Viral vector up-regulation of IDO has a significant consequence on MLR. (A) Five days after viral transduction of either iDCs or mDCs (activated with 20 ng/mL TNF-α, 20 ng/mL IL-1β, 20 ng/mL LPS, and 10 ng/mL PGE2 for 48 hours) with viral vectors, the cells were analyzed for expression of IDO using RT-PCR Southern and Western blots. The density of the IDO bands (as a ratio to the β-actin levels) are shown below each band, as the mean plus or minus SD of 3 experiments. (B) The activity of IDO was determined by measurement of the concentration of tryptophan breakdown product l-kynurenine in the supernatant of virally transduced cells. (C) In order to determine the functional effect of IDO expression, we used variable numbers of virally transduced iDCs (top row) or mDCs (bottom row) as stimulators in an allogeneic MLR performed in the presence (right column) or the absence (left column) of 500 μM 1-MT, an IDO inhibitor. 3H-thymidine incorporation was determined on day 5. The results are the mean plus or minus SD of triplicate cultures.
