Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows

significant potential as a vaccine adjuvant and anti-cancer therapeutic.Different cell types and soluble factors are involved in the induction of IFN-γ. Thus, in addition to IFN-γ-producing cells (T cells, NK cells, and macrophages) and accessory cells (macrophages and dendritic cells) a number of cytokines are known to participate directly or indirectly in the induction of IFN-γ. Among the soluble factors, IL-12 is of special interest, being capable of directly inducing IFN-γ in T and NK cells and promoting T cell differentiation in the IFN-γ-producing Th1 subset. Splenocyte suspensions is prepared from spleens of 6- to 8-wk-old mice by pressing spleens through a wire grid. Pooled cells from three or four animals are  suspended in serum-free DMEM, adjust to a concentration of 107/ml, and place (2 × 106/well) in 96-well plates (Nunc, Roskilde, Denmark). They are then cultured in the presence or the absence of stimulating agents (10 μl/well) at 37°C in a humidified atmosphere containing 8% CO2 for 24 h. Culture supernatants for determination of IFN-γ are stored in aliquots at −80°C until use.IFN-γ in supernatants of splenocyte cultures and in murine plasma is estimated by  ELISA. The limit of IFN-γ detection is 60 pg/ml. IL-12 (p70) in supernatants of macrophage cultures is estimated by ELISA using anti-IL-12 mAb (C17.8) and biotin-labeled anti-IL-12 mAb (C15.6; PharMingen, Hamburg, Germany) as described previously (45). The limit of detection is15 pg/ml.