POTENCIES OF IMMUNOSUPPRESSIVE DRUGS FOR INHIBITION OF SIGNAL 1- AND SIGNAL 2-INDUCED EXPRESSION OF LYMPHOCYTE CELL SURFACE ACTIVATION ANTIGENS USING WHOLE BLOOD ASSAYS

Abstract

202 Whole blood assays of lymphocyte function are more biologically relevant than assays using purified lymphocytes. Therefore, we used these assays to determine the potencies of inhibition of lymphocyte activation by immunosuppressive drugs after stimulation with mitogens that signal through different pathways. Methods: Six different concentrations of cyclosporine (CsA), sirolimus (RAP), mycophenolic acid (MPA) and A77 1726 (A77) were added to the whole blood from Lewis rats. Blood was diluted and stimulated either with Con A (signal 1+2) or PMA+anti-CD28 MoAb (signal 2, P+28). Expression of CD25 (IL-2R), CD71 (transferrin R), CD11a (LFA-1) and CD54 (ICAM) was measured using multicolor flow-cytometry. A rat specific pan-T cell marker (Ox52) was used to identify lymphocytes. Results: The IC50's of inhibition of antigen expression by drugs were: RAP < CsA < MPA < A77 (p<0.05). As expected from studies of inhibition of proliferation with purified cells, RAP more potently inhibited the effects of P+28 than Con A. Unlike previous studies, we found that CsA reproducibly inhibited P+28 activation at lower IC50's than for Con A. For MPA and A77 potencies were greatest for Con A stimulation (p < 0.05). Conclusion: For the first time, the effects of immunosuppressive drugs on mitogen-induced expression of cell surface antigens in whole blood have been determined. All drugs inhibited cell activation. Although other assays have shown that signaling through P+28 is resistant to CsA, our assays showed that CsA does inhibit P+28 signaling. As expected, RAP was a potent inhibitor of P+28. The relative potencies of all drugs were identical to the relative differences in their immunosuppressive doses in vivo. These findings show the advantages of investigating drug mechanisms and of identifying new mechanisms of action of these drugs in whole blood assays rather than in assays using purified cells. (Table)Table