mDCs induced cytotoxic CD8⁺ T cells independently of CD4⁺ T cells whereas MPA-DCs induced lower cytotoxic activity in allogeneic CD8⁺ T cells. Effector cells (E) were incubated at different E:T ratios in the presence of CFSE-labeled PBMC target cells (T) for 7 hours, as described in the cytotoxicity assay section. (A) Target cell lysis was quantified by analyzing 7-AAD staining in CFSE-positive cells. CFSE and 7-AAD histograms from a representative experiment are shown and the percentages of 7-AAD-positive cells are indicated in each histogram. Basal lysis was measured on target cells incubated in the absence of effectors (target alone). (B) To assess the specificity of CD8 cytotoxic function, CD8⁺ mDCs (triangles) or CD8⁺ MPA-DCs (squares) were mixed with PBMCs from the first DC donor (black symbols) or from an unrelated donor (empty symbols) for 7 hours. PBMC lysis was assessed by CFSE/7-AAD staining and the percentage of specific target lysis was calculated as described in “Methods.” Results of 1 of 8 independent experiments are presented for 6 different E:T ratios. (C) Percentage of specific lysis induced by naive (CD45RA⁺) or memory (CD45RO⁺) CD8⁺ T cells cocultured with either mDCs or MPA-DCs was determined using PBMCs from the first DCs donor at a E:T ratio of 1:1. Results of 1 of 3 independent experiments are presented. (D) For intracellular staining, cells were incubated with Golgi Stop solution for the last 5 hours of culture. At the end of coculture, cells were harvested and expression of the CD107a PE, granzyme A PE, granzyme B FITC, and Perforin PE markers was assessed by FACS analysis. Each point represents 1 experiment and the black bar represents the mean of 6 independent experiments. The P value is indicated on each histogram of the figure. *P < .05