Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34 cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34 granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, which could potentially have been induced by IL-12 from CD34 cells. TNF-alpha protein release was significantly increased in CD34 cells incubated with IL-12. No detectable levels of IFN-gamma were noted. Anti-TNF-alpha, but not anti-IFN-gamma, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-alpha, but not IFN-gamma, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-alpha.