Flow cytometric quantitation of calcium-dependent and -independent mitogen-stimulation of T cell functions in whole blood: inhibition by immunosuppressive drugs in vitro

We have optimized assays to measure mitogen-stimulated rat lymphocyte activ ation in whole blood and have used these assays to quantitate the potencies of immunosuppressive drugs with different mechanisms of action. To define the optimal conditions for measuring T cell functions in whole blood, the e ffects of different concentrations of mitogens that activate T cells throug h calcium-dependent and -independent pathways were measured over time. Prol iferation was measured by tritium-labeled thymidine ([H-3]-TdR) incorporati on and by flow cytometric analysis of proliferating cell nuclear antigen (P CNA)/DNA content. Furthermore, we detected the increases in percent express ion of cell-surface activation antigens (CD25, CD134, CD71, CD11a and CD54) . Concanavalin A (Con A) stimulated maximum lymphocyte proliferation and ex pression of T cell surface activations by 72-96 h, which was 48 h later tha n stimulation by phorbol 12-myristate 13-acetate (PMA) plus anti-CD28 monoc lonal antibody (mAb) or PMA plus ionomycin (IONO). Addition of sirolimus, t acrolimus, cyclosporine or the active metabolite of leflunomide, A77 1726, to mitogen-stimulated whole blood produced drug concentration-dependent inh ibitions of lymphocyte proliferation and expression of cell surface activat ion antigen expression. From these data, we determined drug potencies (inhi bitory concentration of 50%, IC50) and drug concentrations causing maximum inhibition of T cell functions (I-max). We developed simple and reproducibl e assays to measure different lymphocyte functions in whole blood cultures. These assays were used to investigate the mechanisms of different immunosu ppressive drugs. These methods can be exploited to measure T cell functions in blood collected from subjects treated with immunosuppressants in vivo. (C) 2001 Elsevier Science B.V. All rights reserved.