Figure 6. ICOS^hiCCR6⁺RORγt⁺ Treg cells are Ag-specific regulatory T cells with potent suppressive activity in vitro.

(A) ICOS^hiCCR6⁺GFP⁻CD4⁺ Th17 cells were isolated seven days after MOG/CFA s.c. immunization of CD45.2⁺Foxp3^GFP reporter mice. In parallel, ICOS^hiCCR6⁺GFP⁺CD4⁺ Treg cells, ICOS^loCCR6⁻GFP⁺CD4⁺ Treg cells, and ICOS^hiCCR6⁻GFP⁺CD4⁺ Treg cells were isolated from MOG/CFA immunized CD45.1⁺CD45.2⁺ Foxp3^GFP reporter mice. Th17 cells were stimulated with MOG peptide (50 μg/ml) in the presence of irradiated T cell-depleted splenocytes (TdS, CD45.1⁺). Three fold serially diluted number of each Treg cells were added to the culture to test the suppressive activity. Three days later, IL-17A, IL-17F or IL-22 production in the culture supernatant was analyzed by ELISA. (B) Foxp3^GFP reporter mice were s.c. immunized with MOG/CFA. Seven days later, MOG-specific cells in indicated populations of dLNs were identified by tetramer staining. (C) ICOS^hiGFP⁻CD4⁺CD45.1⁺ T effector cells (Teff) were labeled with CFSE (2.0 μM) and then were stimulated with splenic CD11c⁺ DCs in the presence or absence of ICOS^hiCCR6⁺GFP⁺CD4⁺CD45.2⁺ Tr17 cells in the indicated Tr17 to Teff ratio. Three days later, FSC value, CFSE dilution, CD25 expression and cytokine production of CD45.1⁺ Teff cells were analyzed by flow cytometry after PMA/Ionomycin restimulation. Data are representatives of at least two independent experiments. *, p<0.05. **, p<0.005. See also Figure S4.