iT reg cells are generated in 1B3.RAG^−/− mouse in the periphery. (a) Flow cytometric staining for Foxp3 expression in CD4⁺CD8⁻ T cells in the thymus and spleen of 5-wk-old 1B3.RAG^−/− (top) or 1B3.RAG^+/− littermates (bottom). Numbers indicate mean ± SD of Foxp3⁺ cells from four individual mice. (b) Foxp3 expression in the CD4⁺CD8⁻ T cells from thymus or pooled spleen+LN of 1B3.RAG^−/− or 1B3.RAG^+/− littermate mice aged 1–3 or 4–6 wk. Each dot represents percentage of Foxp3⁺ cells from an individual mouse and bars represent means. (c) Foxp3 staining in pooled spleen and LN cells from 3.5–4-wk-old mice thymectomized neonatally. Littermates 1B3.RAG^+/− or sham-operated 1B3.RAG^−/− mice were used as controls. A single representative mouse out of five thymectomized mice is shown. (d) Foxp3 staining in Thy1.2⁺ cells in the CD4⁺CD8⁻ subset after intrathymic injection of 10⁷ thymocytes from 1-wk-old 1B3, 1B3.RAG^−/−, WT, or POT (P0-specific TCR-Tg) mice into congenic Thy1.1 host, followed by flow cytometry on day 4. A representative plot is shown on the left. On the right are graphs showing the percentage of Foxp3⁺ cells among CD4⁺CD8⁻ cells (top) and percentage of Thy1.2⁺ cells among total thymocytes (bottom) with bars representing means. Data are representative of two independent experiments. (e) Mixed bone marrow chimeras were generated by mixing (1:1) congenically marked WT and 1B3.RAG^−/− bone marrow. 6 wk after reconstitution, spleen and LN cells were isolated and CD4⁺ cells were stained intracellularly for Foxp3 in respective compartments. Representative plot showing Foxp3 staining in CD4⁺ T cells 1B3.RAG or WT compartments (left) and graph showing pooled data (right) are shown with bar representing mean. Data are representative of two independent experiments.