Induction of cytotoxic T cell function requires sequential action of three different lymphokines

Activation of resting Ly-2' precursor T cells to cytotoxic T lymphocytes (CTL)' requires stimulation by antigen or mitogen in combination with soluble mediators (1 -3).One of these antigen-nonspecific mediators is interleukin 2 (IL 2).IL 2 is released by activated Ly-l+ helper T cells and was distinguished to provide the proliferative signal (4, 5).It was only recently discovered that IL 2 is not the only helper factor required for the cytotoxic response (4-12).In this report, we provide evidence that three different, previously described activities (1 2) are sequentially required in distinct phases of the cytotoxic response.Absorption of Con A-induced spleen cell supernatants (Con A-Lk) on IL 2dependent T cell line cells removes the IL 2 activity, but a factor remains that is required through the first 48 hr of culture and is referred to as T cell cytotoxicity-inducing factor 1 (TCF1) in this report.Another factor is only required in the last 48 hr of culture and is called TCF2.Its activity can be destroyed in Con A-Lk by dialysis at pH 2, a treatment that leaves IL 2 titers unchanged (10-12).It is different from TCFl because it is also present in phorbol myristic acetate-activated EL-4 thymoma cell supernatants that lack TCF1.IL 2 is the third factor and is required at least in the early phase. MATERIALS AND METHODSMice.C3HflIF and DBA/2J were purchased from Bomholtgard, Ry, Denmark and were used at 6 to 10 wk of age.Culture medium.The culture medium used was RPMll640 (GIBCO, Grand Island, NY; powdered media) with Lglutamine (2 x 1 0-3 M) and supplemented anol(4 x M) and gentamicin (50 pg/ml).