In vivo alloactivated T_reg cells do not require granzyme B to suppress T_eff-cell proliferation ex vivo. (A) Experimental protocol for generation of WT and Gzmb^−/− GVHD-activated T_reg cells: 2 × 10⁶ T cells from 129/SvJ WT or Gzmb^−/− FIG reporter mice were injected intravenously in lethally irradiated (900 cGy) Balb/c hosts, together with 2 × 10⁶ bone marrow cells from WT (non-FIG) mice. Four days after transplantation, splenic WT or Gzmb^−/− CD4⁺GFP⁺ T_reg cells were sort-purified and cultured ex vivo with DDAO-SE–stained CD4⁺CD25⁻ T_eff cells for 3 days. T cells were either unstimulated or stimulated with CD3/CD28 beads. (B) Representative flow plot and histogram of T_eff-cell proliferation (ie, loss of DDAO-SE staining) in the presence or absence of CD3/CD28 beads. (C) Dose-dependent inhibition of T_eff-cell proliferation mediated by wild-type or Gzmb^−/− GVHD-activated T_reg cells. (D) Summary graph of normalized data from 3 independent experiments.