1 d mt

The amount of L-kynurenine in culture supernatants was measured with a spectrophotometric analysis, as previously described.^{23 (link)} Briefly, Mo-DC were washed, resuspended in Hanks buffered saline solution supplemented with 500 µM L-tryptophan (Sigma-Aldrich) and incubated (where indicated) with or without the IDO inhibitors 1-MT-D or –L (1 mM, Sigma-Aldrich). Supernatants were harvested after 4 h and mixed with 30% trichloroacetic acid (2:1), vortexed and centrifugated at 8000 g for 5 min. Subsequently, this solution was added to Ehrlich reagent (1:1, Sigma-Aldrich) in a 96-well plate. Triplicate samples were run against a standard curve of defined kynurenine concentrations (0–100 µM; Sigma-Aldrich). Optical density was measured at 490 nm, using a Multiskan EX microplate reader (Thermo Electron Corporation, Vantaa, Finland).

DC were cultured with autologous CD3⁺ T cells (1:10) for 1 day, with or without the IDO inhibitors 1-MT-D or –L (1 mM, Sigma-Aldrich), where indicated. For immunophenotype studies, tri-color immunofluorescence was performed using fluorescein isotiocyanate (FITC)-conjugated anti-human CD4 (clone RPA-T4), phycoerytrin (PE)-conjugated anti-human Foxp3 (clone 206D) and allophycocyanin (APC)-conjugated anti-human CD25 (clone BC96, Biolegend, San Diego, CA). For cell-surface staining, 1×10⁵ cells/100 µl were incubated in the dark for 20 min at 4°C with mAbs in phosPHAte-buffered saline (PBS)-1% bovine serum albumine. Subsequently, for Foxp3 intracellular staining, cells were incubated at room temperature in the dark for 20 min with fix/perm buffer followed by 15 min with perm solution and additional 30 min with the mAb. After 2 washes, samples were analyzed using BD FACSCanto™II equipment (BD Biosciences). A minimum of 10,000 events was collected in list mode on FACSDiva software. To test their suppressive activity, purified CD4⁺CD25⁺ T cells (10⁴/well) were irradiated and added to cultures consisting of CFSE-labeled CD3⁺ T cells (10⁵/well) as responders, stimulated by 1 µg/ml PHA (Sigma-Aldrich). After 5 days, cultures were analyzed using BD FACSCanto™II equipment (BD Biosciences) and the proliferation index was calculated using FCSExpress4 software.^{24 (link)}