Natural CD25⁺CD4⁺ regulatory T cells prevented chronic GVHD. (A) DBA/2 spleen cells were stained with anti-TCRαβ, CD4, and CD25 and then CD4⁺TCRαβ⁺ cells were sorted into CD25⁺ and CD25^-CD4⁺ T cells. (B) CD25^-CD4⁺T cells (0.1 × 10⁶) were stimulated with anti-CD3 mAb and cocultured with titrated numbers of CD25⁺CD4⁺ T cells for 72 hours; the proliferation of CD25^-CD4⁺ T cells was measured by ³H-TdR incorporation. Mean ± SE of triplicate culture, and one representative of 3 replicate experiments are shown. (C) Sublethally irradiated BALB/c mice were injected with CD4⁺ T-cell-depleted DBA/2 spleen cells (CD4-dep alone; 100 × 10⁶), CD4-dep plus CD25^-CD4⁺ T cells (CD25^-; 5 × 10⁶), and CD4-dep plus CD25^- and CD25⁺CD4⁺ T cells (CD25⁺; 2.5 × 10⁶). The development of proteinuria and hair loss in the 3 groups were compared. There were 12 mice in each group. (D) HE staining of skin tissue sections of the recipients given CD4-dep cells, CD4-dep plus CD25^-CD4⁺ T cells, or CD4-dep plus CD25^- and CD25⁺ CD4⁺ T cells 30 days after cell injection. One representative is shown of 4 examined recipients in each group. (E) Seven days after cell injection, yield of donor CD4⁺ T cells from the spleens of recipients given donor CD25^-CD4⁺ T cells and recipients given both CD25^- and CD25⁺ CD4⁺ T cells was measured. Mean ± SE of 4 recipients in each group is shown. (F) Sorted donor CD4⁺ T cells (0.3 × 10⁶/well) from recipient in panel E were cultured in vitro for 5 days without additional stimulation. T-cell proliferation was measured with ³H-TdR incorporation. Mean ± SE of 4 examined recipients in each group is shown. (G) Intracellular staining of IFN-γ in CD4⁺ T cells from the spleens of recipients injected with CD25^-CD4⁺ T cells (bold solid line) or from the recipients injected with both CD25^- and CD25⁺ CD4⁺ T cells (thin solid line) 7 days after cell injection. The isotype control staining was shown as thin dashed line. One representative is shown of 4 examined recipients in each group.