Short-term activation of allogeneic T cells with a CD28-SA in vitro protects from aGVHD while maintaining GVT activity. (A) Increased frequencies and (B) activated phenotype of Foxp3⁺ T_reg cells after CD28-SA stimulation of whole LN cells in vitro. Numbers represent the percentages of Foxp3⁺ cells as indicated by the marker. (C) Lethally irradiated BALB/c mice were reconstituted with 10⁷ C57BL/6 TCD BM cells either alone or together with 5 × 10⁵ CD4⁺ or 4 × 10⁵ T_reg cell–depleted CD4⁺ cells (n = 10) characterized in panel A. P values compare (C) survival curves or (D) clinical scores from animals after transfer of cultured CD4⁺ cells versus T_reg cell–depleted CD4⁺ cells. Data from 2 individual experiments were pooled. BALB/c recipient mice received BCL₁ lymphoma cells followed by TCD BM cells only or TCD BM cells together with either (E) 5 × 10⁵ or (F) 5 × 10⁴ fresh T cells or the same numbers of T cells isolated from whole LN cell cultures stimulated in vitro with a CD28-SA for 4 days. Data from 2 experiments were pooled (n = 12; 5 × 10⁴ T cells [fresh], n = 11), and P values were obtained comparing T-cell recipients with the TCD BM only group. Numbers in parentheses indicate animals with end-stage lymphoma/animals analyzed).