Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay.

Malononitrilamides (MNAs) are a new class of immunomodulatory drug highly effective in in vivo models of allo- and xenotransplantation. Knowledge of their effects on immune cells, however, is limited and has been derived solely from investigations using isolated mononuclear cells. This use of purified cells to investigate drug activity is not ideal, so we have combined the analytical power of flow cytometry with our mitogen-driven, whole blood lymphocyte activation and proliferation assays to investigate the in vitro mechanism of action of MNAs. We first show that MNAs (A77 1726, HMR1279, and HMR1715), as well as brequinar (BQR) and cyclosporine (CsA), effectively inhibit cell activation antigen expression and lymphocyte proliferation. We next show that the inhibitory effects of MNAs and BQR, but not CsA, are reversed by the addition of uridine to the culture. These results suggest that inhibition of pyrimidine biosynthesis may be a mechanism by which MNAs suppress both lymphocyte activation and proliferation since these effects were reversed when uridine nucleotide pools were replenished. This novel finding of suppression of activation antigen expression by MNAs in whole blood expands our understanding of the effects of this new class of drug.