Fig. 1.

Dose-dependent inhibition of ex vivo NK cell proliferation by long-term MPA treatment. a Evaluation of potent MPA dose. Overlay histogram plots show a dose-dependent inhibition of the proliferation of CFSE-labeled NK cells indicated by reduced CFSE fluorescence intensity. The right overlay plot gives an overview of the tested MPA concentrations. Here, 10 µM MPA was the lowest concentration that completely inhibited proliferation. Plots gated on viable CD56⁺CD3⁻ NK cells (red: IL-2-stimulated NK cells + MPA; black: IL-2-stimulated NK cells d9 (positive control); dark gray area: unstimulated NK cells d0 (negative control); light gray area: autofluorescence IL-2-stimulated NK cells). b Inhibition and reversibility of MPA. The proliferation of IL-2-stimulated NK cells was severely inhibited in the first cell divisions by 10 µM MPA. This MPA-mediated inhibition was completely reversed after the removal of MPA from the cell medium on day 3, and these NK cells also reached the 7th cell generation during 9 days of IL-2 stimulation. Plots gated on viable CD56⁺CD3⁻ NK cells. c Effect in absolute cell count. Following an initial decrease in cell count, NK cells showed a mean 2.7-fold expansion. Long-term treatment with 10 µM MPA resulted in an absolute inhibition of proliferation, which correlated with a complete decrease in viable cell count (n = 10, mean ± SD). Plots gated on viable CD56⁺CD3⁻ NK cells. d CD56 surface expression. Unstimulated PB NK cells showed a CD56^dimCD16⁺ (85.8 %) and CD56^brightCD16^dim/− (14.3 %) phenotype. Following IL-2 stimulation, CD56 surface expression became highly up-regulated [mean fluorescence intensity (MFI) 7–73]. This was completely inhibited by long-term MPA incubation (MFI 10). Plots gated on viable CD56⁺CD3⁻ NK cells. Plots show representative results of n = 10 experiments