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

Figure 4

Ex vivo analyses of CD4⁺ T cell contribution to cytokine production. 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. Cells stimulated with Concanavalin A (0.1 μg/mL) were used as positive (+) control. Total splenocytes were incubated for 48 h. Brefeldin A (3 μg/mL) was added during the last 5 h of incubation and CD4⁺ T cells were MACS-isolated from the culture, stained intracellularly for different cytokines, and analyzed by FACS. Data are expressed as means ± SEM (in % of positive cells) from 3 individual experimental infections. indicates statistically significant difference compared to (−) control cells. Twenty thousand gated events were acquired per sample and data analysis was performed using CellQuest software. Histograms were drawn based on PE-control stain and were plotted on logarithmic scales.