Smad3 binding to the foxp3 enhancer is dispensable for the development of regulatory T cells with the exception of the gut

Abstract

Regulatory T cells (T reg cells) are essential for the prevention of autoimmunity throughout life. T reg cell development occurs intrathymically but a subset of T reg cells can also differentiate from naive T cells in the periphery. In vitro, Smad signaling facilitates conversion of naive T cells into T reg cells but results in unstable Foxp3 expression. The TGF-β-Smad response element in the foxp3 locus is located in the CNS1 region in close proximity to binding sites for transcription factors implicated in TCR and retinoic acid signaling. From in vitro experiments it was previously postulated that foxp3 transcription represents a hierarchical process of transcription factor binding in which Smad3 would play a central role in transcription initiation. However, in vitro conditions generate T reg cells that differ from T reg cells encountered in vivo. To address the relevance of Smad3 binding to the CNS1 enhancer in vivo, we generated mice that exclusively lack the Smad binding site (foxp3(CNS1mut)). We show that binding of Smad3 to the foxp3 enhancer is dispensable for T reg cell development in newborn and adult mice with the exception of the gut.

Figure 1.

Deletion of Smad-binding site in foxp3 enhancer/CNS1. (A) Schematic depiction of upstream region of wild-type foxp3 locus and the gene targeting vector. The CNS1 and the position of the Smad-binding site in the wild type or deleted binding site in the foxp3^CNS1mut allele are indicated. (B) Alignment of the wild type and the foxp3^CNS1mut allele at the position of the Smad-binding site, showing the deleted sequence. (C) ChIP analysis of Smad3 binding to the foxp3 CNS1 region. foxp3^WT (sample 1) or foxp3^CNS1mut (sample 2) CD4⁺CD25⁻ splenocytes were not stimulated (0 h) or stimulated for 1 h with TGF-β, anti-CD3, and anti-CD28. Immunoprecipitated DNA was analyzed by PCR. Data are representative of two independent experiments.

Figure 2.

TGF-β–dependent in vitro conversion of naive T cells into T reg cells. (A) Frequencies of Foxp3-expressing cells among CD4⁺ T cells in 2.5-d cultures containing the indicated concentrations of TGF-β have been measured. (B) Conversion rate of foxp3^CNS1mut T cells normalized on conversion rate of foxp3^WT T cells at the indicated TGF-β concentrations. Data are representative of two (0.05 and 2 ng/ml TGF-β) or three (0, 0.1, 0.5, and 1 ng/ml TGF-β) independent experiments. Error bars indicate means ± SEM. ***, P < 0.001; **, P < 0.01.

Figure 3.

T reg cell development in days 4 and 10 neonates. T reg cell frequencies among CD4⁺ cells have been measured in thymi, spleens, and intestines of day 4 (A) and day 10 (B) foxp3^CNS1mut and foxp3^WT mice. Data on day 4 T reg cell frequencies are representative of 6 foxp3^CNS1mut and 10 foxp3^WT mice and two independent experiments. Data on day 10 T reg cell frequencies in thymus and spleen are representative of 10 foxp3^CNS1mut and 13 foxp3^WT males and four independent experiments. Data for day 10 T reg cell frequencies in intestine are representative of six foxp3^CNS1mut and eight foxp3^WT males and two independent experiments. Error bars indicate means ± SEM.

Figure 4.

Analysis of T reg cell frequencies, proliferation rate, and proportion of Helios-expressing T reg cells in adult males. 4-wk-old (A), 8-wk-old (B), and 6-mo-old (C) males have been injected with 1 mg EdU i.p. 16 h before analysis. (A and B) Thymi (thy), spleen (spl), axial/inguinal LN, MLN, PP, LPL, and IEL have been analyzed for frequency of Foxp3⁺ T reg cells among CD4⁺ T cells (top), EdU⁺ (middle), and Helios⁺ (bottom) cells among T reg cells. Data are representative of two to six males and two to three independent experiments per group. (C) Frequencies of T reg cells (left) and EdU⁺ T reg cells (right) were assessed in spleen, PP, LPL, and IEL. Data are representative of 7–10 males and five independent experiments per group. Data for foxp3^WT mice are depicted in black and data for foxp3^CNS1mut mice are depicted in gray. Error bars indicate means ± SEM. *, P < 0.03.

Figure 5.

Analysis of T cell transfer colitis. Naive T cells from foxp3^WT mice (blue) or foxp3^CNS1mut mice (red), respectively, have been transferred into Rag-deficient recipient mice. Body weight was measured (top left) and, at day 45, colitis activity was determined by mini-endoscopy and analyzed in colitis score (top right). Histological analysis of colons at day 45 after transfer showed similar signs of inflammation like in DSS-induced colitis. Data are representative of eight mice and two independent experiments per group. Error bars indicate means ± SEM.