Exosomes (EXOs) derived from tumor cells have been used to stimulate antitumor immune responses. It has been demonstrated that EXO released by tumor cells engineered to express cytokines are of enhanced stimulatory effect on CD8(+) cytotoxic T-lymphocyte (CTL) responses and antitumor immunity. J558 is a mouse myeloma cell line expressing tumor antigen P1A. In this study, we purified EXO(TNF-a), EXO(IL-2), and EXO(IFN-gamma) released by three cytokine-gene (TNF-alpha, IL-2 and IFN-gamma)-engineered J558 (J558(TNF-a), J558(IL-2) and J558(IFN-gamma)) tumor cell lines from their culture supernatants, respectively, by differential ultracentrifugation. These EXOs showed a "saucer" or round shape with a diameter between 50 and 90 nm by electron microscopy and contained EXO-associated proteins, such as LAMP-1 and AIP1, but not lysate-associated protein galectin, by Western blot analysis. EXO displayed expression of molecules (H-2K(d), CD54, and P1A) similarly to, but to a lesser extent to, J558 tumor cells. We then compared the stimulatory effect of these EXOs on P1A-specific CD8(+) CTL responses and antitumor immunity 6 days subsequent to intravenous (i.v.) EXO immunization (30 microg/each BALB/c mouse). We demonstrated that EXO(TNF-alpha) immunization was able to induce more efficient P1A-specific CD8(+) T-cell response accounting for 0.62% of the total CD8(+) T-cell population, using PE-H-2K(d)/P1A peptide and FITC-CD8 staining by flow cytometric analysis then EXO(IL-2) (0.31%) and EXO(IFN-gamma) (0.22%) immunization (P < 0.05), respectively, at day 6 after immunization. EXO(IL-2) and EXO(IFN-gamma) vaccine (i.v. 30 microg/each mouse) only protected 3 of 8 (38%) and 2 of 8 (25%) mice from tumor growth after subcutaneous (s.c.) challenging of immunized mice with J558 tumor cells (0.5 x 10(6) cells/each mouse), whereas EXO(TNF-alpha) immunization protected all 8 of 8 (100%) mice from tumor growth (P < 0.05). Taken together, we demonstrate that EXO(TNF-a) released by engineered J558(TNF-a) tumor cells more efficiently stimulate tumor antigen P1A-specific CD8(+) CTL responses and antitumor immunity than EXO(IL-2) and EXO(IFN-gamma) released by engineered J558(IL-2) and J558(IFN-gamma) tumor cells. Therefore, TNF-alpha-expressing tumor cell-released EXO may represent a more effective EXO-based vaccine in the induction of antitumor immunity.