The absence of DNAM-1 on donor T_reg promotes regulatory function in vivo. (A) Representative FACS plots of the sort strategy on CD25⁺CD8⁻ T cells from naive B6.WT or B6.DNAM-1^−/− mice and Foxp3 expression on sorted cells. (B) 5 × 10⁶ BM from B6.WT mice with or without 0.2 × 10⁶ FACS sort-purified CD25⁺CD4⁺ T cells from B6.WT or B6.DNAM-1^−/− mice were transplanted into lethally irradiated BALB/c mice. Two days later, those recipients were given 0.5 × 10⁶ CD3⁺ T cells from CD25 Ab–treated B6.WT mice. TCD bone marrow from B6.WT donors was transplanted into irradiated BALB/c recipients as non-GVHD controls. Survival data are shown. P < .0001, B6.DNAM-1^−/− T_reg vs no T_reg; P = .0001, B6.WT T_reg vs no T_reg; P = .0011, B6.WT T_reg vs B6.DNAM-1^−/− T_reg. Data shown are combined from 3 replicate experiments (n = 16 T-cell–replete group without T_reg, n = 22 per T-cell–replete group with T_reg, and n = 9 in TCD control). (C) 5 × 10⁶ BM from B6.WT mice with or without 0.4 × 10⁶ FACS sort-purified CD25⁺CD4⁺ T cells from B6.WT or B6.DNAM-1^−/− mice were transplanted into lethally irradiated B6D2F1 mice. Seven days later, those recipients were given 2 × 10⁶ CD3⁺ carboxyfluorescein succinimidyl ester (CFSE)–labeled T cells from CD25 Ab–treated B6 (CD45.1⁺) mice. Four days later, spleens from recipients were analyzed. CD45.1⁺IFN-γ⁺CD4⁺ T cells (CD45.1⁺CD3⁺CD4⁺) (top right) and CD45.1⁺IFN-γ⁺CD8⁺ T cells (CD45.1⁺CD3⁺CD8⁺) (bottom right). Representative FACS plots for CFSE dilution and IFN-γ secretion in CD4⁺ and CD8⁺ T cells from each group are shown. IFN-γ⁺CD4⁺ T cells: P = .016, B6.DNAM-1^−/− T_reg vs no T_reg; P = .029, B6.WT T_reg vs no T_reg; P = .016, B6.WT T_reg vs B6.DNAM-1^−/− T_reg. IFN-γ⁺CD8⁺ T cells; P = .016, B6.DNAM-1^−/− T_reg vs no T_reg; P = .029, B6.WT T_reg vs no T_reg; P = .064, B6.WT T_reg vs B6.DNAM-1^−/− T_reg. n = 4 to 5 per group. Representative data from 1 of 2 experiments is shown.