Fig. 3

Mice with T_reg-specific ectopic expression of Id2 displayed enhanced conversion to ex-Foxp3 T_H17 cells from T_reg cells after induction of EAE. a Schematic representation the mouse model employed. Converging arrows in the lower panel indicate genotyping primers. b Genotyping PCR to detect the presence of Id2-EmGFP transgene in Id2^EmGFPFoxp3^YFP−Cre mice. c Flow cytometry analysis of YFP and EmGFP expression in CD4⁺CD25⁺ T cells from spleen (SP) and peripheral lymph nodes (pLN) of Foxp3^YFP−Cre and Id2^EmGFPFoxp3^YFP−Cre mice (left panel). Id2 expression was assessed between non-T_reg and T_reg cells by intracellular staining (right panel). d Schematic of experimental EAE model. e Representative flow cytometry analysis of Id2 expression between tdTomato⁺YFP⁺ (T_reg) and tdTomato⁺YFP⁻(ex-T_reg) cells in CD4⁺ T cells from 6 to 8 week-old R26TFoxp3^YFP−Cre mice on day 24 after induction of EAE. f Mean clinical scores in mice after induction of EAE (R26TFoxp3^YFP−Cre; n = 12, Id2^EmGFPR26TFoxp3^YFP−Cre; n = 9). g Representative Spinal Cord (SC) sections and hematoxylin and eosin (H&E) staining from EAE induced mice at day 24 after immunization (scale bar, 100 μm). h Flow cytometry analysis and percentages of Foxp3 sufficient (YFP⁺) and deficient (YFP⁻) populations among CD4⁺tdTomato⁺ in SP and SC from R26TFoxp3^YFP−Cre and Id2^EmGFPR26TFoxp3^YFP−Cre mice at day 22–24 after induction of EAE. i Representative flow cytometry analysis and percentages of IFN-γ⁺, IL-17A⁺ and IFN-γ⁺IL-17A⁺ in CD4⁺tdTomato⁺ YFP⁻ or CD4⁺tdTomato⁺ YFP⁺ T cells in total SP and SC cells stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h. NS,  not significant, *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t-test). All data are representative of two independent experiments (error bars, s.d.)