Figure 7. ICOS^hiCCR6⁺RORγt⁺ Treg cells can regulate Th17 cell-mediated CNS autoimmunity.

CD4⁺YFP^hi Th17 cells isolated from MOG/CFA immunized IL-17F^CrexRosa26^YFP fate mapping mice were adoptively transferred into RAG1^−/− mice. CD4⁺YFP⁺ICOS^hiCCR6⁺ cells (Tr17) or CD4⁺YFP⁺ICOS^loCCR6⁻ cells (rTreg) isolated from MOG/CFA immunized Foxp3^YFP-Cre mice were co-transferred with Th17 cells in some recipient mice. All the recipient mice were immunized with MOG/IFA one day after adoptive transfer. Pertussis toxin was injected on the day of immunization and two days later. (A) EAE score was monitored daily. Th17 alone (n=10), Th17 + Tr17 (n=9) and Th17 + rTreg (n=10). (B) The numbers of total cells and CD4⁺ T cells in CNS were analyzed. (C) Representative dot plot of YFP expression in gated CD4⁺CD90⁺ T cells in CNS is shown. (D) Cytokine production in CNS CD4⁺YFP^hi T cells was analyzed after PMA/Ionomycin stimulation. (E) YFP expression in splenic CD4⁺ T cells is shown. (F) YFP^int Treg cell to YFP^hi Th17 fate-mapped cell ratio in spleen was analyzed. (G) Absolute numbers of CD4⁺YFP^hi Th17 fate-mapped cells (Th17fm), CD4⁺YFP^int Treg cells (Treg) and MOG-specific CD4⁺IFN-γ⁺ Th1 cells in spleen are shown. (H) MOG-specific cytokine production by splenic CD4⁺ T cells was analyzed after MOG peptide (50 μg/ml) restimulation. (I–J) IL-17 (I) and IFN-γ (J) production by splenocytes were analyzed by ELISA three days after restimulation with titrated doses of MOG peptide. *, p<0.05. **, p<0.005. N.D. Not detected. Data are combined results (A and F) or representatives (B–E and G–J) of two independent experiments. See also Figure S5.