Vitamin D and its metabolites inhibit activation of both human and murine CD4⁺ and CD8⁺ T cells. (A, C and E) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. (B, D and F) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. (A–F) MACS purified murine or human T cells were CFSE labelled and incubated with increasing concentrations of (A and B) 25-(OH)-vitamin D, (C and D) 1,25-(OH)₂-vitamin D or (E and F) cholecalciferol at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). (A–F) 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 are shown as mean ± SEM; representative plots of two independent experiments; n = 3.