Secondary expansion of polyclonally activated T_reg cells after transfer into the allogeneic host sustains a beneficial T_reg cell/T_conv cell ratio. (A) CFSE dilution among CD4⁺ CD90.1⁺ Foxp3⁺ and Foxp3⁻ donor T cells isolated from spleen (Spl) or mesenteric LNs (mLN) 3 days after transplantation into lethally irradiated BALB/c hosts. The figures represent the percentages of cells per quadrant. (B) Frequencies of Foxp3⁺ cells among CD4⁺ CD90.1⁺ donor T cells 3 and 6 days after transplantation. The bars indicate means ± SD of 3 mice per group. pLN indicates peripheral LNs. The data are representative for 5 experiments with similar results. (C) Direct comparison of suppressive activity of T_reg cells from PBS- or CD28-SA–treated donors in vivo. Either 2.5 (n = 11) or 1.25 × 10⁵ CD4⁺ CD25⁺ T_reg cells (n = 11) from PBS- or CD28-SA–treated mice were transplanted into lethally irradiated BALB/c recipients together with 2.5 × 10⁵ CD4⁺ CD25⁻ cells from mice treated with PBS and 10⁷ C57BL/6 TCD BM cells. Control mice received only TCD BM cells (n = 10) or TCD BM cells together with CD4⁺ CD25⁻ cells (PBS; n = 10). Data from 2 experiments were pooled. P values refer to the comparison of recipients of T_reg cells from either PBS- or CD28-SA–treated donors.