FasL in the transduced cells was functional.

FasL⁺ lin⁻ BMs killed Jurkat T cells in a dose-dependent fashion, and FasL⁺ DCs decreased T-cell proliferative responses. (A) B6.SJL lin⁻ BMs were transduced 3 times to express either the control GFP vector or the vector expressing the GFP-FasL fusion. On day 5 of ex vivo transduction culture, the cells were FACS sorted into (FasL⁺)GFP⁺ or (FasL⁻)GFP⁻ subsets. The (FasL⁺)GFP⁺ subset expressed more than 10-fold higher fluorescence than the (FasL⁻)GFP⁻subset; the lin⁻ BMs with intermediate levels of fluorescence were discarded. Sorted (FasL⁺)GFP⁺cells were mixed in varying ratios with (FasL⁻)GFP⁻ cells for a total of 10⁵ lin⁻ BMs, as indicated, in duplicate wells of a 96-well plate containing 10⁵ PKH-labeled Jurkat cells. At 24 hours later, 7-AAD was added to the cultures, which were then analyzed by FACS for the percent PKH⁺ (Jurkat) cells that had incorporated 7-AAD. Shown are histograms of 7-AAD fluorescence in-gated PKH⁺ (Jurkat) cells. Mixtures containing 1%, 5%, 20%, or 80% (FasL⁺)GFP⁺ lin⁻BMs, indicated by the graph labels, resulted in 5%, 9%, 36%, and 42% dead Jurkat cells, respectively. (B) Function of modified DCs was tested in a standard T-cell proliferation assay. GFP control-transduced or FasL-transduced B6.SJL or BALB/c DCs were generated as described, then irradiated (3000 cGy) and incubated with responder spleen cells (B6, transgenic 2C [on a B6 background] or BALB/c, as indicated in the graph axis labels). Proliferation was determined by incorporation of ³H-thymidine. This plot shows the averages (± SD) of 3 separate experiments. (C) T-cell proliferative responses are shown from experiments in which allogeneic DCs were mixed with syngeneic DCs as stimulators, with either the allogeneic DCs (B6) or the syngeneic DCs (BALB/c) modified to express FasL. In this set of experiments, 10⁶ BALB/c spleen cells were mixed with, from left to right: 10⁵ control B6 DCs; 10⁵FasL⁺ B6 DCs; 5 × 10⁴ control B6 DCs plus 5 × 10⁴ control BALB/c DCs; and 5 × 10⁴control B6 DCs plus 5 × 10⁴ FasL⁺ BALB/c DCs. After 3 days of incubation, 1 μCi (0.037MBq)³H-thymidine was added for 24 hours, then cells were harvested and proliferation was determined by ³H-thymidine incorporation. The results are shown for 2 separate experiments, with triplicates for each condition.