Sequential PBMC stimulation by superantigen-LPS enhances TNF production. (A) Three-dimensional dose-response bar chart showing mean TNF-α by SEB-LPS sequentially stimulated PBMCs from 3 different subjects. Means were derived from triplicate observations at each toxin concentration. Data were representative for those obtained for TSST-1 and SMEZ-1. (B) LPS-induced TNF-α release by PBMCs: effect of duration of SEB pre-exposure. Enhanced TNF-α production required a minimum period of 3 hours of pre-exposure to SEB. (C) LPS-induced TNF-α release by PBMCs following 3 hours of pre-exposure to TSST-1: effect of duration of LPS exposure. Enhanced TNF-α production was maximal 4 hours after LPS stimulation. (D) Reversal in order of exposure (LPS-SEB instead of SEB-LPS) did not produce enhanced observed levels of TNF-α when compared with expected levels. (E-F) HKSA and LTA were unable to prime PBMCs for enhancement of LPS-induced TNF-α production. (G) Enhanced TNF-α production was also seen when TSST-1–primed PBMCs were exposed to lipid A as a secondary stimulant or (H) LPS from Pseudomonas aeruginosa (PSA LPS; threshold concentration one log higher than for E coli LPS or lipid A). All results are mean and SD of triplicate stimulations. Results are representative of 5 separate experiments and were repeated with both TSST-1 and SEB.