Figure 5.

Foxp3^lo T_regP cells are dependent on NFκB1 activation and the Foxp3 regulatory element Cns3. a), Left panels, shown are representative flow cytometry plots for CD4⁺CD8⁻CD73⁻ gated thymocytes antibodies from wild-type and Cd28^−/− mice stained with antibodies to CD25 and Foxp3 (1 experiment, n=4 wild-type and 5 Cd28^−/− mice). Right panels show cumulative data for all mice and depict the relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type and Cd28^−/− mice. b), Shown are representative flow cytometry plots for CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type and Nfkb1^−/− mice stained with antibodies to CD25 and Foxp3 (3 independent experiments, n=19 wild-type and 11 Nfkb1^−/− mice). Right panels show cumulative data for all mice and depict the relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type and Nfkb1^−/− mice c) Top panels, shown are representative flow cytometry plots for CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type, FOXP3-GFP^KIN, and Cns3^−/− mice stained with antibodies to CD25 and Foxp3 (4 independent experiments, n=8 wild-type, 14 Foxp3-GFP^KIN, and 14 Cns3^−/− mice). Right panels show quantification of cumulative data for all mice and depict relative percentage of CD25⁺ T_regP and Foxp3^lo T_regP in CD4⁺CD8⁻CD73⁻ thymocytes from wild-type (black circles), Foxp3-GFP^KIN (black squares), and Cns3^−/− (black triangles) mice. d) Shown are the percentages of CD25⁺Foxp3⁺ mature T_reg cells generated after stimulating sorted CD4⁺CD8⁻CD73⁻CD25⁺ T_regP cells from thymi of Foxp3-GFP^KIN and Cns3^−/− mice for 3 days with 0 U/ml IL2 (black circles, n=5 Foxp3-GFP^KIN and 5 Cns3^−/− mice), 0.2 U/ml IL2 (black squares, n=4 Foxp3-GFP^KIN and 5 Cns3^−/− mice), 1 U/ml IL2 (black triangles, n=5 Foxp3-GFP^KIN and 6 Cns3^−/− mice) and 100 U/ml (open circles, n= Foxp3-GFP^KIN and 5 Cns3^−/− mice) and come from 2 independent experiments. e) Quantification of cumulative data from 2 independent experiments showing the relative percentage of CD25⁺ T_regP, Foxp3^lo T_regP and mature CD25⁺Foxp3⁺ T_reg cells from CD4⁺CD8⁻CD73⁻ gated thymocytes from wild-type (black circles) or EDEL mice, which lack the CaRE4 enhancer (black squares). Data represents 2 independent experiments, n=10 wild-type and 10 EDEL mice. a,b) Data was analyzed by two-sided unpaired t test. c) CD25⁺ T_regP cells (%) and Foxp3^lo T_regP cells (%) were analyzed by one-way ANOVA with Tukey’s multiple comparison test and T_reg cells (%) was analyzed by Kruskal-Wallis test with Dunn’s multiple comparison test. d) Data was analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test. e) Data was analyzed by two-way ANOVA with Sidak multiple comparisons test. All data are displayed as mean ± standard deviation. **P<0.005, ***P<0.0001, ns- not significant.