CD4⁺CD25⁺ regulatory T cells protect mice from developing autoimmunity. Lethally irradiated (900 cGy) Balb/c mice received transplants of TCD B6 BM (10 × 10⁶) plus 3-4 × 10⁵ B6 spleen cells. At 20 days after BMT, mice were killed, and spleen cells (adjusted to yield 0.5 × 10⁶ T cells) were transferred either alone (n = 7) or together with an equivalent number of CD4⁺ CD25⁺ T cells (n = 7) into nonirradiated B6 Rag animals. Mice were killed 42 to 87 days after BMT. (A) Overall pathology score of mice that received transplants of GVHD spleen cells alone (■) or together with Tregs (□). (B) Absolute number of CD3⁺, CD4⁺, and CD8⁺ T cells in the spleens of mice that received transplants of GVHD spleen cells alone (■) or with CD4⁺CD25⁺ Tregs (□). (C,D) Overall cellularity and absolute number of Gr-1⁺ Mac-1⁺ cells in the spleen and bone marrow of mice that received transplants of GVHD spleen cells alone (■) or together with Tregs (□). (E) Absolute number of Thy 1.2⁺ (■) or Thy1.1⁺ (□) T cells in the spleens of mice that received transplants of GVHD spleen cells alone or together with CD4⁺CD25⁺ T cells. Data in Figure 3A-E are presented as the mean plus or minus SEM and are the cumulative results of 2 experiments. (F) Spleen cells, hepatic lymphocytes, and IELs from the colon were isolated from mice with autoimmunity and similarly from animals that had received transplants that were coadministered Tregs and had no evidence of pathological damage. The percentage of foxp3-expressing cells in the gated CD4⁺ T-cell population from each tissue site is depicted by the numbers in the quadrants. Statistics: *P < .05; **P < .01.