CD25⁺T-cell depletion abolished the efficacy of PTCy for recipient mice transplanted with PD-1^−/− graft. (A) Lethally irradiated BDF1 recipients received transplants of 10 × 10⁶ CD25-replete or CD25-depleted T cells from PD-1^+/+ B6 SPs or PD-1^−/− B6 SPs concomitantly with 5 × 10⁶ B6 TCD-BM cells (allogeneic group). The syngeneic group was administered the same amounts of SPs and TCD-BM from BDF1 mice (n = 10 per group). All recipient mice were administered 50 mg/kg of Cy intraperitoneally on day 3 after HSCT. All animals were monitored daily for survival, and GVHD scores were monitored from days 3 to 7 and once per week from day 14. (B) Kaplan-Meier survival curve after HSCT. Solid lines show the outcomes of recipient mice administered CD25-replete T cells, and the dotted line shows the survival of mice in the CD25-depleted group (PD-1^−/− CD25⁺ vs PD-1^−/− CD25⁻, P < .001). (C) Mean GVHD scores of recipient mice (PD-1^−/− CD25⁺ vs PD-1^−/− CD25⁻ on day 14, P < .05). Data from 2 same experiments were combined. Clinical scores are expressed as the mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001.