Reduced accumulation and differentiation of pathogenic CD4⁺ cells in the presence of CD28-SA–activated T_reg cells. CFSE dye dilution among CD4⁺ CD90.1⁺ Foxp3⁻ donor T cells recovered from (A) spleen (Spl) or (B) mesenteric LNs (mLN) 3 and 6 days after transplantation into lethally irradiated BALB/c mice. Numbers indicate the frequencies of CFSE^low cells. Absolute numbers of donor CD4⁺ Foxp3⁻ T cells in Spl, mLN, peripheral LNs (pLN), and liver 3 (C) and 6 (D) days after transplantation. Data from 3 independent experiments were pooled, and bars represent means ± SD of 3 to 9 mice per group. (E) IFNγ expression by CD4⁺ CD90.1⁺ Foxp3⁻ cells isolated from mLN of BALB/c mice 6 days after transplantation. As a control cells from PBS-treated donors were stained for IFNγ expression without restimulation in vitro. (F) The percentages of annexin V⁺ cells among CD4⁺ CD90.1⁺ Foxp3⁻ donor T cells were determined on day 6 after transplantation (PBS, n = 3; mLN, n = 2). (G) Polyclonally activated T_reg cells suppress CD25 expression by Foxp3⁻ donor CD4⁺ cells. Cells were isolated from spleens 4 days after transplantation. Numbers indicate mean fluorescence intensities.