FIGURE 5

AcTreg induce death of Teff in vivo. (a) Representative examples for in vivo killing of CFSE⁺ targets. nu/nu mice were adoptively transferred with different combinations of cells as follows: a mixture of 3 10⁷ Teff previously labeled with a low CFSE fluorescene intensity (0.5 M, CFSE^lowCD4) and 3 10⁷ Teff cells previously labeled with a high CFSE fluorescene intensity (5 M, CFSE^highCD4); a mixture of 3 10⁷ CFSE^lowTeff, 3 10⁷ CFSE^highTeff and 3 10⁶ acTreg; a mixture of 3 10⁷ CFSE^lowCD4T cells pre-treated with DR5-blocking antibody, 3 10⁷ CFSE^highCD4T cells and 3 10⁶ acTreg. Cells of each population were mixed in 500 l PBS for i.v. injection. Samples for submission to the in vivo cytotoxicity assay were acquired by collecting mononuclear cells from spleen, lymph nodes (LN) and blood 18 h after i.v. injection. Ratios were calculated as the percentages of CFSE^low/CFSE^high cells in a total of 1 10⁶ cells. The CFSE-positive target cells from nu/nu mice were determined using FACS. The ratio (R) between the amount of CFSE⁺ and CFSE⁺⁺ cells was calculated. (b) BALB/c nu/nu mice were engrafted with tail skin from C57BL/6 mice and BALB/c mice, and inoculated with different combinations of cells as follows: 3 10⁷ Teff; 3 10⁷ Teff and 3 10⁶ acTreg; 3 10⁷ Teff pre-treated with DR5-blocking antibody; 3 10⁶ acTreg. Skin grafts were transplanted 1 day after cell transfer, and skin rejections were observed more than 100 days after the first grafts. Survival rates were analyzed using the Log-Rank test and all groups, n=8