ALDH⁺ DCs isolated from MLNs and CLNs trigger the differentiation of iTregs. (A) Light-density cells isolated from spleen and from MLNs and CLNs were incubated with ALDEFLUOR and stained for CD11c and MHCII. mDCs from MLNs and CLNs and cDCs from spleen were then sorted into ALDH⁻ and ALDH⁺ fractions. (B) To verify the quality of the sort and assess for the stability of ALDH activity, sorted cells kept in culture for 18 hours were incubated with ALDEFLUOR, stained for CD11c and MHCII, and analyzed by flow cytometry. Dead cells were gated out before analysis using TO-PRO-3 staining. Positioning of the ALDH⁺ gate is based on incubation in the presence of DEAB (not shown). (C-D) Freshly sorted ALDH⁻ and ALDH⁺ mDCs from MLNs and CLNs and cDCs from spleen were incubated with CFSE-labeled OT-II Rag-2^−/− T cells in the presence of the OVA_257-264 peptide (0.06 μg/mL). After 5 days of culture, T cells were analyzed for the expression of Foxp3 and of CCR9. Numbers in outlined areas indicate percentage of cells. Data are representative of at least 3 separate experiments. (D) Comparison of the percentage and the number of converted Foxp3⁺ Tregs induced by ALDH⁻ and ALDH⁺ mDCs from MLNs, CLNs, and cDCs from spleen. Also shown is the effect resulting from the addition of the LE540 (1μM) inhibitor and of exogenous TGF-β (1 ng/mL). Data are representative of 3 independent experiments. Bars represent mean ± SD of triplicate wells within 1 of the 3 independent experiments. *P < .05