Mouse Melanoma Antigen Recognized by Lyt-2~ and L3T4" Cytotoxic T-Lymphocytes1

A mouse melanoma (B16) antigen was investigated at a cellular level by three blocking experiments using monoclonal antimelanoma antibod ies, soluble melanoma antigen, and enzyme-treated B16 melanoma cells as inhibitors. The activity of antimelanoma cytotoxic I -lymphocytes (CTL) was specifically reduced by addition of the mixture of two mono clonal antimelanoma antibodies, one (M2590) recognizing the crossspecies melanoma epitope on Givu(NeuAc) and the other (MS62) reactive with the mouse melanoma-specific epitope on protein molecules. The CTL activity was also blocked by GM« liposome as well as by the soluble antigen. However, 3,000 times more GM3than the soluble melanoma antigen is required to obtain a similar inhibitory effect. When pronasetreated B16 melanoma cells, which have had protein molecules removed but GMJleft intact on the surface, were used as an inhibitor, their blocking activity was greatly reduced but was still partly observed at a high inhibitor/target ratio. These results indicate that the melanoma antigen is not (¡M. itself but is composed of the Givu-protein complex. This finding was also supported by using an interleukin 2-dependent CIL clone whose activity was blocked by both M562 and M2590. Antimelanoma CTL were found to belong to a double-negative I -cell population with Thy-1*, Lyt-2", L3T4" phenotypes. L3T4* T-cells were also demonstrated to be necessary for induction of double negative antimelanoma CTL.