Suppressive properties of Tregs in post septic mice. (A) Representative CD4⁺CD25^high cells used in suppression assays. Before coculture, Tregs were analyzed by FACS for intracellular Foxp3 expression (black line); isotype control (gray line). (B) CD4⁺CD25^high T cells were isolated by FACS from the spleens of naive, sham, and CLP mice, and their suppressive capacities were tested in vitro. CD4⁺CD25⁻ T cells from all groups that underwent FACS were cocultured with naive T cells as a control for the assay. CFDA-SE–labeled cells were examined by flow cytometry, and the magnitude of suppression of CFDA-SE–labeled naive T cells stimulated with anti-CD3/CD28 was calculated with the use of FlowJo software. The graphs represent mean ± SEM of the combined results from 2 to 3 experiments (for sham and CLP at 1, 3, and 15 days after surgery) and 2 independent experiments (for naive mice). #P ≤ .05; ###P ≤ .001 when the suppression activity of post septic Tregs was compared with naive Tregs. *P ≤ .05 and **P ≤ .01 when the suppression activity of post septic Tregs was compared with sham Tregs. (C-E) IFN-γ levels were analyzed in supernatants from cocultures. Data are mean ± SEM of results from 3 independent experiments at each time point. *P ≤ .05 compared with naive T-cell proliferation. (F) Cells from spleens of mice were analyzed for Foxp3 expression. Histogram plots were gated on CD4⁺CD25^high cells, and the mean of fluorescence intensity (MFI) of Foxp3 was analyzed. Control isotype (dashed line), sham (gray area), and post septic (black line). Representative histograms are shown for 3 independent experiments with 3 to 4 mice per group.