(A) The frequency of FACS-sorted IFN-γ⁺ and IL-17⁺ Tregs in healthy control individuals (left) and untreated individuals with RRMS (middle, n = 17) gated on Foxp3⁺ Tregs. Results are shown at right from a purity analysis of the sorted IFN-γ⁺Foxp3⁺ and IFN-γ^–Foxp3⁺ populations from subjects with RRMS used for the methylation analysis described in C. (B) Percentage of IFN-γ⁺Foxp3⁺ and IL-17⁺Foxp3⁺ Tregs (n = 17) as a proportion of total Foxp3⁺ Tregs. (C) Representative example of methylation analysis of the Treg cell-specific demethylated region of the FOXP3 locus in sorted IFN-γ⁺Foxp3⁺ and IFN-γ^–Foxp3⁺ Tregs from subjects with RRMS. An analysis of IFN-γ⁺Foxp3^– memory T cells from subjects with RRMS is shown as a control. (D) Proliferation of responder T cells (Tresp) cultured with ex vivo FACS-sorted Tregs from healthy control subjects and untreated subjects with MS (Treg/Tresp ratio of 1:2) in the presence or absence of an IFN-γ–specific antibody (n = 4). (E) The frequency of IFN-γ⁺ and IL-17⁺ Tregs in healthy control subjects (left) or IFN-β–treated patients with RRMS (right) as assessed by intracellular cytokine staining and FACS analysis. The bar diagram (right) shows the percentage of IFN-γ⁺Foxp3⁺ and IL-17⁺Foxp3⁺ cells as a proportion of total Foxp3⁺ Tregs in healthy controls or IFN-β–treated patients with RRMS (n = 12). Reproduced with permission from Nature Medicine (91).