T cells take up DBP from the environment by macropinocytosis. (A) Representative flow cytometry histograms showing DBP-AF488 fluorescence of naïve T cells and T cells activated for 2 and 4 days and subsequently incubated with 120 nM DBP-AF488 for 12 h at 37 °C. (B) Representative confocal microscopy images illustrating the uptake of DBP-AF488 in activated T cells. DBP-AF488 are shown in green; cell membrane (TCR) in red; nucleus in blue (DAPI stain). (C) Relative megalin, cubilin, GAPDH and RPLP0 mRNA expression in T cells activated from 0-8 days normalized to the respective mRNA expression in naïve T cells. (D-F) Flow cytometry histograms illustrating DBP-AF488 fluorescence of activated T cells incubated with 120 nM DBP-AF488 in the absence or presence of (D) RAP (1 M), (E) unlabelled DBP (12,000 nM), or (F) EIPA (50 M). (G) TCR internalization in activated T cells re-stimulated with 30 nM PDBu for 60 min in the absence or presence of EIPA (50 M). TCR surface expression of non-stimulated cells was set to 100%. (H-I) CD38 expression (H) and IFN-γ production (I) of T cells activated for 3 days with the indicated concentrations of DBP in X-VIVO 15 and X-VIVO 15 supplemented with either 25(OH)D3 (100 nM) or 1,25(OH)2D3 (10 nM). (C, G-I) Data are shown as mean ± SEM from 3 independent experiments.