Stimulant-free preculture in heterologous serum-supplemented medium induces unresponsiveness of T cells to subsequent mitogenic stimulation

Quite often freshly isolated lymphocytes are kept in culture before experim entation for 1 or more days without any stimulus. Most of the rime, culture is supplemented with fetal bovine serum (FBS) which is heterologous to all species except bovine. In the present study, we found that freshly isolate d murine T cells show a good proliferative response to concanavalin A (Con A) and phorbol ester (PMA)/ionomycin in FBS medium, without any detectable background proliferation. However, the cells kept in the same culture witho ut any stimulus for prolonged period of time (referred to as preculture in this report) showed reduced response to Con A and PMA/ionomycin in a time-d ependent manner. Almost a complete loss of response to Con A was observed w ithin 1 day of preculture. However, loss of response to PMA/ionomycin was o bserved only after 2 days of preculture. Interestingly, similar preculture in autologous mouse serum-supplemented media did not cause any loss of the response to these mitogens. The loss of responsiveness of T cells during pr eculture in heterologous serum was irreversible. The heterologous serum-ind uced unresponsiveness of T cells to these mitogens was also prevented by ad ding Calphostin C, a specific protein kinase C (PKC) inhibitor, during prec ulture in heterologous serum. These results showed that prolonged stimulant -free preculture in heterologous serum induces irreversible unresponsivenes s of T cells to mitogens through the down regulation of T cell receptor sig naling pathway, which can be prevented by autologous serum or a PKC inhibit or, J. Cell. Biochem. 77:44-49, 2000. (C) 2000 Wiley-Liss, Inc.