Normal peripheral blood mononuclear cells (PBMC) were co-cultured with a human lung cancer cell line (LC89) transduced with the interleukin-2 (IL-2), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) genes to evaluate the capacity of the engineered cells to: allow survival of CD3+ and CD56+ cells, generate cytotoxic effectors with HLA class I restricted and unrestricted antitumor activity, and interfere in the molecular organization of the CD3/T-cell receptor associated signal transduction machinery. When PBMC were cultured up to 3 weeks with IL-2 releasing LC89 cells (LC89/IL-2), the number of viable CD3+ and CD56+ lymphocytes was much greater than in cultures with parental cells or with LC89 cells transduced with the other cytokine genes. After 1 week of coculture, a variable degree of restricted and unrestricted killing directed against different targets was observed. When the cultures were prolonged up to 3 weeks, LC89/IL-2 cells induced a marked increase in specific cytotoxic activity, which was coupled to a further enhancement of unrestricted lytic function. In the presence of LC89/IL-7 cells the degree of specific lysis remained unchanged, whereas unrestricted effectors were markedly decreased. No cytotoxic activity could be induced by LC89/GM-CSF and LC89/TNF-alpha cells in the few lymphocytes surviving after 3 weeks of culture. Coculture of parental LC89 cells with PBMC was consistently associated with a downmodulation in the expression of the CD3 zeta chain, as well as of the tyrosine kinases p56ick and ZAP-70. On the contrary, LC89/IL-2 cells, and not LC89 cells transduced with the IL-7, GM-CSF, or TNF-alpha gene, were capable of reverting the immunosuppressive effect exerted by the tumor cells. This protective effect could be maintained in cultures prolonged up to 4 weeks. When the same cultures were set up in Transwell, ie, with a membrane separation between cancer cells and PBMC, the expression of the CD3 zeta chain and of the p56ick and ZAP-70 tyrosine kinases remained unchanged under all culture conditions, indicating that the downmodulation of T-cell signal transduction molecules requires a direct cell to cell contact. These results show that transfer of the IL-2 gene into the DNA of human cancer cells promotes both restricted and unrestricted antitumor activity, and is capable of restoring and maintaining the expression of molecules involved in the process of T-cell mediated tumor cell recognition, thus underlining the potential role of the IL-2 gene in the design of vaccination protocols with cytokine gene transduced cancer cells.