Absence of Tregs in donor inocula abrogates the GVHD protective activity of PTCy. (A-B) C3H.SW recipients were lethally conditioned (1050 cGy TBI) before injection of T cell–depleted (TCD) B6 CD45.1⁺ bone marrow and GVH inocula from either B6 or B6.Foxp3^RFP CD45.2⁺ donors. PTCy was administered on days +3 and +4 at a dose of 33 mg/kg intraperitoneally (IP) as previously described.²¹  All animals were monitored twice weekly for GVHD and daily for survival until day +60, when the experiments were terminated. After the death of the last mouse in a particular group, GVHD scores at that time were carried forward in the graph depictions until the end of the experiment. (A) Mean GVHD scores of B6→C3H.SW chimeras that received GVH inocula composed of 2.2 × 10⁶ CD4⁺ and CD8⁺ T cells from B6 CD45.2⁺ donors with or without CD4⁺ CD25^hi cells. (B) Mean GVHD scores of chimeras that received GVH inocula comprised of 2.2 × 10⁶ B6 CD4⁺ and CD8⁺ T cells from Foxp3^RFP donors, with or without CD4⁺ Foxp3⁺ T cells (depleted populations contained <1.0% RFP⁺ cells). (n = 5-7/group). (C) Mean GVHD scores of B6→BALB/B chimeras. Lethally irradiated (775 cGy) BALB.B mice were transplanted with 10⁷ TCD BM cells from B6 CD45.1⁺ donors and 12 × 10⁶ splenocytes and cutaneous lymph node (CLN) cells from B6.Foxp3^GFP (CD45.2⁺) donors that were replete or depleted of Foxp3^GFP+ T cells via sorting. PTCy (200 mg/kg IP) was administered on day +3 post-alloBMT. Mice were scored on day +7 post-alloBMT and every 4 days thereafter until the termination of the experiments at day +60. The data represent 2 independent experiments with a total of 10 animals per group.