GT inhibits functional CD4⁺ and CD8⁺ T-cell responses. PBMCs from 5 healthy donors were stimulated with SEB, CMV lysates (to stimulate CD4⁺ T cells), or a CMV pp65 peptide mixture (to stimulate CD8⁺ T cells). Escalating doses of GT were added ranging from 50 ng/mL to 500 ng/mL. Functional T-cell responses following SEB and CMV stimulation were assessed by cytokine flow cytometry by gating on CD4⁺ or CD8⁺ T cells co-expressing CD69 and intracellular IFNγ. Panel A shows a typical example of CFC FACS plots after stimulation with SEB and addition of GT. Panel B displays the effects of GT on the activation of CD4⁺ cells. P values (Mann-Whitney) are shown versus the no-GT sample. Panel C displays the effects on activation of CD8⁺ T cells. A decrease in the frequency of cytokine-producing SEB-activated T cells was found when GT was added to the culture in increasing concentrations. P values (Mann-Whitney) are shown versus the no-GT sample. GT-induced suppression was most pronounced at concentrations equal to 300 ng/mL for CD4⁺ and equal to 400 ng/mL for CD8⁺ T cells; at these concentrations functional activation was minimal. Similar results (data not shown) were seen following CMV antigen stimulation and when TNFα production was used as an analysis end point. Panel D illustrates a representative experiment illustrating gliotoxin-induced suppression of cytokine production (IFNγ, left panels; and TNFα, right panels) in both CD4⁺ and CD8⁺ T cells following SEB stimulation. Error bars indicate SEM; ^*P < .05.