APG101 inhibits CD95-mediated apoptosis but does not prevent T-cell proliferation or cytotoxicity in vitro. (A) Spleen cells from B6 mice were incubated with medium or recombinant CD95L plus enhancer in the presence or absence of increasing concentrations of APG101. After 24 hours cells were stained for CD4⁺ and CD8⁺ expression and apoptosis was determined in both T-cell populations by measuring 7-AAD–positive cells. Data present the mean of 3 independent experiments with triplicates ± SD. (B) CFSE-labeled B6-derived spleen cells were stimulated with medium or irradiated B6D2F1-derived allogeneic spleen cells in the presence or absence of increasing concentrations of APG101. After 6 days, proliferation of CD4⁺ and CD8⁺ T cells was measured. Data represent the mean of triplicates ± SD of 1 representative experiment of 3 experiments performed. (C) H-2^d–specific CTLs were established by coincubation of B6-derived spleen cells with irradiated B6D2F1 spleen cells in the presence or absence of APG101 (100 ng/mL; 1st stim). After each week, living cells were isolated and restimulated with irradiated B6D2F1 spleen cells (2nd stim, 3rd stim) in the presence or absence of APG101. Six days after each stimulation, H-2^d–specific cytotoxicity of CTLs was tested on P815 (H-2^d) or EL4 (H-2^b) target cells in a chromium release assay. Data are representative for 1 experiment of 3 done.