Soluble factors secreted by U937 cells inhibit Th1 cytokine production. (A) FACS analysis of intracellular cytokines (γ-IFN and IL-4, IL-2, and IL-10) was performed on PBMNCs that had been stimulated with PMA/ionomycin in the absence or presence of U937 cells separated by a 0.4-μm pore membrane. PBMNCs stimulated in the absence of U937 cells were almost exclusively producing γ-IFN and IL-2. Those activated in the presence of U937 cells showed a much-reduced number producing γ-IFN and IL-2. (B) The same experiment in the presence of reagents to either block potential Th1 inhibitors (neutralizing antibodies to TGF-β, IL-10, or [not shown here] indomethacin to block PGE₂) or overcome them (IL-2 and IL-12) showed all had no effect when added concurrently with U937 cells. (C) PBMNCs were incubated with IL-2 for 4 hours before the addition of U937 cells and were then analyzed for γ-IFN production by intracellular staining as described above. Preincubation with IL-2 overcame the effect of the leukemia-derived inhibitory factor on γ-IFN production.