Figure 5: Deletion of Nr4a2 attenuates iTreg induction.
From: The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells
(a) Effects of Nr4a2-knockout on CD4+T cells differentiation. iTreg induction was carried out at the two different TGF-β concentrations indicated. Naïve CD4+T cells from Nr4a2+/+Lck-Cre+ or Nr4a2fl/flLck-Cre+ mice were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or IL-17 expressions were analysed. (b) Quantification of the results in a of the iTreg condition at 0.2 and 0.5 ng ml−1 TGF-β. Top: ratio of the Foxp3+ fractions. Bottom: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. The value of Nr4a2+/+Lck-Cre+ cells at 0.2 ng ml−1 TGF-β was set to 1. Data are representative of three independent experiments (mean±s.d. in triplicate). Filled bars: Nr4a2+/+Lck-Cre+ cells; open bars: Nr4a2fl/flLck-Cre+ cells. (c–f) Effects of the ex vivo deletion of Nr4a2 on Th and iTreg differentiation. Isolated naïve CD4+ T cells from Nr4a2+/+ERT2-Cre+ (c, d) or Nr4a2fl/flERT2-Cre+ (e, f) mice were treated (d, f) or untreated (c, e) with 4-hydroxy tamoxifen for 6 h in RPMI1640 10% FBS at 37 °C. Cells were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or Il-17a expression were analysed. (g) Body weight of Nr4a2+/+Lck-Cre+ (open circles) or Nr4a2fl/flLck-Cre+ (filled squares) mice treated with 1.5% DSS. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). (h) Histological analysis of the experiments of (g) at day 11. Top: hematoxylin and eosin staining of colon sections. Scale bar, 100 μm. Bottom: histological score of colitis. Data are representative of two independent experiments, each with five mice per group (vertical bars: median of the colitis scores). (i) Analysis of colon and caecum lamina proprial CD4+ T cells from the experiments in g at day 11, examining IFN-γ and IL-17a expression (top) and Foxp3 expression (bottom). (j) Quantification of results in i. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). Filled bars: Nr4a2+/+Lck-Cre+ mice; open bars: Nr4a2fl/flLck-Cre+ mice. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. *P<0.05, **P<0.01 (two-tailed Student's t-test).
