In vivo ablation of donor Foxp3⁺ T cells abolishes PTCy protection against GVHD. (A) Experimental schema. BALB/B recipients after lethal conditioning (775 cGy) received 10⁷ TCD BM (B6 CD45.1⁺) and GVH inocula of 12 × 10⁶ splenocytes and CLN cells from Foxp3^DTR or Foxp3^WT B6 CD45.2⁺ donors. Designated groups received DT (50 ng/g IP) or PBS on days 0 and +1. PTCy (200 mg/kg IP) was administered to designated groups on day +3 post-alloBMT. All animals were monitored daily for survival and scored twice weekly for GVHD on day +7 post-alloBMT and every 4 days thereafter until day +60, when experiments were terminated. (B) Mean GVHD scores and survival. Combined results of 3 independent experiments are shown. (C) Frequency of cytokine-producing, inocula-derived CD4⁺CD45.2⁺ T cells in spleens of surviving chimeras on day +28. (D) Frequency (left panel) and total numbers (right panel) of CD4⁺CD45.2⁺Foxp3⁺ T cells in the spleens of surviving chimeras on day +28. Data are representative of 3 independent experiments, with a minimum of 4 animals per group. The results are presented as the mean ± standard error of the mean.