OX40 or GITR triggering on T reg's impedes T reg–mediated inhibition of GVHD. (A) Lethally irradiated BALB/c mice were reconstituted with 2 × 10⁵ C57BL/6 T-cell–depleted BM cells in the absence (▪) or presence (▴) of 5 × 10⁵ CD4⁺CD25^- cells. Some mice also received 3 × 10⁵ CD4⁺CD25⁺ cells and were left untreated (○) or injected intraperitonally with 600 μg of anti-OX40 (•) or anti-GITR (^*) mAb immediately after transplantation (6 mice/group). The same experiment was repeated as in panel A, treating the mice intraperitonally with 300 μg(•), 600 μg(□), or 1200 μg (^*) of anti-OX40 (B) or anti-GITR antibody (B-C). Results are shown in 2 separate panels (B-C) for clarity. Additional experiments were performed (D) in which mAbs were given to T reg's in vitro with anti-OX40 (•) or anti-GITR (^*) mAbs (30 μg/mL) and washed before their injection with BM and effector T cells: 2 groups of T reg's pretreated with control isotype rat IgG1 (□) or rat IgG2a (○) were added (6 mice/group). All mice were monitored daily for signs of GVHD and lethality was recorded. One representative experiment, for both in vivo and in vitro treatment with mAbs, out of 2 with similar results is shown.