LEF-M interferes with DC differentiation. (a) Monocytes were cultured for 5 days with granulocyte–macrophage colony-stimulating factor (GM-CSF; 50 ng/ml) plus IL-4 (10 ng/ml) in the absence or presence of 150 μmol/l of the active metabolite of leflunomide (LEF-M). Subsequently, surface marker expression was determined using fluorescence-activated cell sorting (FACS) analysis. Open profiles with dotted line represent staining pattern with an isotype control antibody, open profiles with fine line indicate the staining pattern of differentiated control dendritic cells (DCs) stained with the indicated mAbs, whereas solid grey profiles show staining of DCs differentiated in the presence of LEF-M. (b) Myeloid precursor cells differentiated in the presence of LEF-M are resistant to maturation. Cells were treated as described above and then stimulated with lipopolysaccharide (LPS; 100 ng/ml) for 48 hours. Open profiles with dotted line represent staining pattern with an isotype control antibody, open profiles with fine line indicate staining of activated control DCs, and solid grey profiles show staining of DCs differentiated in the presence of LEF-M and subsequently exposed to LPS. Data are representative of at least four independent experiments. (c,d) The effects of LEF-M on DC differentiation are independent of pyrimidine depletion. The respective change in mean flourescence intensity (MFI) are shown (c) after the differentiation phase for CD40 and CD1a and (d) after subsequent maturation with 100 ng/ml LPS for CD40 and CD83 with and without 50 μmol/l uridine. White bars represent control DCs, and black bars indicate LEF-M-treated cells. Shown are mean percentage control responses ± standard error of the mean, calculated from five to eight independent experiments. Student's t-tests were calculated for control versus LEF-M-treated DCs and for LEF-M-treated DCs versus without uridine addition, as indicated. *P < 0.05, **P < 0.01.