Reconstitution of PTCy-treated Treg-ablated recipients with DT-insensitive Tregs rescues chimeras from lethal GVHD. (A) Experimental design. Chimeras were constructed as in Figure 2 and PTCy was administered on day +3 post-alloBMT. DT (50 ng/g) was given on days +4 and +5. On day +4 post-alloBMT, groups received PBS (controls) or 2 × 10⁵ tTregs purified from day +28 PTCy-treated chimeras as described in Figure 3 or pre-transplant Tregs from B6.Foxp3^RFP donors. (B) Survival was monitored twice weekly until day +60, when the experiment was terminated. (C) Mice were scored for GVHD on day +7 and every +4 days subsequently until day +60. Data shown represent 2 independent experiments with at least 10 animals per group.