helper T cells (TFH cells), the primers we used to amplify Bcl6 were specific for Bcl6b, not Bcl6. We have now traced back the results and have redone experiments that demonstrate that both Bcl6 and Bcl6b are modified in the same way and to a similar extent in antigen-specific and non–antigen-specific TFH cells induced both in vivo and in vitro. In the original studies we inadvertently mixed up primers specific for Bcl6b (but not for Bcl6) with those specific for Bcl6. After closer inspection, we found that this error was made when the wrong set of primers was ordered and labeled "Bcl6 #2" in our laboratory but the primers were in fact specific for Bcl6b. We used both sets of primers in the original studies of gene expression on antigen-specific TFH cells analyzed immediately after isolation ( in Nat. Immunol.10, 375–384 (2009), and Addendum Fig. 1). Hence, the expression of both Bcl6 and Bcl6b seemed to be three- to fourfold higher in pigeon cytochrome c (PCC)-specific TFH cells than in naive CD4+ helper T cells. Figure 1: Upregulation of both Bcl6 and Bcl6b in antigen-specific TFH cells analyzed immediately after isolation. Quantitative PCR analysis of Bcl6 mRNA and Bcl6b mRNA in naive helper T cells (Va11+Vb3+CD44loCD62Lhi) or PCC-specific TFH cells (PI−B220− CD11b−CD8−Va11+Vb3+CD44hiCD62LloCXCR5hi) sorted from draining lymph nodes on day 7 after subcutaneous priming with PCC in Ribi adjuvant (2 × 103 cells), followed by cDNA synthesis and amplification with Platinum SYBR Green (details as for in the original study (Nat. Immunol., 375–384 (2009))); results are presented in arbitrary units (AU) relative to the expression of β2-microglobulin mRNA, set as 1. Other antigen-specific helper T cell subsets are sorted on the basis of differences in the expression of CD62L and CXCR5 as described in detail in the original study (Nat. Immunol., 375–384 (2009)). * Full size image (46 KB) * Figures/tables index * Next figure As these genes are structurally related, we were concerned that the primers for Bcl6b may have sufficiently cross-reacted with Bcl6 and perhaps amplified the latter gene in T cells. Thus, we sorted antigen-specific TFH cells immediately after isolation and amplified random hexamer–primed cDNA with both sets of primers (Addendum Fig. 2). Both gene products were amplified and produced PCR products of different sizes (the predicted sizes for the different gene products). We then sequenced these products to demonstrate they belonged to the different genes as presented. Thus, both primers amplified the expected PCR products in both naive helper T cells and antigen-specific TFH cells. Figure 2: Primers for Bcl6 and Bcl6b amplify the expected PCR products from naive helper T cells and TFH cells. PCR analysis of Bcl6 and Bcl6b in naive helper T cells or antigen-specific TFH cells (5 × 103) sorted (as described in Addendum Fig. 1) on day 8 of the memory response to PCC; cDNA synthesized from RNA by random-hexamer priming was analyzed by 40 cycles of PCR with both sets of primers (Addendum Fig. 1), then primers were removed and PCR products were analyzed directly with a BigDye Terminator Cycle Sequencing kit on a 3130 Genetic Analyzer. * Full size image (18 KB) * Previous figure * Figures/tables index * Next figure To explore this issue further, we sorted non–antigen-specific TFH cells on the basis of cell surface phenotype and amplified DNA from these cells by RT-PCR without further treatment (Addendum Fig. 3). These experiments also showed that both Bcl6 and Bcl6b had similar basal expression in naive helper T cells and a similar change of expression in CXCR5+ TFH cells. Figure 3: Equivalent upregulation of Bcl6 and Bcl6b in CXCR5+ TFH cells assessed immediately after isolation. Reverse transcription and quantitative PCR analysis of Bcl6 and Bcl6b in naive helper T cells (PI−CD8−CD11b−CD4+CD44loCD62LhiCXCR5−) or TFH cells (PI−CD8−CD11b−CD4+CD44hiCD62LloCXCR5+) from the draining lymph nodes of mice immunized with Ribi adjuvant only and sorted, results are presented in arbitrary units relative to the expression of β-actin mRNA. * Full size image (23 KB) * Previous figure * Figures/tables index * Next figure In our more recent study on antigen presentation by plasma cells (Nat. Immunol.11, 1110–1118 (2010)), we reported that plasma cells negatively regulate the TFH program in vitro and in vivo. Our conclusions were based on expression of the genes encoding interleukin 21 (IL-21) and Bcl-6, as measured by reverse transcription and quantitative PCR. The original primer mix-up continued in the early phase of these studies. However, the error was sporadic and these more recent studies reported on Bcl6 and not Bcl6b, as outlined below (Addendum Table 1). Table 1: Addendum Table 1 Induction and inhibition of Bcl6 or Bcl6b in TFH cells Full table * Figures/tables index Hence, we concluded that antigen presentation by plasma cells inhibits the expression of mRNA for IL-21 and Bcl-6 by in vivo–derived, endogenous, antigen-specific TFH cells (, second panel, in Nat. Immunol.11, 1110–1118 (2010)). Furthermore, the adoptive transfer of antigen-pulsed plasma cells significantly inhibited the expression of mRNA for both IL-21 and Bcl-6 on antigen-specific TFH cells (, in Nat. Immunol.11, 1110–1118 (2010)) without affecting the expression of mRNA for IL-4 or the transcription factor GATA-3 in vivo. Therefore, this provided evidence of antigen-specific negative regulation of the TFH program by plasma cells in vivo. We have also repeated the in vitro experiments using dendritic cells (DCs) or plasma cells as antigen-presenting cells to evaluate the expression of Bcl6 and Bcl6b after in vitro induction of TFH cell activity (Addendum Fig. 4). These studies demonstrated equivalent induction of both Bcl6 and Bcl6bin vitro with DCs as antigen-presenting cells and naive helper T cells with transgenic expression of the 5C.C7 αβ T cell antigen receptor (αβTCR) as antigen-specific helper T cells in these cultures. Consistent with the overall conclusions of our earlier study (Nat. Immunol.11, 1110–1118 (2010)) plasma cells with antigen did not induce Bcl6 or Bcl6b in 5C.C7 αβTCR–transgenic helper T cells in vitro. Figure 4: Selective upregulation of Bcl6 and Bcl6b in antigen-specific TFH cells with the use of DCs but not PCs for antigen presentation in vitro. Quantitative PCR analysis of Bcl6 and Bcl6b in cells derived from CD11c+ DCs or IgM−CD138+ plasma cells (PC) sorted ex vivo with protein antigen and cultured for 4 d in vitro together with cytosolic dye CFSE–labeled naive CD4+ PCC-specific 5C.C7 αβTCR–transgenic helper T cells, followed by sorting of CD44hi 5C.C7 αβTCR–transgenic helper T cells that diluted CFSE, for RNA extraction, cDNA synthesis and SYBR Green–based quantitative PCR amplification of Bcl6 and Bcl6b (details as for Fig. 4 in the original study (Nat. Immunol., 1110–1118 (2010))); results are presented in arbitrary units relative to β2-microglobulin mRNA expression. * Full size image (75 KB) * Previous figure * Figures/tables index We did these new experiments without secondary in vitro transcription or specific synthesis of target cDNA and preamplification by PCR to evaluate the extent of signal to be expected for Bcl6 after in vitro activation of TFH cells. The tenfold induction of both Bcl6 and Bcl6b in these studies is more in line with the expected results and the results of other studies using standard culturing conditions to induce TFH cell activity. These data are also more in line with the expression of Bcl6 and Bcl6b on antigen-specific TFH cells derived in vivo we have reported in all our studies. We believe that the starting cell population, in particular the primary culture stimulus, atypical secondary culture conditions and secondary amplification of the assay all contributed to the more enhanced signals we reported before for Bcl6b ( in Nat. Immunol.11, 1110–1118 (2010)). In conclusion, we mixed up primers for Bcl6 amplification and in parts of our published work inadvertently reported on Bcl6b, not Bcl6. Bcl6b is a distinct gene but remains a structurally and functionally related member of the Bcl6 gene family. We have now been able to revisit this issue and have demonstrated that both Bcl6 and Bcl6b seemed to be coexpressed to a similar extent in naive helper T cells. Both gene products were similarly enhanced in TFH cells that emerged in vivo or were derived in vitro. Furthermore, both genes seemed to be inhibited similarly by antigen presentation by plasma cells. Hence, we believe that this addendum will rectify the mistake made in the initial studies and clarify the coexpression of Bcl6 and Bcl6b in antigen-specific TFH cells. Additional data Author Details * Nicolas Fazilleau Search for this author in: * NPG journals * PubMed * Google Scholar * Louise J McHeyzer-Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Hugh Rosen Search for this author in: * NPG journals * PubMed * Google Scholar * Michael G McHeyzer-Williams Search for this author in: * NPG journals * PubMed * Google Scholar - Plasma cells negatively regulate the follicular helper T cell program
- Nat Immunol 12(4):362-363 (2011)
Nature Immunology | Addendum Plasma cells negatively regulate the follicular helper T cell program * Nadége Pelletier * Louise J McHeyzer-Williams * Kurt A Wong * Eduard Urich * Nicolas Fazilleau * Michael G McHeyzer-WilliamsJournal name:Nature ImmunologyVolume: 12,Pages:362–363Year published:(2011)DOI:doi:10.1038/ni0411-362bPublished online21 March 2011 Figures at a glance * Figure 1: Upregulation of both Bcl6 and Bcl6b in antigen-specific TFH cells analyzed immediately after isolation. Quantitative PCR analysis of Bcl6 mRNA and Bcl6b mRNA in naive helper T cells (Va11+Vb3+CD44loCD62Lhi) or PCC-specific TFH cells (PI−B220− CD11b−CD8−Va11+Vb3+CD44hiCD62LloCXCR5hi) sorted from draining lymph nodes on day 7 after subcutaneous priming with PCC in Ribi adjuvant (2 × 103 cells), followed by cDNA synthesis and amplification with Platinum SYBR Green (details as for in the original study (Nat. Immunol., 375–384 (2009))); results are presented in arbitrary units (AU) relative to the expression of β2-microglobulin mRNA, set as 1. Other antigen-specific helper T cell subsets are sorted on the basis of differences in the expression of CD62L and CXCR5 as described in detail in the original study (Nat. Immunol., 375–384 (2009)). See larger * Figure 2: Primers for Bcl6 and Bcl6b amplify the expected PCR products from naive helper T cells and TFH cells. PCR analysis of Bcl6 and Bcl6b in naive helper T cells or antigen-specific TFH cells (5 × 103) sorted (as described in Addendum Fig. 1) on day 8 of the memory response to PCC; cDNA synthesized from RNA by random-hexamer priming was analyzed by 40 cycles of PCR with both sets of primers (Addendum Fig. 1), then primers were removed and PCR products were analyzed directly with a BigDye Terminator Cycle Sequencing kit on a 3130 Genetic Analyzer. See larger * Figure 3: Equivalent upregulation of Bcl6 and Bcl6b in CXCR5+ TFH cells assessed immediately after isolation. Reverse transcription and quantitative PCR analysis of Bcl6 and Bcl6b in naive helper T cells (PI−CD8−CD11b−CD4+CD44loCD62LhiCXCR5−) or TFH cells (PI−CD8−CD11b−CD4+CD44hiCD62LloCXCR5+) from the draining lymph nodes of mice immunized with Ribi adjuvant only and sorted, results are presented in arbitrary units relative to the expression of β-actin mRNA. See larger * Figure 4: Selective upregulation of Bcl6 and Bcl6b in antigen-specific TFH cells with the use of DCs but not PCs for antigen presentation in vitro. Quantitative PCR analysis of Bcl6 and Bcl6b in cells derived from CD11c+ DCs or IgM−CD138+ plasma cells (PC) sorted ex vivo with protein antigen and cultured for 4 d in vitro together with cytosolic dye CFSE–labeled naive CD4+ PCC-specific 5C.C7 αβTCR–transgenic helper T cells, followed by sorting of CD44hi 5C.C7 αβTCR–transgenic helper T cells that diluted CFSE, for RNA extraction, cDNA synthesis and SYBR Green–based quantitative PCR amplification of Bcl6 and Bcl6b (details as for Fig. 4 in the original study (Nat. Immunol., 1110–1118 (2010))); results are presented in arbitrary units relative to β2-microglobulin mRNA expression. See larger Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.11, 1110–1118 (2010); published online 31 October 2010; addendum published after print 8 March 2011 It has been called to our attention that in a series of studies on the development of follicular helper T cells (TFH cells), the primers we used to amplify Bcl6 were specific for Bcl6b, not Bcl6. We have now traced back the results and have redone experiments that demonstrate that both Bcl6 and Bcl6b are modified in the same way and to a similar extent in antigen-specific and non–antigen-specific TFH cells induced both in vivo and in vitro. In the original studies we inadvertently mixed up primers specific for Bcl6b (but not for Bcl6) with those specific for Bcl6. After closer inspection, we found that this error was made when the wrong set of primers was ordered and labeled "Bcl6 #2" in our laboratory but the primers were in fact specific for Bcl6b. We used both sets of primers in the original studies of gene expression on antigen-specific TFH cells analyzed immediately after isolation ( in Nat. Immunol.10, 375–384 (2009), and Addendum Fig. 1). Hence, the expression of both Bcl6 and Bcl6b seemed to be three- to fourfold higher in pigeon cytochrome c (PCC)-specific TFH cells than in naive CD4+ helper T cells. Figure 1: Upregulation of both Bcl6 and Bcl6b in antigen-specific TFH cells analyzed immediately after isolation. Quantitative PCR analysis of Bcl6 mRNA and Bcl6b mRNA in naive helper T cells (Va11+Vb3+CD44loCD62Lhi) or PCC-specific TFH cells (PI−B220− CD11b−CD8−Va11+Vb3+CD44hiCD62LloCXCR5hi) sorted from draining lymph nodes on day 7 after subcutaneous priming with PCC in Ribi adjuvant (2 × 103 cells), followed by cDNA synthesis and amplification with Platinum SYBR Green (details as for in the original study (Nat. Immunol., 375–384 (2009))); results are presented in arbitrary units (AU) relative to the expression of β2-microglobulin mRNA, set as 1. Other antigen-specific helper T cell subsets are sorted on the basis of differences in the expression of CD62L and CXCR5 as described in detail in the original study (Nat. Immunol., 375–384 (2009)). * Full size image (48 KB) * Figures/tables index * Next figure As these genes are structurally related, we were concerned that the primers for Bcl6b may have sufficiently cross-reacted with Bcl6 and perhaps amplified the latter gene in T cells. Thus, we sorted antigen-specific TFH cells immediately after isolation and amplified random hexamer–primed cDNA with both sets of primers (Addendum Fig. 2). Both gene products were amplified and produced PCR products of different sizes (the predicted sizes for the different gene products). We then sequenced these products to demonstrate they belonged to the different genes as presented. Thus, both primers amplified the expected PCR products in both naive helper T cells and antigen-specific TFH cells. Figure 2: Primers for Bcl6 and Bcl6b amplify the expected PCR products from naive helper T cells and TFH cells. PCR analysis of Bcl6 and Bcl6b in naive helper T cells or antigen-specific TFH cells (5 × 103) sorted (as described in Addendum Fig. 1) on day 8 of the memory response to PCC; cDNA synthesized from RNA by random-hexamer priming was analyzed by 40 cycles of PCR with both sets of primers (Addendum Fig. 1), then primers were removed and PCR products were analyzed directly with a BigDye Terminator Cycle Sequencing kit on a 3130 Genetic Analyzer. * Full size image (18 KB) * Previous figure * Figures/tables index * Next figure To explore this issue further, we sorted non–antigen-specific TFH cells on the basis of cell surface phenotype and amplified DNA from these cells by RT-PCR without further treatment (Addendum Fig. 3). These experiments also showed that both Bcl6 and Bcl6b had similar basal expression in naive helper T cells and a similar change of expression in CXCR5+ TFH cells. Figure 3: Equivalent upregulation of Bcl6 and Bcl6b in CXCR5+ TFH cells assessed immediately after isolation. Reverse transcription and quantitative PCR analysis of Bcl6 and Bcl6b in naive helper T cells (PI−CD8−CD11b−CD4+CD44loCD62LhiCXCR5−) or TFH cells (PI−CD8−CD11b−CD4+CD44hiCD62LloCXCR5+) from the draining lymph nodes of mice immunized with Ribi adjuvant only and sorted, results are presented in arbitrary units relative to the expression of β-actin mRNA. * Full size image (22 KB) * Previous figure * Figures/tables index * Next figure In our more recent study on antigen presentation by plasma cells (Nat. Immunol.11, 1110–1118 (2010)), we reported that plasma cells negatively regulate the TFH program in vitro and in vivo. Our conclusions were based on expression of the genes encoding interleukin 21 (IL-21) and Bcl-6, as measured by reverse transcription and quantitative PCR. The original primer mix-up continued in the early phase of these studies. However, the error was sporadic and these more recent studies reported on Bcl6 and not Bcl6b, as outlined below (Addendum Table 1). Table 1: Addendum Table 1 Induction and inhibition of Bcl6 or Bcl6b in TFH cells Full table * Figures/tables index Hence, we concluded that antigen presentation by plasma cells inhibits the expression of mRNA for IL-21 and Bcl-6 by in vivo–derived, endogenous, antigen-specific TFH cells (, second panel, in Nat. Immunol.11, 1110–1118 (2010)). Furthermore, the adoptive transfer of antigen-pulsed plasma cells significantly inhibited the expression of mRNA for both IL-21 and Bcl-6 on antigen-specific TFH cells (, in Nat. Immunol.11, 1110–1118 (2010)) without affecting the expression of mRNA for IL-4 or the transcription factor GATA-3 in vivo. Therefore, this provided evidence of antigen-specific negative regulation of the TFH program by plasma cells in vivo. We have also repeated the in vitro experiments using dendritic cells (DCs) or plasma cells as antigen-presenting cells to evaluate the expression of Bcl6 and Bcl6b after in vitro induction of TFH cell activity (Addendum Fig. 4). These studies demonstrated equivalent induction of both Bcl6 and Bcl6bin vitro with DCs as antigen-presenting cells and naive helper T cells with transgenic expression of the 5C.C7 αβ T cell antigen receptor (αβTCR) as antigen-specific helper T cells in these cultures. Consistent with the overall conclusions of our earlier study (Nat. Immunol.11, 1110–1118 (2010)) plasma cells with antigen did not induce Bcl6 or Bcl6b in 5C.C7 αβTCR–transgenic helper T cells in vitro. Figure 4: Selective upregulation of Bcl6 and Bcl6b in antigen-specific TFH cells with the use of DCs but not PCs for antigen presentation in vitro. Quantitative PCR analysis of Bcl6 and Bcl6b in cells derived from CD11c+ DCs or IgM−CD138+ plasma cells (PC) sorted ex vivo with protein antigen and cultured for 4 d in vitro together with cytosolic dye CFSE–labeled naive CD4+ PCC-specific 5C.C7 αβTCR–transgenic helper T cells, followed by sorting of CD44hi 5C.C7 αβTCR–transgenic helper T cells that diluted CFSE, for RNA extraction, cDNA synthesis and SYBR Green–based quantitative PCR amplification of Bcl6 and Bcl6b (details as for Fig. 4 in the original study (Nat. Immunol., 1110–1118 (2010))); results are presented in arbitrary units relative to β2-microglobulin mRNA expression. * Full size image (78 KB) * Previous figure * Figures/tables index We did these new experiments without secondary in vitro transcription or specific synthesis of target cDNA and preamplification by PCR to evaluate the extent of signal to be expected for Bcl6 after in vitro activation of TFH cells. The tenfold induction of both Bcl6 and Bcl6b in these studies is more in line with the expected results and the results of other studies using standard culturing conditions to induce TFH cell activity. These data are also more in line with the expression of Bcl6 and Bcl6b on antigen-specific TFH cells derived in vivo we have reported in all our studies. We believe that the starting cell population, in particular the primary culture stimulus, atypical secondary culture conditions and secondary amplification of the assay all contributed to the more enhanced signals we reported before for Bcl6b ( in Nat. Immunol.11, 1110–1118 (2010)). In conclusion, we mixed up primers for Bcl6 amplification and in parts of our published work inadvertently reported on Bcl6b, not Bcl6. Bcl6b is a distinct gene but remains a structurally and functionally related member of the Bcl6 gene family. We have now been able to revisit this issue and have demonstrated that both Bcl6 and Bcl6b seemed to be coexpressed to a similar extent in naive helper T cells. Both gene products were similarly enhanced in TFH cells that emerged in vivo or were derived in vitro. Furthermore, both genes seemed to be inhibited similarly by antigen presentation by plasma cells. Hence, we believe that this addendum will rectify the mistake made in the initial studies and clarify the coexpression of Bcl6 and Bcl6b in antigen-specific TFH cells. Additional data Author Details * Nadége Pelletier Search for this author in: * NPG journals * PubMed * Google Scholar * Louise J McHeyzer-Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Kurt A Wong Search for this author in: * NPG journals * PubMed * Google Scholar * Eduard Urich Search for this author in: * NPG journals * PubMed * Google Scholar * Nicolas Fazilleau Search for this author in: * NPG journals * PubMed * Google Scholar * Michael G McHeyzer-Williams Search for this author in: * NPG journals * PubMed * Google Scholar
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