DCs differentiated in the presence of LEF-M exhibit reduced T-cell stimulatory capacity. (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 presence or absence of the indicated concentrations of LEF-M. Dendritic cells (DCs) differentiated in the presence of the active metabolite of leflunomide (LEF-M) are labelled 'LEF-M DCs' in the figure. The cells were extensively washed, irradiated (3000 rad) and subsequently co-cultured with 1 × 105 purified allogeneic T cells at the indicated ratios. (b) To determine maturation sensitivity, DCs differentiated in the presence or absence of LEF-M were exposed to 100 ng/ml lipopolysaccharide for an additional 48 hours. Then, the cells were employed as allogeneic stimulators, as described above. DNA synthesis was assessed at day 5. The standard deviation of the counts/min (cpm) for the respective triplicates was generally below 20%. Shown are the means of at least eight independent experiments.