Figure 4. Analysis of conditioned HCECs. A: Determination of direct alloresponse of conditioned HCECs toward T-cells is shown. T-cells were either unstimulated or stimulated using IFN-γ or TNF-α or a combination of both. No significant T-cell proliferation could be obtained. HCECs need stimulation of IFN-γ to upregulate class II molecules. As this co-occurred with an upregulation of IDO, a significant induction of T-cell proliferation could not be detected. However, using 1-MT to inhibit IDO, a good proliferation of T-cells up to more than 3×10³ cpm was seen (Lane 6 and 10). B: The effect of IDO-induced tryptophan metabolites on the proliferation of effector cells (PBMCs) is shown in this panel. Mature DCs were incubated (1×10⁴ cells/well) for 72 h with allogeneic PBMC cells (1×10⁵ cells/well) in three different T-cell culture supernatants of HCECs stimulated with 500 ng/ml IFN-γ, 100 U/ml TNF-α, or 500 ng/ml IFN-γ + 100 U/ml TNF-α. The positive control consisted of culturing PBMC cells with allogeneic DCs in cell culture supernatant of unstimulated HCECs, and the negative control consisted of PBMCs or mDCs alone in different pools of HCECs. T-cell proliferation was determined by ³[H]-Thymidin incorporation (cpm) after three days of culturing. We can show that the conditioned medium taken from HCECs stimulated with IFN-γ and IFN-γ/TNF-α led to a significant suppression of T-cell proliferation. C: Re-supplementation of the conditioned medium as described in B using L-tryptophan is shown. To determine whether the low tryptophan or the produced L-kynurenine was responsible for the reduction in T-cell proliferation, we supplemented the pre-conditioned supernatant to L-tryptophan. Only a slight yet significant restoration of the T-cell proliferation up to 4×10³ cpm could be registered. However, the T-cell proliferation did not reach the level as shown in B as L-Kyn was not depleted from the supernatant. D: Apoptosis assay using flow cytometry for Annexin V was used to determine dose-dependant toxicity of L-kynurenine toward HCECs. We did see a slight and insignificant increase in apoptosis supplementing the HCEC supernatant to L-kynurenine at 10, 20, and 40 µmol/ml.