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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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Related in: MedlinePlus

Nr4a2 stabilizes Foxp3 expression in Tregs.(a) Top: CD4+ and CD8+ compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. (b) Quantification of the results in a. Ratio of YFP+/YFP– in total Foxp3+ 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.). (c) Left: CFSE-labeled CD4+CD25+YFP+ Tregs from Nr4a2+/+Foxp3Yfp-Cre/+or Nr4a2fl/flFoxp3Yfp-Cre/+ mice, cultured under neutral condition with 20 ng ml−1 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: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (d) Proliferation and survival status of cells in c. Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (e) Rag2– mice were co-transferred with 3×105 CD4+CD25+YFP+Ly5.2+Tregs from Nr4a2fl/flFoxp3Yfp-Cre mice and 3×105 CD4+CD25+Ly5.1+ wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. (f) Quantification of the results in e. Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1+ fractions; open bars: cells in Ly5.1− fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). (g) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4+ T cells, or of naïve CD4+ T cells stimulated under the iTreg condition. (h) 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. (i) 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 (c, h, i) or two (d) independent experiments (mean±s.d. of triplicate). *P< 0.05, **P<0.01 (two-tailed Student's t-test).
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f6: Nr4a2 stabilizes Foxp3 expression in Tregs.(a) Top: CD4+ and CD8+ compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. (b) Quantification of the results in a. Ratio of YFP+/YFP– in total Foxp3+ 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.). (c) Left: CFSE-labeled CD4+CD25+YFP+ Tregs from Nr4a2+/+Foxp3Yfp-Cre/+or Nr4a2fl/flFoxp3Yfp-Cre/+ mice, cultured under neutral condition with 20 ng ml−1 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: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (d) Proliferation and survival status of cells in c. Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (e) Rag2– mice were co-transferred with 3×105 CD4+CD25+YFP+Ly5.2+Tregs from Nr4a2fl/flFoxp3Yfp-Cre mice and 3×105 CD4+CD25+Ly5.1+ wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. (f) Quantification of the results in e. Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1+ fractions; open bars: cells in Ly5.1− fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). (g) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4+ T cells, or of naïve CD4+ T cells stimulated under the iTreg condition. (h) 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. (i) 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 (c, h, i) or two (d) independent experiments (mean±s.d. of triplicate). *P< 0.05, **P<0.01 (two-tailed Student's t-test).

Mentions: Next, we analysed the roles of Nr4a2 in Tregs by breeding Nr4a2-floxed mice with Foxp3YFP-Cre mice3637 to knockout Nr4a2 specifically in Tregs. We confirmed the specific deletion of Nr4a2 in Tregs (Supplementary Fig. S6a). Nr4a2fl/flFoxp3YFP-Cre mice were apparently normal, disease free and without differences in the activation status of CD4+ T cells (Supplementary Fig. S6b). In the first experiment, we compared the relative sizes of Nr4a2-sufficient and Nr4a2-deficient Tregs population in Nr4a2+/+Foxp3YFP-Cre/+, heterozygous Nr4a2fl/+Foxp3YFP-Cre/+ and homozygous Nr4a2fl/flFoxp3YFP-Cre/+ females (foxp3 is on X chromosome). The YFP+/YFP– ratio in Nr4a2+/+Foxp3YFP-Cre/+ mice was near 1:1 in both thymus and periphery (Fig. 6a,b). However, in Nr4a2fl/flFoxp3YFP-Cre/+ mice, ratios of YFP+/YFP− were suboptimal (∼0.3 in thymus and ∼0.1 in periphery, Fig. 6a,b), indicating that Tregs were not properly maintained on deletion of Nr4a2. The YFP+/YFP− ratio in Nr4a2fl/+Foxp3YFP-Cre/+ mice was near ∼0.8 in thymus, and ∼0.4 in periphery, indicating that Tregs are not properly maintained even by the heterozygous deletion of Nr4a2 (Fig. 6b). Importantly, the ratio of YFP+/YFP− in Nr4a2fl/flFoxp3YFP-Cre/+ mice decreased with age (Supplementary Fig. S6c), supporting the idea that the disequilibrium is caused by the accelerated reduction of the Nr4a2-deficient Tregs in the periphery, rather than the reduced positive selection of Tregs in the thymus. Another possibility which could explain this disequilibrium might be reduced export of Nr4a2-deficient Tregs, although we think it less probable, because the reduced export is expected to result in an increase in the number of Nr4a2-deficient Tregs in the thymus. Deletion of Nr4a1 did not affect the YFP+/YFP− ratio in Tregs (Fig. 6a,b and Supplementary Fig. S6d), indicating that Nr4a1 does not contribute to Treg homoeostasis; this corresponds well with the observation that Nr4a1-transduction did not induce Foxp3 (Fig. 3a). The CpG methylation status of CNS2, an important determinant of Treg lineage stability, was not perturbed in Nr4a2fl/flLck-Cre+ mice (Supplementary Fig. S6e), suggesting that Nr4a2 is not essential for the demethylation of CNS2 during nTreg development. This was consistent with our forced expression study (Fig. 2d).


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 stabilizes Foxp3 expression in Tregs.(a) Top: CD4+ and CD8+ compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. (b) Quantification of the results in a. Ratio of YFP+/YFP– in total Foxp3+ 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.). (c) Left: CFSE-labeled CD4+CD25+YFP+ Tregs from Nr4a2+/+Foxp3Yfp-Cre/+or Nr4a2fl/flFoxp3Yfp-Cre/+ mice, cultured under neutral condition with 20 ng ml−1 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: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (d) Proliferation and survival status of cells in c. Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (e) Rag2– mice were co-transferred with 3×105 CD4+CD25+YFP+Ly5.2+Tregs from Nr4a2fl/flFoxp3Yfp-Cre mice and 3×105 CD4+CD25+Ly5.1+ wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. (f) Quantification of the results in e. Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1+ fractions; open bars: cells in Ly5.1− fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). (g) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4+ T cells, or of naïve CD4+ T cells stimulated under the iTreg condition. (h) 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. (i) 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 (c, h, i) or two (d) independent experiments (mean±s.d. of triplicate). *P< 0.05, **P<0.01 (two-tailed Student's t-test).
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f6: Nr4a2 stabilizes Foxp3 expression in Tregs.(a) Top: CD4+ and CD8+ compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. (b) Quantification of the results in a. Ratio of YFP+/YFP– in total Foxp3+ 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.). (c) Left: CFSE-labeled CD4+CD25+YFP+ Tregs from Nr4a2+/+Foxp3Yfp-Cre/+or Nr4a2fl/flFoxp3Yfp-Cre/+ mice, cultured under neutral condition with 20 ng ml−1 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: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (d) Proliferation and survival status of cells in c. Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: Nr4a2fl/flFoxp3Yfp-Cre cells; crosses: Nr4a2+/+Foxp3Yfp-Cre cells. (e) Rag2– mice were co-transferred with 3×105 CD4+CD25+YFP+Ly5.2+Tregs from Nr4a2fl/flFoxp3Yfp-Cre mice and 3×105 CD4+CD25+Ly5.1+ wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. (f) Quantification of the results in e. Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1+ fractions; open bars: cells in Ly5.1− fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). (g) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4+ T cells, or of naïve CD4+ T cells stimulated under the iTreg condition. (h) 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. (i) 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 (c, h, i) or two (d) independent experiments (mean±s.d. of triplicate). *P< 0.05, **P<0.01 (two-tailed Student's t-test).
Mentions: Next, we analysed the roles of Nr4a2 in Tregs by breeding Nr4a2-floxed mice with Foxp3YFP-Cre mice3637 to knockout Nr4a2 specifically in Tregs. We confirmed the specific deletion of Nr4a2 in Tregs (Supplementary Fig. S6a). Nr4a2fl/flFoxp3YFP-Cre mice were apparently normal, disease free and without differences in the activation status of CD4+ T cells (Supplementary Fig. S6b). In the first experiment, we compared the relative sizes of Nr4a2-sufficient and Nr4a2-deficient Tregs population in Nr4a2+/+Foxp3YFP-Cre/+, heterozygous Nr4a2fl/+Foxp3YFP-Cre/+ and homozygous Nr4a2fl/flFoxp3YFP-Cre/+ females (foxp3 is on X chromosome). The YFP+/YFP– ratio in Nr4a2+/+Foxp3YFP-Cre/+ mice was near 1:1 in both thymus and periphery (Fig. 6a,b). However, in Nr4a2fl/flFoxp3YFP-Cre/+ mice, ratios of YFP+/YFP− were suboptimal (∼0.3 in thymus and ∼0.1 in periphery, Fig. 6a,b), indicating that Tregs were not properly maintained on deletion of Nr4a2. The YFP+/YFP− ratio in Nr4a2fl/+Foxp3YFP-Cre/+ mice was near ∼0.8 in thymus, and ∼0.4 in periphery, indicating that Tregs are not properly maintained even by the heterozygous deletion of Nr4a2 (Fig. 6b). Importantly, the ratio of YFP+/YFP− in Nr4a2fl/flFoxp3YFP-Cre/+ mice decreased with age (Supplementary Fig. S6c), supporting the idea that the disequilibrium is caused by the accelerated reduction of the Nr4a2-deficient Tregs in the periphery, rather than the reduced positive selection of Tregs in the thymus. Another possibility which could explain this disequilibrium might be reduced export of Nr4a2-deficient Tregs, although we think it less probable, because the reduced export is expected to result in an increase in the number of Nr4a2-deficient Tregs in the thymus. Deletion of Nr4a1 did not affect the YFP+/YFP− ratio in Tregs (Fig. 6a,b and Supplementary Fig. S6d), indicating that Nr4a1 does not contribute to Treg homoeostasis; this corresponds well with the observation that Nr4a1-transduction did not induce Foxp3 (Fig. 3a). The CpG methylation status of CNS2, an important determinant of Treg lineage stability, was not perturbed in Nr4a2fl/flLck-Cre+ mice (Supplementary Fig. S6e), suggesting that Nr4a2 is not essential for the demethylation of CNS2 during nTreg development. This was consistent with our forced expression study (Fig. 2d).

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