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The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.

Sekiya T, Kashiwagi I, Inoue N, Morita R, Hori S, Waldmann H, Rudensky AY, Ichinose H, Metzger D, Chambon P, Yoshimura A - Nat Commun (2011)

Bottom Line: Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.

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Nr4a2 is highly expressed in Treg subsets and directly induces Foxp3.(a) Top: Nr4a2 protein expression in Tregs (CD4+CD25+) and in the Tconv (CD4+CD25–) population. Bottom: quantitative PCR (qPCR) analysis of Nr4a2 expression in CD4+ T cell subsets, normalized against Hprt. (b) 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. (c) 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 domain28, 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+ T cells in the gated area. (d) 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−1 on TGF-β or Nr4a2-mediated Foxp3 induction. (e) 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. (f) ChIP assay of Nr4a2 in the Foxp3 promoter and enhancer regions in naive CD4+ 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+T cells in the gated area. Data shown in (a, b, d, e) are representative of three independent experiments (mean and s.d. of triplicate).
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f1: Nr4a2 is highly expressed in Treg subsets and directly induces Foxp3.(a) Top: Nr4a2 protein expression in Tregs (CD4+CD25+) and in the Tconv (CD4+CD25–) population. Bottom: quantitative PCR (qPCR) analysis of Nr4a2 expression in CD4+ T cell subsets, normalized against Hprt. (b) 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. (c) 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 domain28, 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+ T cells in the gated area. (d) 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−1 on TGF-β or Nr4a2-mediated Foxp3 induction. (e) 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. (f) ChIP assay of Nr4a2 in the Foxp3 promoter and enhancer regions in naive CD4+ 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+T cells in the gated area. Data shown in (a, b, d, e) are representative of three independent experiments (mean and s.d. of triplicate).

Mentions: We confirmed that the expression levels of Nr4a2 were higher in Foxp3+ Tregs than in Foxp3− T cells at both the protein and mRNA levels (Fig. 1a). Nr4a2 was low in naïve T cells, but was transiently induced by TCR stimulation (Fig. 1b). Nr4a2 was undetectable in Th1, Th2, Th17 or iTreg cells differentiated in vitro (Supplementary Fig. S2).


The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.

Sekiya T, Kashiwagi I, Inoue N, Morita R, Hori S, Waldmann H, Rudensky AY, Ichinose H, Metzger D, Chambon P, Yoshimura A - Nat Commun (2011)

Nr4a2 is highly expressed in Treg subsets and directly induces Foxp3.(a) Top: Nr4a2 protein expression in Tregs (CD4+CD25+) and in the Tconv (CD4+CD25–) population. Bottom: quantitative PCR (qPCR) analysis of Nr4a2 expression in CD4+ T cell subsets, normalized against Hprt. (b) 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. (c) 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 domain28, 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+ T cells in the gated area. (d) 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−1 on TGF-β or Nr4a2-mediated Foxp3 induction. (e) 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. (f) ChIP assay of Nr4a2 in the Foxp3 promoter and enhancer regions in naive CD4+ 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+T cells in the gated area. Data shown in (a, b, d, e) are representative of three independent experiments (mean and s.d. of triplicate).
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Related In: Results  -  Collection

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f1: Nr4a2 is highly expressed in Treg subsets and directly induces Foxp3.(a) Top: Nr4a2 protein expression in Tregs (CD4+CD25+) and in the Tconv (CD4+CD25–) population. Bottom: quantitative PCR (qPCR) analysis of Nr4a2 expression in CD4+ T cell subsets, normalized against Hprt. (b) 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. (c) 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 domain28, 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+ T cells in the gated area. (d) 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−1 on TGF-β or Nr4a2-mediated Foxp3 induction. (e) 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. (f) ChIP assay of Nr4a2 in the Foxp3 promoter and enhancer regions in naive CD4+ 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+T cells in the gated area. Data shown in (a, b, d, e) are representative of three independent experiments (mean and s.d. of triplicate).
Mentions: We confirmed that the expression levels of Nr4a2 were higher in Foxp3+ Tregs than in Foxp3− T cells at both the protein and mRNA levels (Fig. 1a). Nr4a2 was low in naïve T cells, but was transiently induced by TCR stimulation (Fig. 1b). Nr4a2 was undetectable in Th1, Th2, Th17 or iTreg cells differentiated in vitro (Supplementary Fig. S2).

Bottom Line: Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.

Show MeSH
Related in: MedlinePlus