Figure 4: Neutralization of IL-10 increases IFN-γ and TNF-α production by CD3⁺ versus CD3⁻ cells in response to B. pseudomallei.

PBMCs of healthy seropositive individuals (n = 8) were incubated with 10 μg/ml E. coli LPS or killed B. pseudomallei (30:1 ratio) versus medium alone and in the presence or absence of 3 μg/ml of anti-IL-10 mAb. IFN-γ (a), TNF-α (b) and IL-6 (c) production was measured by ELISA from collected supernatant after 48 hours in vitro. Each symbol represents data from an individual. The values from the same individual in the presence or absence of anti-IL-10 mAb condition are joined by a line. To identify the cellular source of cytokines, PBMCs were incubated with killed B. pseudomallei (30:1 ratio) for 20 hours and assayed by flow cytometry for intracellular IFN-γ and TNF-α versus cell surface expression of CD3. A profile of one representative donor, gated initially on lymphocytes (d) and monocytes (e) by FSC/SSC shows the frequency of IFN-γ and TNF-α producing cells respectively. Statistical significance was determined using paired T test; ns, non significant, *p < 0.05, **p < 0.01 and ***p < 0.001. IL-6 statistical significance was determined from 3 out of 6 responding donors.