Critical role for CCR8 expression by T_regs in the prevention of GVHD. (A) Expression of CCR7, CCR4, and CCR8 was evaluated using qRT-PCR in naïve T_regs, activated T_regs, naïve CD4⁺CD25^– T_cons, and activated CD4⁺ T_cons. Fold differences in expression relative to naïve CD4⁺CD25^– T_cons are depicted. (B-C) B6D2 recipients were lethally irradiated to 950 rads on day –1 and then administered 3 × 10⁶ TCD BM cells from WT B6 donors +/− 4 × 10⁶ CD25-depleted whole (CD4⁺ and CD8⁺) splenic T_cons from either WT B6 or CCR4^−/− B6 donors on day 0 by tail vein injection. Recipient animals were followed for survival (B) and scored for GVHD twice weekly (C) using a validated clinical scoring system.²¹  Animals were assigned a score from 0 to 2 for each of 5 GVHD parameters: weight loss, activity, fur ruffling, kyphosis, and skin lesions. Scores ranged from 0 (minimum) to 10 (maximum). Error bars depict standard error of the mean. Data are combined from 2 separate transplant experiments; n = 7 animals per group. (D-E). B6D2 recipients were lethally irradiated to 950 rads on day –1 and then administered 3 × 10⁶ TCD BM cells from WT B6 donors +/− 4 × 10⁶ CD25-depleted splenic T_cons from either WT B6 or CCR8^−/− B6 donors on day 0. Data are combined from 2 separate transplant experiments; n = 8 animals per group. (F-G). B6D2 recipients were lethally irradiated on day –1 and then administered 3 × 10⁶ TCD BM cells from WT B6 donors +/− 1 × 10⁶ column-purified CD4⁺CD25⁺ T_regs from WT B6 or CCR4^−/− B6 mice on day 0. Then 4 × 10⁶ CD25-depleted T_cons from WT B6 donors were administered on day +2; n = 4 animals per group. (H-I) B6D2 recipients were lethally irradiated on day –1 and then administered 3 × 10⁶ TCD BM cells from WT B6 donors +/− 1 × 10⁶ column-purified CD4⁺CD25⁺ T_regs from WT B6 or CCR8^−/− B6 mice on day 0. Then 4 × 10⁶ CD25-depleted T_cons from WT B6 donors were administered on day +2. Data are combined from 2 separate transplant experiments; n = 4 BM, 8 BM/T_con, 8 BM/T_con/WT T_reg, 10 BM/T_con/CCR8^−/− T_reg. (H) *P = .013 for overall survival comparison between BM/T_con/WT T_reg and BM/T_con/CCR8^−/− T_reg groups by Fisher’s exact test. (I) *P = .003 for GVHD score comparison between BM/T_con/WT T_reg and BM/T_con/CCR8^−/− T_reg groups on transplant day +70 by the Mann-Whitney Test.