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

Abstract

We have optimized assays to measure mitogen-stimulated rat lymphocyte activation 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 effects of different concentrations of mitogens that activate T cells through calcium-dependent and -independent pathways were measured over time. Proliferation was measured by tritium-labeled thymidine ([³H]-TdR) incorporation and by flow cytometric analysis of proliferating cell nuclear antigen (PCNA)/DNA content. Furthermore, we detected the increases in percent expression of cell-surface activation antigens (CD25, CD134, CD71, CD11a and CD54). Concanavalin A (Con A) stimulated maximum lymphocyte proliferation and expression of T cell surface activations by 72–96 h, which was 48 h later than stimulation by phorbol 12-myristate 13-acetate (PMA) plus anti-CD28 monoclonal antibody (mAb) or PMA plus ionomycin (IONO). Addition of sirolimus, tacrolimus, cyclosporine or the active metabolite of leflunomide, A77 1726, to mitogen-stimulated whole blood produced drug concentration-dependent inhibitions of lymphocyte proliferation and expression of cell surface activation antigen expression. From these data, we determined drug potencies (inhibitory concentration of 50%, IC₅₀) and drug concentrations causing maximum inhibition of T cell functions (I_max). We developed simple and reproducible assays to measure different lymphocyte functions in whole blood cultures. These assays were used to investigate the mechanisms of different immunosuppressive drugs. These methods can be exploited to measure T cell functions in blood collected from subjects treated with immunosuppressants in vivo.

Introduction

Investigations of in vitro mechanisms of action of immunosuppressants on lymphocytes have most commonly used transformed cell lines or mononuclear cells purified from peripheral blood and cultured with heterologus serum. These test systems are not ideal, since transformed cell lines differ from normal immune cells, and since purification methods cause selective loss of different cell populations and disturb normal cell–cell interactions (De Groote et al., 1993). Assays of lymphocyte function are preferred, since selective cell loss does not occur and interactions among immune cells and their products are all retained (Chen et al., 1999). Since drugs partition between plasma and formed elements in blood, testing the effects of drugs in a whole blood matrix more faithfully represents their actions in the circulation. Plasma–drug binding can differ among species, so it is important to avoid heterologus sera (Metcalfe et al., 1992). Finally, whole blood assays are more rapid and require small sample volumes compared to methods that rely on lymphocytes purified from peripheral blood Bloemena et al., 1989, Bocchieri et al., 1995.

Whole blood cultures have been used to measure immune cell function Bloemena et al., 1989, Doldi et al., 1985, Gregory et al., 1987a, Gregory et al., 1987b, Leroux et al., 1985, Pauly et al., 1973, Shifrine et al., 1978, Wilson et al., 1991 and recent papers describe the use of tritium-labeled thymidine ([³H]-TdR) incorporation to measure lymphocyte proliferation Fasanmade and Jusko, 1995, Silva and Morris, 1998, Silva et al., 1996, Silva et al., 1997, Silva et al., 1998. Methods have also been developed to measure cytokine production in whole blood (De Groote et al., 1992). Since lymphocyte cell surface proteins that are receptors for cytokines and other growth factors and adhesion molecules are crucial for lymphocyte activation (Gaines et al., 1996), it is important to understand how immunosuppressive drugs affect the expression of these molecules. Therefore, we have begun to develop assays that measure not only lymphocyte proliferation and intracellular cytokine synthesis in mitogen stimulated whole blood, but we have also devised methods to quantitate the expression of cell surface activation antigens Gummert et al., 1999c, Klupp et al., 2000, Slauson et al., 1999. The methods presented in these papers were limited by the use of one mitogen, single color flow cytometric analysis or were preliminary reports of methods using multicolor flow cytometry.

The present report extends our development of methods to measure T cell function in whole blood by optimizing techniques for both calcium-dependent (concanavalin A (Con A) and phorbol 12-myristate 13-acetate (PMA)+ionomycin (IONO)) and calcium-independent (anti-CD28 mAb (CD28)+PMA) mitogens and by assessing the expression of the following T cell surface antigens using three-color flow cytometry: CD11a, CD25, CD54, CD71 and CD134. Additionally, we assessed lymphocyte proliferation by measuring [³H]-TdR incorporation. We also used flow cytometric analysis to measure lymphocyte proliferation by detecting cells positive in the S/G₂M-phase of the cell cycle by measuring the expression of proliferating cell nuclear antigen (PCNA), an auxiliary cyclin protein necessary for DNA polymerase and maximally expressed in mid S-phase (Celis et al., 1984).

After we had optimized these methods for quantitating mitogen-stimulated T cell functions in whole blood, we exploited these assays to compare the suppression of these T cell functions by different immunosuppressants. The effects of one Gummert et al., 1999c, Silva et al., 1996, Silva et al., 1998, Slauson et al., 1999, Wiegers et al., 1993 or more Ferron and Jusko, 1998, Piekoszewski et al., 1994, Silva et al., 1997 immunosuppressants on the suppression of immune functions in whole blood have been described. However, these studies measured only incorporation of [³H]-TdR, used only single color flow cytometric or investigated inhibition of one mitogenic signalling pathway by immunosuppressants.

By using different mitogens and measuring a variety of immune cell functions, we were able to determine the potencies (inhibitory concentration of 50%, IC₅₀) and efficacies (maximal inhibition, I_max) of immunosuppressants for suppression of immune functions that had not been measured or compared for these drugs. To evaluate the ability of our assays to measure immune suppression, we chose to investigate the effects of the following immunosupressants with distinct mechanisms of immunosuppressive actions (Gummert et al., 1999b): cyclosporin A (CsA) and tacrolimus (TRL) (inhibitors of calcineurin), sirolimus (SRL, rapamycin; inhibitor of mammalian target of rapamycin), and A77 1726, the active metabolite of leflunomide (inhibitor of dihydroorotate dehydrogenase, DHO-DH). We found these drugs shared some but differed in other in vitro effects on mitogen-stimulated T cell functions measured by our optimized whole blood assays.