Splenic DCs (CD11c⁺) from post septic mice potently induce Treg conversion. (A) Sham and post septic mice were administrated 0.8 mg/mL BrdU in the drinking water beginning 20 hours before the surgery, and at 48 hours the mice were injected intraperitoneally with 1 mg of BrdU per mouse. At day 3 after surgery, spleen cells were stained for CD4 and Foxp3, and the absolute numbers of CD4⁺Foxp3⁺BrdU⁺ cells were compared between sham and post septic groups from data obtained by flow cytometry. Data represent the mean ± SEM of 3 mice per group. *P ≤ .05, when sham mice are compared with post septic mice. (B-E) Naive CD4⁺Foxp3⁻ T cells isolated from Foxp3^eGFP mice underwent FACS and were cultured in Treg-polarizing conditions or not (ie, no transforming growth factor-β [TGF-β] served as negative control) with post septic or sham splenic DCs (CD11c⁺MHCII⁺ or CD8α⁺) or macrophages (CD11b⁺CD11c⁻). The percentage of converted CD4⁺Foxp3⁺ T cells is shown (B). (C) Dot plots gated on viable CD4⁺GFP⁺ T cells show Foxp3 expression (FJK-16s) after culture in the presence of splenic DCs CD11c⁺ from sham or post septic mice; numbers in the upper quadrants indicate percentages of CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ T-cell populations. (D) Percentage of conversion was analyzed on viable CD4⁺ T cells, and this panel shows the Foxp3 expression in the presence of post septic or sham splenic DCs incubated with blocking anti–IL-10 or IgG control (both 100 μg/mL). All converted Tregs expressed CD25. Data shown are representative of 2 independent experiments (n = 3-5 mice per group) with similar results. Data using DCs (CD11c⁺) are representative of 3 independent experiments with similar results. (E) The measurement of IFN-γ, KC, and IL-17 by Bioplex on the supernatants derived from the cell-culture conditions are shown as described in panel B.