Absence of CCR8 does not impair T_reg activation or suppressive function. (A-B) B6D2 mice were lethally irradiated on day −1 and then transplanted with 3 × 10⁶ WT B6 TCD BM cells with either 1 × 10⁶ WT eGFP⁺ B6 or CCR8^−/− eGFP⁺ B6 T_regs on day 0. Then 4 × 10⁶ WT eGFP^– B6 T_cons were administered on day +2. On transplant day +7, recipient mice were euthanized, and their spleens disrupted. Donor T_regs were then examined for activation markers using a Foxp3⁺eGFP⁺ gate. Representative flow cytometry plots following l-selectin (A) and CD44 (B) staining are depicted. In addition, the mean percentages of l-selectin^low and CD44^high cells are depicted graphically; n = 9 total recipients per group. The spleens were pooled into groups of 3 to maximize the number of donor events, and mean percentages of the groups compared using the Mann-Whitney Test. (C) 1.5 × 10⁶ freshly isolated CD4⁺CD25⁺ WT or CCR8^−/− T_regs were labeled with CFSE and transplanted with 3 × 10⁶ unlabeled WT B6 TCD BM cells into irradiated B6D2 recipients. On transplant day +5, recipient mice were euthanized, their spleens disrupted, and donor T_regs examined for CFSE positivity by flow cytometry using a K^d–, Foxp3⁺ gate. (D) CD4⁺CD25⁺ T_regs were isolated from WT or CCR8^−/− B6 mice and then cultured for 72 hours at varying ratios with 5 × 10⁴ CD4⁺CD25^– WT B6 responder cells and 5 × 10⁴ irradiated TCD B6D2 stimulator splenocytes. During the last 16 to 20 hours of incubation, 0.037 MBq (1 µCi) of [³H]thymidine was added to each well, and [³H]thymidine incorporation measured by scintillation counting.