Figure 6: 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^+/+Foxp3^Yfp-Cre/+or Nr4a2^fl/flFoxp3^Yfp-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: Nr4a2^fl/flFoxp3^Yfp-Cre cells; crosses: Nr4a2^+/+Foxp3^Yfp-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: Nr4a2^fl/flFoxp3^Yfp-Cre cells; crosses: Nr4a2^+/+Foxp3^Yfp-Cre cells. (e) Rag2^– mice were co-transferred with 3×10⁵ CD4⁺CD25⁺YFP⁺Ly5.2⁺Tregs from Nr4a2^fl/flFoxp3^Yfp-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).