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Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

Maruyama T, Li J, Vaque JP, Konkel JE, Wang W, Zhang B, Zhang P, Zamarron BF, Yu D, Wu Y, Zhuang Y, Gutkind JS, Chen W - Nat. Immunol. (2010)

Bottom Line: We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells).Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter.Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

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Id3−/− T cells fails to generate Foxp3+ Treg cells in response to TGF-βa, Quantitative analysis of Foxp3, present as mRNA expression relative to HPRT (mean ± s.d. of duplicate wells) in naïve CD4+CD25− T cells overnight after TCR stimulation with or without TGF-β. Data shown is of one experiment representative of at least five. b, Flow cytometry of naïve CD4+CD25− T cells cultured with TCR (with or without TGF-β) for three days. Numbers in the quadrants indicate Foxp3+ Treg cells. Each plot is of one experiment representative of five. c, Percent Foxp3+ Treg cells (mean ± s.d. of five experiments) in cultured cells in a. ** P < 0.01. d,e, Flow cytometry of CD4+CD25− T cells cultured with indicated reagents for 3-5 days. Numbers in the quadrants indicate CD25+Foxp3+ Treg cells of Id3+/+ (top row) or Id3−/− (bottom row) mice. Each plot is of one experiment representative of at least two. RA, retinoic acid.
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Figure 2: Id3−/− T cells fails to generate Foxp3+ Treg cells in response to TGF-βa, Quantitative analysis of Foxp3, present as mRNA expression relative to HPRT (mean ± s.d. of duplicate wells) in naïve CD4+CD25− T cells overnight after TCR stimulation with or without TGF-β. Data shown is of one experiment representative of at least five. b, Flow cytometry of naïve CD4+CD25− T cells cultured with TCR (with or without TGF-β) for three days. Numbers in the quadrants indicate Foxp3+ Treg cells. Each plot is of one experiment representative of five. c, Percent Foxp3+ Treg cells (mean ± s.d. of five experiments) in cultured cells in a. ** P < 0.01. d,e, Flow cytometry of CD4+CD25− T cells cultured with indicated reagents for 3-5 days. Numbers in the quadrants indicate CD25+Foxp3+ Treg cells of Id3+/+ (top row) or Id3−/− (bottom row) mice. Each plot is of one experiment representative of at least two. RA, retinoic acid.

Mentions: Next, we determined whether the Id3 deficiency affected the generation of Foxp3+ Treg cells in vitro. We cultured peripheral naïve CD4+CD25−Foxp3− T cells with CD3- and CD28-specific antibodies in the presence of TGF-β1 to induce Foxp3+ Treg cells 9. TGF-β1 induced profoundly lower levels of Foxp3 mRNA and protein in naïve Id3−/− compared to WT CD4+CD25− T cells (Fig. 2a-c) over a range of TGF-β1 concentrations, although the defect was partially reversed with high doses of TGF-β1 (Supplementary Fig. 3). Addition of exogenous IL-2 did not correct the defect, eliminating the lack of IL-2 as the cause (Fig. 2d). Inclusion of retinoic acid (RA) in the cultures only slightly increased the percentage of Id3−/− Foxp3+ T cells, whereas RA exhibited the reported synergistic effect on TGF-β1-dependent Foxp3+ Treg differentiation in WT CD4+ T cells29 (Fig. 2e). Analysis of proximal Smad activation downstream of TGF-β1 signaling revealed no significant difference in TGF-β receptor-activated Smad2 and 3 phosphorylation (represented by P-Smad 2) between Id3−/− and WT CD4+ T cells following TGF-β1 treatment (Supplementary Fig. 4a). Smad7, an inhibitory Smad that antagonizes Smad2- and 3-mediated TGF-β signaling30, was lower in Id3−/−compared to WT CD4+ T cells following TGF-β1 treatment (Supplementary Fig. 4b), ruling-out Smad7 as a factor in causing reduced Foxp3-induction in Id3−/− cells. Id3 is therefore a critical component of TGF-β1 mediated Foxp3+ Treg generation, but it likely functions downstream of P-Smad2- and P-Smad3-mediated signal pathways.


Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

Maruyama T, Li J, Vaque JP, Konkel JE, Wang W, Zhang B, Zhang P, Zamarron BF, Yu D, Wu Y, Zhuang Y, Gutkind JS, Chen W - Nat. Immunol. (2010)

Id3−/− T cells fails to generate Foxp3+ Treg cells in response to TGF-βa, Quantitative analysis of Foxp3, present as mRNA expression relative to HPRT (mean ± s.d. of duplicate wells) in naïve CD4+CD25− T cells overnight after TCR stimulation with or without TGF-β. Data shown is of one experiment representative of at least five. b, Flow cytometry of naïve CD4+CD25− T cells cultured with TCR (with or without TGF-β) for three days. Numbers in the quadrants indicate Foxp3+ Treg cells. Each plot is of one experiment representative of five. c, Percent Foxp3+ Treg cells (mean ± s.d. of five experiments) in cultured cells in a. ** P < 0.01. d,e, Flow cytometry of CD4+CD25− T cells cultured with indicated reagents for 3-5 days. Numbers in the quadrants indicate CD25+Foxp3+ Treg cells of Id3+/+ (top row) or Id3−/− (bottom row) mice. Each plot is of one experiment representative of at least two. RA, retinoic acid.
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Figure 2: Id3−/− T cells fails to generate Foxp3+ Treg cells in response to TGF-βa, Quantitative analysis of Foxp3, present as mRNA expression relative to HPRT (mean ± s.d. of duplicate wells) in naïve CD4+CD25− T cells overnight after TCR stimulation with or without TGF-β. Data shown is of one experiment representative of at least five. b, Flow cytometry of naïve CD4+CD25− T cells cultured with TCR (with or without TGF-β) for three days. Numbers in the quadrants indicate Foxp3+ Treg cells. Each plot is of one experiment representative of five. c, Percent Foxp3+ Treg cells (mean ± s.d. of five experiments) in cultured cells in a. ** P < 0.01. d,e, Flow cytometry of CD4+CD25− T cells cultured with indicated reagents for 3-5 days. Numbers in the quadrants indicate CD25+Foxp3+ Treg cells of Id3+/+ (top row) or Id3−/− (bottom row) mice. Each plot is of one experiment representative of at least two. RA, retinoic acid.
Mentions: Next, we determined whether the Id3 deficiency affected the generation of Foxp3+ Treg cells in vitro. We cultured peripheral naïve CD4+CD25−Foxp3− T cells with CD3- and CD28-specific antibodies in the presence of TGF-β1 to induce Foxp3+ Treg cells 9. TGF-β1 induced profoundly lower levels of Foxp3 mRNA and protein in naïve Id3−/− compared to WT CD4+CD25− T cells (Fig. 2a-c) over a range of TGF-β1 concentrations, although the defect was partially reversed with high doses of TGF-β1 (Supplementary Fig. 3). Addition of exogenous IL-2 did not correct the defect, eliminating the lack of IL-2 as the cause (Fig. 2d). Inclusion of retinoic acid (RA) in the cultures only slightly increased the percentage of Id3−/− Foxp3+ T cells, whereas RA exhibited the reported synergistic effect on TGF-β1-dependent Foxp3+ Treg differentiation in WT CD4+ T cells29 (Fig. 2e). Analysis of proximal Smad activation downstream of TGF-β1 signaling revealed no significant difference in TGF-β receptor-activated Smad2 and 3 phosphorylation (represented by P-Smad 2) between Id3−/− and WT CD4+ T cells following TGF-β1 treatment (Supplementary Fig. 4a). Smad7, an inhibitory Smad that antagonizes Smad2- and 3-mediated TGF-β signaling30, was lower in Id3−/−compared to WT CD4+ T cells following TGF-β1 treatment (Supplementary Fig. 4b), ruling-out Smad7 as a factor in causing reduced Foxp3-induction in Id3−/− cells. Id3 is therefore a critical component of TGF-β1 mediated Foxp3+ Treg generation, but it likely functions downstream of P-Smad2- and P-Smad3-mediated signal pathways.

Bottom Line: We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells).Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter.Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

Show MeSH
Related in: MedlinePlus