T reg cell–depleted GF mice developed severe inflammatory disease. SPF and GF Foxp3^DTR mice were treated with either PBS or DT and analyzed at day 8 after the start of the treatment. (A) The numbers of cells in the spleen and LNs in the indicated mice (n = 7–16 per group). (B and C) Flow cytometric analyses of the cellular composition of LNs in the indicated mice (n = 7–16 per group). (B) Percentage of cells expressing the indicated surface markers. (C) Mean fluorescence intensity (MFI) of the indicated co-stimulatory molecules on CD11c⁺ MHC class II^high DCs. (D) Splenocytes isolated from the indicated mice were stimulated in vitro with anti-CD3 and anti-CD28, and production of the indicated cytokine was measured by flow cytometry. Frequencies of the cytokine-producing cells among CD4⁺ or CD8⁺ T cells were determined (n = 3–9 per group). (E) The serum cytokine levels (n = 5–8 per group) were measured by Luminex multiplex bead cytokine assay. (A–E) Error bars represent SD.