Figure 3

CD3⁺ T cells produce low ex vivo levels of IFN-γ and show poor activation in response to S. suis systemic infection.

Mice were infected intraperitoneally with a dose of 5 × 10⁷ CFU of S. suis wild-type strain P1/7 (n = 3 per group × 3 individual experimental infections). Spleens were harvested 6 h post-infection and total splenocytes plated at 5 × 10⁶ cells/well. After 6 h of incubation, gentamycin was added to the culture to prevent cell toxicity. Non-stimulated cells from mock-infected animals served as negative control for basal expression (C−). Total splenocytes were incubated for 14 h or 48 h with Brefeldin A (3 μg/ml) added during the last 5 h of incubation. Cells were harvested and intracellularly stained for (a,b) IFN-γ, (c) TNF-α or surface stained for (d) CD69 in combination with several surface markers for multi-parametric FACS analysis. (a,d) Representative data from 3 different experimental infections based on CD3⁺ population or total splenic population (Void). (b,c) Number of either IFN-γ⁺ or TNF-α⁺ cells within the CD3⁺ population or within total splenic population (All) at 48 h. Data are expressed as mean ± SEM from 3 different experimental infections. FACS was performed using a FACSCanto II instrument. Fifty thousand gated events were acquired per sample and data analysis was performed using FACSDiva™ software. Fluorescence Minus One (FMO) control staining was performed for proper analysis and gating of target cells. *P < 0.05, indicates statistically significant difference compared to negative control cells (C−).