Vitamin D high excess supplementation enhances activation of peripheral T cells and promotes their encephalitogenic differentiation. (A and B) Splenocytes were isolated and T-cell activation and differentiation was analysed by FACS 9 weeks after vitamin D diet onset and 15 days post immunization (representative plots of two independent experiments; data given as median; n = 5–7). (C–E) Splenic T cells were MACS purified from wild-type mice 50 days post-immunization and 17 weeks after vitamin D diet onset. (C) T cells were CFSE labelled and incubated in anti-CD3/anti-CD28 pre-coated wells for 72 h. T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions in the three groups are shown according to this setup as mean ± SEM; n = 3–5. (D and E) Differentiation of T cells into Th1- and Th17-secreting T cells was analysed by quantification of IFN-γ and IL-17 in culture supernatants by ELISA (data given as mean ± SEM; n = 3–5).