The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells
Identifieur interne : 000016 ( Pmc/Curation ); précédent : 000015; suivant : 000017The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells
Auteurs : Takashi Sekiya [Japon] ; Ikkou Kashiwagi [Japon] ; Naoko Inoue [Japon] ; Rimpei Morita [Japon] ; Shohei Hori [Japon] ; Herman Waldmann [Royaume-Uni] ; Alexander Y. Rudensky [États-Unis] ; Hiroshi Ichinose [Japon] ; Daniel Metzger [France] ; Pierre Chambon [France] ; Akihiko Yoshimura [Japon]Source :
- Nature Communications [ 2041-1723 ] ; 2011.
Abstract
Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms. Although the Forkhead transcription factor Foxp3 defines the Treg cell lineage and functions, the molecular mechanisms of Foxp3 induction and maintenance remain elusive. Here we show that Foxp3 is one of the direct targets of Nr4a2. Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications. Ectopic expression of Nr4a2 imparts Treg-like suppressive activity to naïve CD4+ T cells by inducing Foxp3 and by repressing cytokine production, including interferon-γ and interleukin-2. Deletion of Nr4a2 in T cells attenuates induction of Tregs and causes aberrant induction of Th1, leading to the exacerbation of colitis. Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both
Url:
DOI: 10.1038/ncomms1272
PubMed: 21468021
PubMed Central: 3104557
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uniqKey="Kashiwagi I" first="Ikkou" last="Kashiwagi">Ikkou Kashiwagi</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Microbiology and Immunology, Keio University School of Medicine</institution>, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Inoue, Naoko" sort="Inoue, Naoko" uniqKey="Inoue N" first="Naoko" last="Inoue">Naoko Inoue</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Microbiology and Immunology, Keio University School of Medicine</institution>, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Morita, Rimpei" sort="Morita, Rimpei" uniqKey="Morita R" first="Rimpei" last="Morita">Rimpei Morita</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Microbiology and Immunology, Keio University School of Medicine</institution>, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Hori, Shohei" sort="Hori, Shohei" uniqKey="Hori S" first="Shohei" last="Hori">Shohei Hori</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Research Unit for Immune Homeostasis, Research Center for Allergy and Immunology, RIKEN</institution>, Yokohama 230-0045,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Waldmann, Herman" sort="Waldmann, Herman" uniqKey="Waldmann H" first="Herman" last="Waldmann">Herman Waldmann</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Sir William Dunn School of Pathology</institution>, Oxford, OX1 3RE,<country>UK</country>.</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Rudensky, Alexander Y" sort="Rudensky, Alexander Y" uniqKey="Rudensky A" first="Alexander Y." last="Rudensky">Alexander Y. Rudensky</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center</institution>, New York, New York 10065,<country>USA</country>.</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ichinose, Hiroshi" sort="Ichinose, Hiroshi" uniqKey="Ichinose H" first="Hiroshi" last="Ichinose">Hiroshi Ichinose</name><affiliation wicri:level="1"><nlm:aff id="a5"><institution>Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology</institution>, Yokohama 226-8501,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Metzger, Daniel" sort="Metzger, Daniel" uniqKey="Metzger D" first="Daniel" last="Metzger">Daniel Metzger</name><affiliation wicri:level="1"><nlm:aff id="a6"><institution>Department of Functional Genomics Institut de Génétique et Biologie Moléculaire et Cellulaire</institution>, 67404 Illkirch,<country>France</country>.</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Chambon, Pierre" sort="Chambon, Pierre" uniqKey="Chambon P" first="Pierre" last="Chambon">Pierre Chambon</name><affiliation wicri:level="1"><nlm:aff id="a6"><institution>Department of Functional Genomics Institut de Génétique et Biologie Moléculaire et Cellulaire</institution>, 67404 Illkirch,<country>France</country>.</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Yoshimura, Akihiko" sort="Yoshimura, Akihiko" uniqKey="Yoshimura A" first="Akihiko" last="Yoshimura">Akihiko Yoshimura</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Microbiology and Immunology, Keio University School of Medicine</institution>, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a7"><institution>Japan Science and Technology Agency (JST), CREST</institution>, Chiyoda-ku, Tokyo 102-0075,<country>Japan</country>.</nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Nature Communications</title><idno type="eISSN">2041-1723</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms. Although the Forkhead transcription factor Foxp3 defines the Treg cell lineage and functions, the molecular mechanisms of Foxp3 induction and maintenance remain elusive. Here we show that Foxp3 is one of the direct targets of Nr4a2. Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications. Ectopic expression of Nr4a2 imparts Treg-like suppressive activity to naïve CD4<sup>+</sup> T cells by inducing Foxp3 and by repressing cytokine production, including interferon-γ and interleukin-2. Deletion of Nr4a2 in T cells attenuates induction of Tregs and causes aberrant induction of Th1, leading to the exacerbation of colitis. Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both <italic>in vitro</italic> and <italic>in vivo</italic>. Thus, Nr4a2 has the ability to maintain T-cell homoeostasis by regulating induction, maintenance and suppressor functions of Tregs, and by repression of aberrant Th1 induction.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Sakaguchi, S" uniqKey="Sakaguchi S">S. Sakaguchi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ochs, H D" uniqKey="Ochs H">H. D. Ochs</name></author><author><name sortKey="Ziegler, S F" uniqKey="Ziegler S">S. F. Ziegler</name></author><author><name sortKey="Torgerson, T R" uniqKey="Torgerson T">T. R. Torgerson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bennett, C L" uniqKey="Bennett C">C. L. Bennett</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Godfrey, V L" uniqKey="Godfrey V">V. L. Godfrey</name></author><author><name sortKey="Wilkinson, J E" uniqKey="Wilkinson J">J. E. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type="epub">2041-1723</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21468021</article-id><article-id pub-id-type="pmc">3104557</article-id><article-id pub-id-type="pii">ncomms1272</article-id><article-id pub-id-type="doi">10.1038/ncomms1272</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4<sup>+</sup> T cells</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Sekiya</surname><given-names>Takashi</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kashiwagi</surname><given-names>Ikkou</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Inoue</surname><given-names>Naoko</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Morita</surname><given-names>Rimpei</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Hori</surname><given-names>Shohei</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Waldmann</surname><given-names>Herman</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Rudensky</surname><given-names>Alexander Y.</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Ichinose</surname><given-names>Hiroshi</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Metzger</surname><given-names>Daniel</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Chambon</surname><given-names>Pierre</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Yoshimura</surname><given-names>Akihiko</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a7">7</xref></contrib><aff id="a1"><label>1</label><institution>Department of Microbiology and Immunology, Keio University School of Medicine</institution>, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,<country>Japan</country>.</aff><aff id="a2"><label>2</label><institution>Research Unit for Immune Homeostasis, Research Center for Allergy and Immunology, RIKEN</institution>, Yokohama 230-0045,<country>Japan</country>.</aff><aff id="a3"><label>3</label><institution>Sir William Dunn School of Pathology</institution>, Oxford, OX1 3RE,<country>UK</country>.</aff><aff id="a4"><label>4</label><institution>Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center</institution>, New York, New York 10065,<country>USA</country>.</aff><aff id="a5"><label>5</label><institution>Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology</institution>, Yokohama 226-8501,<country>Japan</country>.</aff><aff id="a6"><label>6</label><institution>Department of Functional Genomics Institut de Génétique et Biologie Moléculaire et Cellulaire</institution>, 67404 Illkirch,<country>France</country>.</aff><aff id="a7"><label>7</label><institution>Japan Science and Technology Agency (JST), CREST</institution>, Chiyoda-ku, Tokyo 102-0075,<country>Japan</country>.</aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>yoshimura@a6.keio.jp</email></corresp></author-notes><pub-date pub-type="ppub"><month>04</month><year>2011</year></pub-date><pub-date pub-type="epub"><day>05</day><month>04</month><year>2011</year></pub-date><volume>2</volume><fpage>269</fpage><lpage></lpage><history><date date-type="received"><day>13</day><month>01</month><year>2011</year></date><date date-type="accepted"><day>09</day><month>03</month><year>2011</year></date></history><permissions><copyright-statement>Copyright © 2011, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.</copyright-statement><copyright-year>2011</copyright-year><copyright-holder>Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</ext-link></license-p></license></permissions><abstract><p>Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms. Although the Forkhead transcription factor Foxp3 defines the Treg cell lineage and functions, the molecular mechanisms of Foxp3 induction and maintenance remain elusive. Here we show that Foxp3 is one of the direct targets of Nr4a2. Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications. Ectopic expression of Nr4a2 imparts Treg-like suppressive activity to naïve CD4<sup>+</sup> T cells by inducing Foxp3 and by repressing cytokine production, including interferon-γ and interleukin-2. Deletion of Nr4a2 in T cells attenuates induction of Tregs and causes aberrant induction of Th1, leading to the exacerbation of colitis. Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both <italic>in vitro</italic> and <italic>in vivo</italic>. Thus, Nr4a2 has the ability to maintain T-cell homoeostasis by regulating induction, maintenance and suppressor functions of Tregs, and by repression of aberrant Th1 induction.</p></abstract><abstract abstract-type="web-summary"><p><inline-graphic id="i1" xlink:href="ncomms1272-i1.jpg"></inline-graphic> Regulatory T cells are characterized by the expression of Foxp3, however, how the expression of this protein is controlled is unclear. Here, the authors show that the nuclear orphan receptor, Nr4a2, is a transcriptional activator of Foxp3, and suggest that it is required for the function of regulatory T cells.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Nr4a2 is highly expressed in Treg subsets and directly induces Foxp3.</title><p>(<bold>a</bold>) Top: Nr4a2 protein expression in Tregs (CD4<sup>+</sup>CD25<sup>+</sup>) and in the Tconv (CD4<sup>+</sup>CD25<sup>–</sup>) population. Bottom: quantitative PCR (qPCR) analysis of Nr4a2 expression in CD4<sup>+</sup> T cell subsets, normalized against Hprt. (<bold>b</bold>) Naïve T cells were incubated under TCR stimulation with anti-CD3, CD28 antibodies. The protein level at each indicated time point was analysed by western blotting. Bottom: qPCR results depict the induction of Nr4a2 by TCR stimulation. Data are normalized against Hprt. (<bold>c</bold>) Effects of expression of Nr4a2 (indicated by GFP expression, which is synergistically expressed by IRES in the eMIGR1 vector) and its transactivation-deficient mutant Nr4a2-ΔN, which lacks the N terminus 237aa of the transactivation domain<xref ref-type="bibr" rid="b28">28</xref>, on Foxp3 expression eMIGR1, an empty vector, was used as a negative control. Foxp3 induction was detected with an anti-human CD2 (hCD2) antibody, which recognizes membranous hCD2 protein coexpressed with endogenous Foxp3 in the Foxp3-hCD2-hCD52-KI mice. Numbers in FACS plots represent percentages of CD4<sup>+</sup> T cells in the gated area. (<bold>d</bold>) Effects of blocking TGF-β signalling with a chemical inhibitor (SB431542, at 5 or 20 μM) or TGF-β neutralizing antibodies, at 5 or 20 μg ml<sup>−1</sup> on TGF-β or Nr4a2-mediated Foxp3 induction. (<bold>e</bold>) Foxp3 promoter and enhancer-driven luciferase reporter constructs were transfected into the 293 cell line. Regions with consensus binding sequences for Nr4a are indicated by open ovals. (<bold>f</bold>) ChIP assay of Nr4a2 in the <italic>Foxp3</italic> promoter and enhancer regions in naive CD4<sup>+</sup> T cells and Tregs. Cell lysates were immunoprecipitated with anti-Nr4a2 (open bars) or control IgG (filled bars). Values are presented as the percentage of corresponding input with the standard deviation (s.d.) of three independent experiments of triplicate. Numbers in FACS plots represent percentages of CD4<sup>+</sup>T cells in the gated area. Data shown in (<bold>a</bold>, <bold>b</bold>, <bold>d</bold>, <bold>e</bold>) are representative of three independent experiments (mean and s.d. of triplicate).</p></caption><graphic xlink:href="ncomms1272-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Nr4a2 induces Treg-like active histone modifications in <italic>Foxp3</italic> regulatory regions.</title><p>(<bold>a</bold>–<bold>c</bold>) Native ChIP assay of <italic>Foxp3</italic> promoter (left), CNS1 (middle) and CNS2 (right) enhancers was performed with antibodies against modified histones (<bold>a</bold>: anti-acetylated H4, open bars; <bold>b</bold>: anti-tri-methylated H3K4, dotted bars; <bold>c</bold>: anti-tri-methylated H3K27, striped bars) and control IgG (filled bars). Naïve T cells, Tregs, mock-transduced cultured cells and sorted GFP<sup>+</sup> cells from the population of eMIGR1 or eMIGR1-Nr4a2 retrovirus-transduced cells were analysed. Values are depicted as the percentage of corresponding input. Data are representative of two independent experiments (mean and s.d. of triplicate). (<bold>d</bold>) Bisulphite sequencing revealed that Nr4a2 can not mediate the demethylation of the CpG island in the CNS2 region of the <italic>foxp3</italic> locus. Sequencing was performed against the bisulphite-treated genomic DNA from naïve T cells, Tregs, sorted GFP<sup>+</sup> cells from the eMIGR1 retrovirus-transduced cells and sorted GFP<sup>+</sup>Foxp3<sup>+</sup> cells from the eMIGR1-Nr4a2 retrovirus-transduced cells. Ten CG sites in the CNS2 locus, located at the indicated positions, were analysed. Top: closed and open circles indicate methylated and unmethylated CG sites, respectively. Bottom: data shown in top panels were quantitated, and shown as percentages of methylation at each position. Data are pooled from two independent experiments. Foxp3<sup>+</sup> cells were sorted with APC-conjugated anti-hCD2 antibody unless otherwise indicated.</p></caption><graphic xlink:href="ncomms1272-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Nr4a families vary in their ability to induce Foxp3 and to repress IFN-γ.</title><p>(<bold>a</bold>) Top row: effects of Nr4a1, Nr4a2 and Nr4a3 expression on Foxp3 expression. Bottom row: effects of Nr4a1, Nr4a2 and Nr4a3 expression on IFN-γ expression. eMIGR or eMIGR-Nr4a retrovirus-transduced cells were restimulated with phorbol myristate acetate (PMA) and ionomycine at 60 h after infection for 8 h. (<bold>b</bold>, <bold>c</bold>) Quantification of the results of <bold>a</bold>. (<bold>d</bold>) Effects of Nr4a2 transductions on Foxp3 expression (top row) and IFN-γ repression (bottom row) under Th1 and Th17 conditions. (<bold>e</bold>) Quantification of the results of the eMIGR-Nr4a2 retrovirus-transduced cells in <bold>d</bold>. (<bold>f</bold>) Schematic representation of Nr4a2, Nr4a3 and the chimeric protein of Nr4a3 in which the N-terminal of Nr4a3 is replaced with that of Nr4a2 (Nr4a2-a3 chimera). DBD, DNA-binding domain; LBD, ligand-binding domain. (<bold>g</bold>) Foxp3 induction (top row) and IFN-γ repression (bottom row) by Nr4a2, Nr4a3 and Nr4a2-a3 chimera. Cells were transduced with eMIGR1- or eMIGR1-Nr4a-retorviruses, and Foxp3 induction and IFN-γ repression were analysed. (<bold>h</bold>) Transcriptional activities as monomers of Nr4a2, Nr4a3 and Nr4a2-a3 chimera were demonstrated using NBRE-luc in 293T cells. (<bold>i</bold>) Quantification of the result in <bold>g</bold>. Numbers in FACS plots represent percentages of CD4<sup>+</sup> T cells in the gated area. Data in (<bold>b</bold>, <bold>c</bold>, <bold>e</bold>, <bold>h</bold>, <bold>i</bold>) are representative of three independent experiments (mean and s.d. of triplicate). *<italic>P</italic>< 0.05 and **<italic>P</italic><0.01 (two-tailed Student's <italic>t</italic>-test).</p></caption><graphic xlink:href="ncomms1272-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Nr4a2 induces suppression activity <italic>in vitro</italic> by upregulating CD25 and repressing IL-2.</title><p>(<bold>a</bold>) qPCR analysis of the effects of Nr4a2 expression on the expression of Treg effector genes. mRNA was prepared from naïve T cells (columns 1 and 2), Tregs (columns 3 and 4), the GFP<sup>+</sup> fraction of eMIGR1 retrovirus-transduced cells (columns 5, 6, 11 and 12), and the GFP<sup>+</sup>Foxp3<sup>–</sup> (columns 7, 8, 13 and 14) or GFP<sup>+</sup>Foxp3<sup>+</sup> (columns 9, 10, 15 and 16) fraction of eMIGR1-Nr4a2 retrovirus-transduced cells, which were incubated with (even-numbered columns) or without (odd-numbered columns) PMA/ionomycine for 2 h. Retrovirus-transduced cells were sampled at 48 h (blue bars, columns 5 to 10) and 84 h (green bars, columns 11 to 16) after infection. Expression levels of the indicated mRNA, are presented relative to the <italic>Hprt</italic> expression. Data are representative of three independent experiments (mean and s.d. of triplicate). (<bold>b</bold>) <italic>In vitro</italic> suppression assay. Top: suppression of CFSE-labeled naïve CD4<sup>+</sup> T cells (responder, Tresp) by Tregs (open squares), the GFP<sup>+</sup> fraction of eMIGR1 retrovirus-transduced cells (open circles), the GFP<sup>+</sup>Foxp3<sup>+</sup> fraction of eMIGR1-Nr4a2 retrovirus-transduced cells (filled triangles), or the GFP<sup>+</sup>Foxp3<sup>+</sup> fraction of eMIGR1-Nr4a2 retrovirus-transduced cells supplemented with IL-2 (5 ng ml<sup>−1</sup>, crosses). Cells were cultured for 96 h with anti-CD3/anti-CD28-coated beads. The ratio of responder cells with more than three divisions are presented. Bottom: results in the top panel at the 1:1 ratio of regulatory cells/Tresp are presented. Numbers in FACS plots represent percentages of CD4<sup>+</sup> T cells in the gated area. Data are representative of three independent experiments (mean and s.d. of triplicate). **<italic>P</italic><0.01 (two-tailed Student′s <italic>t</italic>-test).</p></caption><graphic xlink:href="ncomms1272-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Deletion of Nr4a2 attenuates iTreg induction.</title><p>(<bold>a</bold>) Effects of Nr4a2-knockout on CD4<sup>+</sup>T cells differentiation. iTreg induction was carried out at the two different TGF-β concentrations indicated. Naïve CD4<sup>+</sup>T cells from <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> mice were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or IL-17 expressions were analysed. (<bold>b</bold>) Quantification of the results in <bold>a</bold> of the iTreg condition at 0.2 and 0.5 ng ml<sup>−1</sup> TGF-β. Top: ratio of the Foxp3<sup>+</sup> fractions. Bottom: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3<sup>+</sup> fractions. The value of <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> cells at 0.2 ng ml<sup>−1</sup> TGF-β was set to 1. Data are representative of three independent experiments (mean±s.d. in triplicate). Filled bars: <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> cells; open bars: <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> cells. (<bold>c</bold>–<bold>f</bold>) Effects of the <italic>ex vivo</italic> deletion of Nr4a2 on Th and iTreg differentiation. Isolated naïve CD4<sup>+</sup> T cells from <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>ERT2-Cre</italic><sup><italic>+</italic></sup> (<bold>c</bold>, <bold>d</bold>) or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>ERT2-Cre</italic><sup><italic>+</italic></sup> (<bold>e</bold>, <bold>f</bold>) mice were treated (<bold>d</bold>, <bold>f</bold>) or untreated (<bold>c</bold>, <bold>e</bold>) with 4-hydroxy tamoxifen for 6 h in RPMI1640 10% FBS at 37 °C. Cells were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or Il-17a expression were analysed. (<bold>g</bold>) Body weight of <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> (open circles) or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> (filled squares) mice treated with 1.5% DSS. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). (<bold>h</bold>) Histological analysis of the experiments of (<bold>g</bold>) at day 11. Top: hematoxylin and eosin staining of colon sections. Scale bar, 100 μm. Bottom: histological score of colitis. Data are representative of two independent experiments, each with five mice per group (vertical bars: median of the colitis scores). (<bold>i</bold>) Analysis of colon and caecum lamina proprial CD4<sup>+</sup> T cells from the experiments in <bold>g</bold> at day 11, examining IFN-γ and IL-17a expression (top) and Foxp3 expression (bottom). (<bold>j</bold>) Quantification of results in <bold>i</bold>. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). Filled bars: <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> mice; open bars: <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Lck-Cre</italic><sup><italic>+</italic></sup> mice. Numbers in FACS plots represent percentages of CD4<sup>+</sup> T cells in the gated area. *<italic>P</italic><0.05, **<italic>P</italic><0.01 (two-tailed Student's <italic>t</italic>-test).</p></caption><graphic xlink:href="ncomms1272-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>Nr4a2 stabilizes Foxp3 expression in Tregs.</title><p>(<bold>a</bold>) Top: CD4<sup>+</sup> and CD8<sup>+</sup> compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. (<bold>b</bold>) Quantification of the results in <bold>a</bold>. Ratio of YFP<sup>+</sup>/YFP<sup>–</sup> in total Foxp3<sup>+</sup> cells. Filled bars: results of thymus; open bars: results of spleen plus lymph nodes. Data are pooled from three independent experiments, with six mice from each genotype total, aged 11- to 13 weeks (mean±s.d.). (<bold>c</bold>) Left: CFSE-labeled CD4<sup>+</sup>CD25<sup>+</sup>YFP<sup>+</sup> Tregs from <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre/+</italic></sup>or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre/+</italic></sup> mice, cultured under neutral condition with 20 ng ml<sup>−1</sup> IL-2, thymidine (2 mM) and z-VAD-fmk (10 μM) were analysed for Foxp3 expression at the time indicated. YFP-fluorescences were bleached by fixation/permeabilization. Right: quantification of the results in left. Open squares: <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> cells; crosses: <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> cells. (<bold>d</bold>) Proliferation and survival status of cells in <bold>c</bold>. Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> cells; crosses: <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> cells. (<bold>e</bold>) Rag2<sup>–</sup> mice were co-transferred with 3×10<sup>5</sup> CD4<sup>+</sup>CD25<sup>+</sup>YFP<sup>+</sup>Ly5.2<sup>+</sup>Tregs from <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> mice and 3×10<sup>5</sup> CD4<sup>+</sup>CD25<sup>+</sup>Ly5.1<sup>+</sup> wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. (<bold>f</bold>) Quantification of the results in <bold>e</bold>. Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3<sup>+</sup> fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1<sup>+</sup> fractions; open bars: cells in Ly5.1<sup>−</sup> fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). (<bold>g</bold>) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4<sup>+</sup> T cells, or of naïve CD4<sup>+</sup> T cells stimulated under the iTreg condition. (<bold>h</bold>) Left: coimmunoprecipitation of Flag-Runx1 and T7-Nr4a in 293T cell lysates. Right: quantification of the result in left. Intensity of the anti-T7 blot of the anti-Flag immunoprecipitates, normalized with the anti-T7 input. (<bold>i</bold>) Top: schematic representation of the reporter construct. Nr4a-binding site and the Runx1-binding sites are indicated. Bottom: results of the luciferase assay. Numbers in FACS plots represent percentages of cells in the gated area. Data are representative of three (<bold>c</bold>, <bold>h</bold>, <bold>i</bold>) or two (<bold>d</bold>) independent experiments (mean±s.d. of triplicate). *<italic>P</italic>< 0.05, **<italic>P</italic><0.01 (two-tailed Student's <italic>t</italic>-test).</p></caption><graphic xlink:href="ncomms1272-f6"></graphic></fig><fig id="f7"><label>Figure 7</label><caption><title>Attenuated <italic>in vitro</italic> and <italic>in vivo</italic> suppressive activity of Nr4a2-deficient Tregs.</title><p>(<bold>a</bold>) FACS analysis of the indicated protein levels in YFP<sup>+</sup>Foxp3<sup>+</sup> and YFP<sup>–</sup>Foxp3<sup>+</sup> Tregs of <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre/+</italic></sup> or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre/+</italic></sup> mice. For Foxp3, CD25, CTLA-4 and GITR expression levels in non-Treg cells are represented by black lines, as a negative value. Data are representative of three independent experiments (means of duplicate are shown for the positive fractions of Annexin V and Ki67 staining). (<bold>b</bold>) Comparison of mRNA levels between Tregs in <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> and <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> mice. Expression levels of the indicated mRNA, are presented relative to <italic>Hprt</italic> expression. Open bars: <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> Tregs; closed bars: <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> Tregs. (<bold>c</bold>) Top: <italic>in vitro</italic> suppression assay with CFSE-labeled wild-type CD4<sup>+</sup>CD25<sup>–</sup> cells as responders and CD4<sup>+</sup>CD25<sup>+</sup>YFP<sup>+</sup> Tregs from <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> (open squares) or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> (closed triangles) mice as suppressors. Ratios of responder cells with more than three divisions are presented. Bottom: results in the top panel at 1:1 Treg/Tresp ratio. (<bold>d</bold>) Body weight of <italic>Rag2</italic><sup>–</sup> mice injected with 5×10<sup>5</sup> naïve Ly5.1<sup>+</sup>CD4<sup>+</sup> T cells alone or in combination with 3×10<sup>5</sup> CD4<sup>+</sup>CD25<sup>+</sup>YFP<sup>+</sup>Ly5.2<sup>+</sup> Tregs from <italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> or <italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> mice. Data are pooled from two independent experiments, with seven mice from each sample set total. The ranges of body weights (relative body weights to day 0) of each group at day 21 are Tnaive only: 0.69–0.76; Tnaive+<italic>Nr4a2</italic><sup><italic>+/+</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> Tregs: 0.96–1.08; Tnaive+<italic>Nr4a2</italic><sup><italic>fl/fl</italic></sup><italic>Foxp3</italic><sup><italic>Yfp-Cre</italic></sup> Tregs: 0.76–0.88. (<bold>e</bold>) Expression of Foxp3 and IFN-γ in CD4<sup>+</sup> T cells from the spleen and lymph nodes (left panels), and from the colon and caecum lamina propria (right panels) in <italic>Rag2</italic><sup>–</sup> recipients presented in <bold>d</bold> at day 21. (<bold>f</bold>) Frequencies of IFN-γ-expressing cells in Foxp3<sup>–</sup>CD4<sup>+</sup> T cells and Foxp3<sup>+</sup>CD4<sup>+</sup> T cells from the spleen and lymph nodes of <italic>Rag2</italic><sup>–</sup> recipients presented in <bold>d</bold> at day 21. Numbers in FACS plots represent percentages of CD4<sup>+</sup> T cells in the gated area. Data in <bold>b</bold>, <bold>c</bold> are representative of three independent experiments (mean and s.d. of triplicate). *<italic>P</italic><0.05 (two-tailed Student's <italic>t</italic>-test).</p></caption><graphic xlink:href="ncomms1272-f7"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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