- We have analyzed and compared in detail the malignant phenotypes of, the immune mechanisms induced by, and the immunotherapeutic potentials of B16-F10.9 melanoma cells manipulated by gene transfer to express syngeneic H-2K b molecules or to secrete the cytokines interleukin 2 (IL-2) or IL-6. Local tumor growth in the footpad of transduced cells is mainly retarded by expression of H-2K b and IL-2 genes and less by expression of IL-6. Mice given injections intrafootpad of tumorigenic doses of transduced clones manifested significantly reduced postsurgical spontaneous metastasis. After i.v. inoculation, mice given injections of F10.9-K b expressors did not develop experimental lung metastases; mice given injections of F10.9-IL-6 secretors developed reduced metastatic loads; whereas mice given injections of F10.9-IL-2 secretors developed high loads of lung metastases. On the basis of injections into nude mice, in vivo depletions of CD4 + , CD8 + , and NK1.1 + cells, and in vitro CTL and natural killer (NK) assays, we show that all F10.9-modified cells induce CD8 + tumor-specific CTL activity and that F10.9-IL-2 secretors also induce nonspecific NK/lymphokine-activated killer cell activity. Vaccinations with F10.9-modified cells were capable of significantly reducing metastatic spread from small established F10.9 footpad tumors. However, in mice carrying pre-established lung metastases, a highly therapeutic effect was achieved only when H-2K b expressors and IL-2 secretors were combined in vaccination, whereas individual vaccines or other combinations had marginal effects. This higher efficiency of the combined vaccine is due to the combined effect of efficient CTL induction and NK/lymphokine-activated killer cell activity as concluded from depletion of CD8 + and NK1.1 cells during immunotherapy. Thus, the cure of established metastasis can be achieved by the synergistic effects of vaccination with class I and IL-2-transduced tumor cells.