Hypercalcaemia increases proliferation and encephalitogenic differentiation 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, F, G and H) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. (A and B) MACS purified murine or human T cells were CFSE labelled and incubated with increasing calcium concentrations 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). 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. MACS purified (C) murine or (D) human T cells were incubated with concentrations of ionized (free) calcium equivalent to concentrations measured in serum or culture medium. After 1 h, T cells were stained with Fluo-3 AM and Fura Red AM and calcium flux was evaluated by FACS. Representative calcium flux is shown left and area under the curve is depicted on the right (data given as mean ± SEM; (C) pooled plots from two independent experiments; n = 5; (D) pooled plots from three independent experiments; n = 8). MACS purified (E) murine or (F) human T cells were incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 1–3 h (murine T cells) or 3–20 h (human T cells). Total RNA was isolated, transcribed into cDNA and analysed by qPCR (data given as mean ± SEM; (E) pooled plots from two independent experiments; n = 4, (F) pooled plots from two independent experiments; n = 6–7). Number of CD4⁺ (G) and CD8⁺ (H) T cells in bottom chamber after 16 h migration over inflamed human BBB-ECs (modified Boyden chamber assay; left: total number of cells; right: number of cytokine-producing cells), following activation of T cells in the presence of various calcium concentrations. One million activated human T lymphocytes were seeded (data given as mean ± SEM; n = 6 different T-cell donors, two different BBB-EC preparations).