Activation of T and B Lymphocytes in Vitro

Abstract

Abstract The biologic and metabolic conditions required for optimal production of a nonantigen-specific biologically active factor termed allogeneic effect factor (AEF) have been explored in greater depth. AEF is produced in mixed lymphocyte cultures (MLC) of in vivo alloantigen-activated T cells (ATC) and the respective target cells. In these studies, we describe conditions for production of AEF in serum-free media supplemented with appropriate concentrations of 2-mercaptoethanol, Ficoll, or gelatin. Analysis of the cellular components required for AEF production revealed that AEF is not produced by macrophages nor are macrophages required for optimal production of AEF in vitro. The production of AEF requires the participation of T cells of the Ly-1+, Ly-2, 3- phenotype in the effector cell population and target cells of appropriate specificity differences determined by H-2 haplotype; Mls locus differences are neither essential nor effective in inducing AEF production in this secondary MLC system. Investigation of the possible contribution of target cell products to the active components of AEF involved the use of glutaraldehyde-fixed normal spleen cells of lipopolysaccharide-induced blasts as stimulators in the MLC. Although it was found that such fixed cells could serve as effective stimulators of AEF production, the finding that 7 to 10% of radiolabeled surface membrane proteins were shed from target cells despite glutaraldehyde fixation precluded a definitive conclusion on the role of target cell products in AEF activity. Finally, metabolic inhibitors were used to explore the metabolic processes required of the participating cells; such studies demonstrated that neither proliferation nor DNA synthesis is required for generation of active AEF, whereas protein synthesis, glycolysis, and electron transfer are required.