Group B Streptococcus Induces a Robust IFN-γ Response by CD4⁺ T Cells in an In Vitro and In Vivo Model

Figure 3

Ex vivo analyses of cellular sources of IFN-γ during GBS infection. C57BL/6 mice were injected intraperitoneally with a dose of 10⁷ CFU of wild-type GBS serotype III strain COH-1 ( per group × 3 individual experimental infections). Spleens were harvested 6 h after infection and total splenocytes plated at 5 × 10⁶ cells/well. After 4 h of incubation, the bacteriostatic agent chloramphenicol (12 μg/mL) was added to the culture to prevent cell toxicity. Nonstimulated cells from mock-infected animals served as negative (−) control for basal expression. Total splenocytes were incubated for 48 h with Brefeldin A (3 μg/mL) added during the last 5 h of incubation. Cells were harvested and intracellularly stained for IFN-γ (a) or surface-stained for CD69 (b) in combination with several surface markers for multiparametric FACS analysis. Representative data from 3 different experimental infections based on CD3⁺ population or total splenic population (Void). (c) Number of TNF-α⁺ cells within the CD3⁺ population or total splenic population (All). Data are expressed as means ± SEM from 3 different experimental infections; indicates statistically significant difference compared to (−) control cells. Fifty thousand gated events were acquired per sample and data analysis was performed using FACSDiva™ software.

(a)

(b)

(c)