CD4⁺CD25⁺ regulatory T cells are present in Tgf-β1^-/- mice but are reduced in number in Tgf-β1^-/-/CD28^-/- mice. (A) Percentages of CD4⁺CD25⁺ T cells in spleens are shown. Each histogram is representative of independent results from 8 mice with either the Tgf-β1^+/+/CD28^+/+ or the Tgf-β1^-/-/CD28^+/+ genotype, 6 Tgf-β1^-/-/CD28^-/- mice, and 4 Tgf-β1^+/+/CD28^-/- mice. (B) Percentages of CD4⁺CD25⁺ T cells in total spleen cells are shown (numbers of mice per genotype same as those described for panel A). P < .005 between Tgf-β1^-/-/CD28^+/+ and Tgf-β1^-/-/CD28^-/- genotypes using Student t test. (C) Expression of intracellular CTLA-4, GITR, α_E integrin in CD4⁺CD25^- T cells and CD4⁺CD25⁺ T cells of spleen from Tgf-β1^+/+ mice and Tgf-β1^-/- mice (1 representative plot shown; 3 mice evaluated for each genotype). (D) Percentages of CD69⁺ cells in the CD4⁺CD25⁺ T-cell subset of splenocytes from Tgf-β1^+/+ mice and Tgf-β1^-/- mice. (E) Foxp3 mRNA expression in CD4⁺CD25^- T cells and CD4⁺CD25⁺ T cells of Tgf-β1^+/+ mice and Tgf-β1^-/- mice obtained by real-time PCR. Error bars (B,D) indicate SD.