Treatment with LEF-M during maturation of immature DCs leads to a differentially affected phenotype. Monocytes were cultured for 5 days with granulocyte–macrophage colony-stimulating factor (GM-CSF; 50 ng/ml) plus IL-4 (10 ng/ml). (a) On day 5 these immature dendritic cells (DCs; 5 × 105/ml) were activated with lipopolysaccharide (LPS; 100 ng/ml) in the absence or presence of 150 μmol/l of the active metabolite of leflunomide (LEF-M) for 48 hours. Surface marker expression was determined by fluorescence-activated cell sorting analysis. Open profiles with dotted line represent the staining pattern with an isotype control, open profiles with fine line indicate the staining pattern of DC exposed to LPS with the indicated monoclonal antibodies, and solid grey profiles show staining of DCs matured in the presence of LEF-M. The results shown are representative of five independent experiments. (b,c) Effect of LEF-M on maturation-associated clustering of DCs; immature DCs were stimulated with LPS in the (panel b) absence or (panel c) presence of 150 μmol/l LEF-M. After 8 hours of cultivation, cells were analyzed by inspecting photomicrographs obtained by light microscopy. Similar results were obtained in four additional experiments. MHC, major histocompatibility complex.