Reduced stability of Foxp3 in <i>TRAF6</i>-deficient Treg cells

<p>(A) Foxp3 expression in freshly isolated CD4⁺ T cells from the spleen, mesenteric lymph node (LN) and thymus of the indicated mice. Foxp3 levels were measured using YFP-fluorescence. Data are representative of five independent experiments with similar results. The numbers of YFP⁺ Tregs in each organ are shown in right graphs (n=5). (B) Expression of KLRG1 was measured in CD4⁺Foxp3(YFP)⁺ gated cells. (C) <i>In </i><i>vitro</i> suppression assay. FACS purified 5×10⁴ CD4⁺CD25^-CD62L⁺CD44^- naïve T cells from WT mice were stimulated for 72 hours with 10⁵ T-cell depleted spleen cells and anti-CD3 (2C11) (1µg/ml), in the absence or presence of CD3⁺CD4⁺Foxp3(YFP) + Treg cells from WT (diamond) or cKO (square) mice as indicated concentrations. Cells were pulsed with 0.5 µCi of ³H-thymidine for the final 16 hours before being harvested. Error bars indicate SD; n=3. (D) <i>In </i><i>vivo</i> suppression assay. 3 x 10⁵ naïve T cells from the LN of CD45.1⁺ WT mice together with or without 1.5 x 10⁵ Tregs (CD4⁺YFP ⁺ RFP⁺) from the LN of CD45.2 Foxp3 ^YFP-CreROSA26^RFP TRAF6^+/+ (WT-Tregs) or CD45.2 Foxp3 ^YFP-CreROSA26^RFP TRAF6^fl/fl (cKO-Tregs) were transferred into Rag2^-/- mice. Body weight changes were measured daily. Data are representative from four independent experiments. *<i>p</i><0.05 **<i>p</i><0.01; Representative histology of the colon is shown in right panels. Magnification x200 (E) Flow cytometric analysis of Foxp3-YFP and CD45.1 expression on CD3⁺CD4⁺ T cells from the LN in Rag2^-/- mice. Data are representative from four independent experiments. Quantification of the data of the fraction of YFP ⁺ CD45.1^- cells (remaining Foxp3 ⁺ Tregs) is shown in the right panel. **<i>p</i>< 0.01.