CCR8 ^−/− T_regs undergo enhanced apoptosis. (A-B) B6D2 recipients were lethally irradiated on day −1 and then administered 3 × 10⁶ TCD BM cells from WT B6 donors plus 1 × 10⁶ column-purified CD4⁺CD25⁺ T_regs from WT eGFP⁺ B6 or CCR8^−/− eGFP⁺ B6 mice on day 0. Then 4 × 10⁶ CD25-depleted eGFP^– T_cons from WT B6 donors were administered on day +2. On transplant day +14, recipient animals were each administered 250 µL of BrdU (Invitrogen) by intraperitoneal injection. After 3 hours, these mice were euthanized, their spleens disrupted, and the cells stained with an anti-BrdU antibody. Donor T_regs within the recipient spleen were then evaluated for BrdU uptake by flow cytometry using an eGFP⁺Foxp3⁺ gate. (A) Representative flow cytometry plots of a WT and CCR8^−/− T_reg recipient are depicted. (B) The percentages of BrdU⁺ T_regs are depicted graphically; n = 5 mice per treatment group. (C-D) B6D2 recipients were lethally irradiated on day −1 and then administered 3 × 10⁶ TCD BM cells from WT eGFP^– B6 donors plus 1 × 10⁶ column-purified CD4⁺CD25⁺ T_regs from WT eGFP⁺ B6 or CCR8^−/− eGFP⁺ B6 mice on day 0. Then 4 × 10⁶ CD25-depleted eGFP^– T_cons from WT B6 donors were administered on day +2. On transplant day +10, recipient mice were euthanized, and their spleens disrupted. Donor T_regs within the recipient spleen were then examined for markers of apoptosis and cell viability by flow cytometry using an eGFP⁺Foxp3⁺ gate. A commercially available amine reactive viability dye was used, as standard propidium iodide staining is not compatible with the membrane permeabilization step required for intracellular Foxp3 staining. (C) Representative flow cytometry plots of a WT and CCR8^−/− T_reg recipient are depicted. (D) The percentages of dead or dying donor T_regs (Annexin V^high and/or Viability Dye^high) are depicted graphically; n = 3 mice per treatment group. *P = .003 by Student t test.