There are currently extensive studies relating to cancer vaccines using tumor cells engineered to express immunogenes and cancer gene therapy using adenovirus (AdV)-mediated gene transfer. In this study, a mouse tumor cell line, VKCK, was cotransfected with genes coding for tumor necrosis factor-alpha (TNF-alpha) and costimulatory B7-1 molecule to enhance immunogenicity. The transfectant cell line VKCK-TNF-alpha/B7-1 showed reduced tumorigenicity and tumor regression. Its inoculation further induced protective immunity; both CD4+ and CD8+ T cells were involved in the induction phase, whereas only CD8+ T cells mediated the effector phase. Susceptible mice bearing VKCK tumors developed a T helper type 2-dominant response, whereas resistant mice with VKCK-TNF-alpha/B7-1 tumor regression developed a T helper type 1-dominant response to VKCK, indicating that the tumor regression was related to a shift in the cytokine profile of the host from type 2 to type 1. Vaccination of VKCK-TNF-alpha/B7-1 cells inhibited tumor formation derived from a single dose of 3 x 10(6) VKCK cells and eradicated 3-day tumors but not 10-day tumors. AdV-mediated TNF-alpha gene transfer by intratumoral injection of AdV-TNF-alpha significantly inhibited tumor growth but failed to eradicate any well-established tumors. However, combinational immunotherapy with vaccination of VKCK-TNF-alpha/B7-1 cells and AdV-mediated TNF-alpha gene transfer not only significantly inhibited tumor growth but also eradicated 10-day VKCK tumors in three of eight mice. Therefore, the present study may be useful not only in understanding the mechanisms responsible for an efficient antitumoral immunity, but also in establishing a more effective immunotherapeutic approach for cancer patients.