The enhancement of proinflammatory responses to LPS by superantigens in PBMCs is a reproducible and specific cellular phenomenon. (A-B) TNF-α production by PBMCs stimulated with superantigen-endotoxin in expanded donor population. Cells were pre-exposed to 1 ng/mL superantigen (A, TSST-1, n = 21; B, SEB, n = 15) for 3 hours, then coincubated with LPS (1 ng/mL) for an additional 4 hours. Median and 10th, 25th, 75th, and 90th centiles are shown. ^*Represents significant supra-additive superantigen-endotoxin interaction; P < .001 for both superantigens. (C) IL-1β and (D) IL-6 cytokine induction by SEB-LPS compared with that expected from individual toxins was also supra-additive (P = .008). Results are shown for each individual donor (D1-D8). (E) IFN-γ production in response to SEB-LPS was not supra-additive from that predicted by levels induced by individual toxins. (F) In comparison with control PBMCs (incubated either alone or sequentially with SEB and LPS), SEB-exposed donor PBMCs retained hyperresponsiveness to LPS even when supernatants were removed and replaced with fresh medium (bars marked “D”). Supernatants from PBMCs pre-exposed to SEB did not directly transfer priming (ie, immediate enhanced LPS responsiveness) to naive recipient PBMCs (bars marked “R”). (G) Cycloheximide (cyclo) inhibited enhanced production of TNF-α by superantigen-endotoxin. Error bars reflect standard deviation of triplicate wells.