Factors in AMLR culture supernatant which mediate the cytotoxic T-cell response to hapten-altered self: interaction with macrophages.

Supernatants from autologous mixed lymphocyte reaction (AMLR) cultures mediated the cytotoxic T-cell response in a system containing T cells and mitomycin C-treated, TNP-modified syngeneic thymocytes. No cytotoxic activity developed in the absence of the AMLR supernatants. The removal of thymic adherent cells abrogated the effect of the AMLR supernatants in the generation of cytotoxic cells. AMLR helper activity was restored following the addition of small numbers of splenic adherent cells. These results led to speculation that the AMLR supernatant interacted with accessory cells. Examination of the supernatant revealed significant levels of colony stimulating factor (CSF), and interferon-gamma (IFN-gamma). Functionally, incubation of the AMLR supernatant with P388D1, a macrophage tumor line, resulted in the production of interleukin 1 (IL-1). CSF is a major inducer of IL-1 synthesis. In addition, the AMLR supernatant induced the expression of Ia antigens on the P388D1 cells. IFN-gamma has been reported to mediate this activity. Although both lymphokines were present and appeared to have an effect on macrophages, it was unknown if both were required for cytotoxic T-cell development in this system. Samples of AMLR supernatant were incubated for 10 min at various temperatures. The ability of the AMLR supernatants to mediate cytotoxic T-cell activation was sensitive to incubation at 80 or 90 degrees C. Interestingly, at these temperatures CSF activity in the bone marrow colony-forming cell assay was enhanced. In separate experiments, AMLR culture supernatant was dialyzed against a pH 2 buffer. The induction of IA antigens by these supernatants was sensitive to dialysis at this pH. The similarity with IFN-gamma provides further evidence that this activity was mediated by IFN-gamma in the AMLR supernatants. The ability of the AMLR supernatants to mediate the cytotoxic T-cell response to altered self was also pH 2 sensitive. In contrast, IL-1 inducing capability and bone marrow colony growth, both CSF activities, were not reduced by pH 2 dialysis. Taken together, these data demonstrate a primary role for an IFN-like molecule, present in the AMLR supernatant, on cytotoxic T-cell activation. CSF involvement in this response is suggested by its IL-1 inducing activity, but cannot be definitively proven in the present study.