Figure 2. Functional activity of IDO was determined by measuring the concentration of L-kynurenine and L-tryptophan in the tissue culture supernatant of pooled HCECs. HCECs were treated with 500 ng/ml IFN-γ, 100 U/ml TNF-α, 500 ng/ml IFN-γ+100 U/ml TNF-α, or without any of the cytokines. After 72 h, the cell culture supernatants were harvested, and the concentrations of L-kynurenine (A) and L-tryptophan (B) were detected by HPLC. The difference between the IFN-γ or cocktail stimulated HCECs versus the unstimulated or TNF-α only stimulated HCECs was highly significant (p<0.03). The chromatograms (C) show the total free L-kynurenine (top) and L-tryptophan (bottom) of stimulated HCECs. As indicated by arrows, the retention times are different regarding L-kynurenine and L-tryptophan. Interestingly, below L-kynurenine, no further downstream metabolites of the IDO pathway were detected. Furthermore, no L-kynurenine (0.0 µmol) was detected in the unstimulated group or in the TNF-α stimulated group. The bars shown do represent the mean±standard deviation (SD).