Effects of IL-21 on FoxP3⁺ T-cell generation. Lethally irradiated B10.BR recipients were injected with 10⁷ T cell–depleted bone marrow and (10⁶ CD4⁺ (▴, ▵) or CD4⁺25⁻ (▾, ▿) T cells from wt (▴,▾) or IL-21^−/− (▵, ▿) mice and monitored for survival (A; P = .001 for wt vs IL-21^−/− groups). Lethally irradiated recipients were injected with 2 × 10⁶ wt of IL-21^−/− CD25⁻ T cells and lamina propria (LP) cells were isolated from the colon on day 14, stained with anti-CD4 and anti-FoxP3, and analyzed by FACS. The frequency of FoxP3-expressing CD4⁺ cells was determined (B; P = .002). Subgroups of mice described in panel A were killed on day 30 and sections from frozen tissue blocks were analyzed for expression of CD4 (green) and FoxP3 (red) by confocal microscopy (C). Lethally irradiated recipients were injected with 2 × 10⁶ FoxP3⁻ T cells from FoxP3-GFP knock-in mice. Subgroups were treated with irrelevant IgG or anti–IL-21 Ab. The frequency of FoxP3-expressing CD4⁺ spleen cells (D; P = .06) or CD4⁺LP T cells (E; P = .004) was determined. Data were obtained from 1 experiment each with 8 (A), 6 (B), 4 (C), and 6 (D-E) mice per group.