Polyclonally activated T_reg cells protect from aGVHD. Lethally irradiated BALB/c mice were reconstituted with 10⁷ C57BL/6 TCD BM cells either alone (n = 4) or together with 10⁶ CD4⁺ cells from PBS-treated mice (n = 5) or from CD28-SA–treated donors (250 μg/mouse; n = 6) or 8 × 10⁵ CD4⁺ CD25⁻ cells from CD28-SA–treated mice (n = 9). (A) The percentages of animals surviving over time and (B) the mean clinical scores of recipient animals are depicted. P values refer to the comparison of recipients of CD4⁺ versus CD4⁺ CD25⁻ cells from CD28-SA–treated donors. The experiment has been repeated twice with similar result. (C) Increased frequencies of Foxp3⁺ cells among donor CD4⁺ cells 3 days after CD28-SA treatment compared with PBS-treated mice. The numbers indicate mean percentages ± SD of 5 independent analyses. (D) Representative hematoxylin and eosin stainings of small bowel (sb) and large bowel (lb) sections obtained from mice 6 days after transplantation show reduced aGVHD-associated pathology in recipients of CD4⁺ T cells from CD28-SA–treated donors. Original magnification ×200 with LEICA DMIRE2 microscope, N Plan L 20×/0.40 objective lens, LEICA DFC300 FX camera, and LEICA IM50 Image Manager as the acquisition software. Adobe Photoshop CS3 was used to adjust for brightness and contrast. (E) Histopathologic changes were scored on small and large bowel sections as detailed in “Histology.” Cumulative scores are depicted as means ± SD of 3 mice per group (CD28-SA, day 3; n = 2).