Fig. 4.

Transfection of CD4⁺CD25⁺Foxp3⁺CD127⁻ regulatory T cells with Foxp3 siRNA is associated with recovery of splenocytes proliferative capacity in septic mice. Murine splenocytes were harvested 24 h after the induction of a polymicrobial septic challenge (cecal ligation and puncture model: CLP, n = 17) or from sham/control animals (Sh, n = 17). Immediately after harvesting, 2 × 10⁶ cells were transfected with 2 µM Foxp3 siRNA or control siRNA or left untouched. a Cells were stained using 3-color flow cytometry staining (CD4-PECy7–CD25-PE–Foxp3-APC) 16 h after transfection or not with siRNA. Results are presented as mean ± SEM [black bar: septic mice, light gray bar: septic mice after transfection with Foxp3 siRNA, dark gray bar: septic mice after transfection with control (CTRL) siRNA]. b After overnight incubation, cells were stained with PKH26 and cultured in 96-well plates in RPMI with or without concanavalin A (0.1 mg/ml, 1 × 10⁶ cells/ml) for 5–6 days. Proliferation was assessed by flow cytometric measurement of the decrease in PKH26 (yellow) fluorescence on viable cells selected based on SSC/FCS characteristics. Results are presented as mean ± SEM of proliferation ratio [i.e. (percentage of cells with decreased fluorescence/percentage of highly fluorescent cells) × 100] measured on splenocytes from sham animals (open bar) or from mice after CLP transfected or not (black bar) with Foxp3 (light-gray bar) or control (CTRL) siRNA (medium-gray bar). The Student t test was used for comparison between groups (*P < 0.05) and the Wilcoxon Signed Ranked test was used for comparison between paired values (^#P < 0.05) 682