CD8⁺FoxP3⁺ T_reg attenuate GVHD mortality after BMT. (A) Recipient B6D2F1 mice were lethally irradiated and 24 hours later transplanted with BM from B6.FoxP3-GFP mice and T cells from both B6.FoxP3-GFP and DEREG mice in the following combinations: (1) WT CD4⁺ T cells and DEREG CD8⁺ T cells, (2) DEREG CD4⁺ T cells and WT CD8⁺ T cells, or (3) DEREG CD4⁺ T cells and DEREG CD8⁺ T cells. *P < .05, B6 (CD8⁺ WT + CD4⁺ DEREG) versus B6 (CD8⁺ DEREG + CD4⁺ DEREG). **P < .01, B6 (CD8⁺ DEREG + CD4⁺ WT) versus B6 (CD8⁺ DEREG + CD4⁺ DEREG). Survival data are shown and are combined from 4 duplicate experiments (CD4⁺FoxP3⁺ deplete, n = 42; CD8⁺FoxP3⁺ deplete, n = 39; CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ deplete, n = 39; TCD no depletion, n = 20). (B) BALB/c recipients were lethally irradiated and transplanted with BM and T cells from B6.FoxP3-GFP donors 24 hours later. At day 7 after transplantation, LN and spleens were removed, and CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ cells sorted on B6.FoxP3-GFP and CD4 or CD8. Sorted FoxP3⁺ populations (3.5 × 10⁴) were transplanted with BM from B6.WT donors into lethally irradiated BALB/c recipients. Whole CD3⁺ T cells (7.5 × 10⁵) were injected 2 days later. *P < .05, BALB/c (+ CD8⁺FoxP3⁺) versus BALB/c (+ CD4⁺FoxP3⁺). ***P < .001, BALB/c versus BALB/c (+ CD8⁺FoxP3⁺). Survival data are shown and are combined from 2 duplicate experiments (no FoxP3⁺, n = 13; CD8⁺FoxP3⁺, n = 17; CD4⁺FoxP3⁺, n = 12; no FoxP3⁺ TCD, n = 6). (C) Recipients were transplanted as in panel B; however, CD8⁺Foxp3⁺ were sorted from FoxP3.LuciDTR-4 donors (luciferase, GFP, and the DT receptor-driven of the FoxP3 promoter, labeled as B6.FoxP3-luc⁺). Recipients were imaged at days 2, 7, and 14 after transfer, with images shown contrasting recipients that did or did not receive T_reg.