In vitro induction of polyclonal killer T cells with 2-mercaptoethanol and the essential role of macrophages in this process

Abstract

Killer T cells against allogeneic and syngeneic tumor cells were generated in vitro by the addition of 2-mercaptoethanol (2-ME) to the murine spleen cell culture in the absence of any antigenic stimulation. The maximum activity of T cell-mediated cytotoxicity (CMC) induced with 2-ME was observed on day 4 of culture and the induction of CMC was completely inhibited by the addition of inhibitor of DNA synthesis, hydroxyurea, or cytosin arabinoside. CMC induced with 2-ME was specifically inhibited by the addition of unlabeled target cells to the 51Cr-release assay system. These results indicated that killer T cells were generated in the presence of 2-ME as a result of nonspecific polyclonal activation of precursors into cytotoxic effector cells and that they recognized target cells with antigen-specific recognition receptors. Spleen cells deprived of adherent cells showed impaired induction of CMC with 2-ME. The addition of peritoneal exudate macrophages to splenic T cells restored this response. The result indicated that macrophages were essential for the induction of CMC with 2-ME. The possibility that the function of macrophages was mediated by soluble factor(s) released from macrophages was demonstrated by the separate culture of splenic T cells and macrophages in double-chambered, Marbrook-type vessels and by the addition of supernatants from macrophage cultures to splenic T cells. 2-ME and soluble factor(s) released from macrophages seemed to be required for the activation of precursors into killer cells.