Antigen-specific de novo generation of CD4⁺Foxp3⁺ iT_regs from CD4⁺Foxp3^– T cells in Sp and MLNs during the induction of oral tolerance. (A-D) Sp KJ1-26⁺Foxp3^EGFP− T cells were transferred into WT mice (5 per group) that had been treated with CpG ODN (B-D), and the animals were subsequently fed PBS (none) or OVA protein the day after the adoptive transfer. (A) Immunofluorescent microscopic analysis of horizontal sections, stained as indicated, from Sp or MLNs on day 11 after the adoptive transfer. Image acquisition information: BIOREVO BZ-9000 fluorescence microscope (KEYENCE); immunofluorescence, 10×, 10×/0.25 objective lenses; room temperature; no imaging medium; Alexa Fluor 488, R-phycoerythrin, Alexa Fluor 647, DAPI fluorochromes; BZ-II Analyzer acquisition software (KEYENCE); JPEG, Preview 3.0.9 (Apple Inc). (B-D) Expression of Foxp3^EGFP among gated KJ1-26⁺ T cells in Sp (B-C) and MLNs (B,D) on day 11 after the adoptive transfer was analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the proportion of Foxp3^EGFP+ cells among gated KJ1-26⁺ T cells in each quadrant (B) and are the percentage of positive cells (C-D). *P < .01 compared with untreated mice. Data are the mean ± SD, and the results are representative of 4 independent experiments.