Patient plasma inhibition of IFN-γ–induced pSTAT-1 production and RNA expression. Normal PBMC incubated in 10% control or patient plasma at time points specified in Figure 1 were left unstimulated or stimulated with IFN-α or IFN-γ for 15 minutes. CD14⁺ cells were assayed for intracellular pSTAT-1 by flow cytometry. (A) pSTAT-1 in CD14⁺ cells, solid gray, unstimulated; red line, IFN-γ stimulated; blue line, IFN-α stimulated. Representative example of 1 of 3 experiments performed for each patient. (B) To determine the relative inhibitory effect of plasma on IFN-γ induced pSTAT-1 production, a stimulation index for each plasma (ratio of the geometric mean channel for stimulated to unstimulated) was calculated and then graphed as a percentage of the stimulation index seen in the presence of normal plasma. Error bars represent the SEM seen for 3 separate experiments performed for each series of patient plasma. (C) PBMC obtained from healthy donors (n = 4 experiments per series of patient plasmas) were cultured in the presence of normal or patient plasma (10%) and stimulated with IFN-γ (400U/mL) for 3 hours. Target gene expression was evaluated by real time PCR and values are mean fold induction (± SD) relative to the unstimulated cells. GAPDH was used as normalization control. Fold-induction of IFN-γ–induced gene expression in presence of subject plasma was calculated as a percentage of the fold-induction expression seen for the same PBMCs in the presence of normal plasma. Plasma from patient 4 inhibited IFN-γ induced gene expression when stimulated with IFN-γ 400U/mL at all time points, and so the experiment was repeated using IFN-γ at a higher concentration of 3000U/mL.