Expression of IRF8 and TYROBP regulate the acquisition and loss of IDO competence in human DCs. (A) RT-PCR analysis of IRF8 and TYROBP gene expression in human DCs matured with LPS (IRF8 analysis) or CD40L (TYROBP), both treated with gene-specific siRNA for different times. Control cells were treated with negative control siRNA (nc siRNA). HCST (coding for human DAP10) and IRF1 gene expression was assayed as specificity control for TYROBP and IRF8 siRNA, respectively. (B) Kynurenine levels in supernatants of human DCs subjected to treatment with nc or gene-specific (indicated) siRNA for 24 hours, followed by overnight incubation with medium alone, IFN-γ, or hCTL0-4-Ig. LPS-matured DCs (IDO⁺ DCs) were subjected to IRF8 gene silencing, whereas CD40L-matured DCs (IDO^- DCs) were subjected to TYROBP gene silencing. Results (mean ± SD) are from 1 of 2 representative experiments. (C) Proliferative response of human CD4⁺ T lymphocytes in response to allogeneic DCs at 4 days. MLR was established with T lymphocytes purified from allogeneic donors and DCs subjected to siRNA treatment as in panel B, followed by overnight incubation with medium alone or hIFN-γ. Cocultures were also established in the presence of 20 μM 1-MT. Results are expressed as mean cpm ± SD in 1 of 3 representative experiments. Radiolabel incorporation by DCs or T cells cultured alone was less than 1000 and less than 3000 cpm, respectively, and these values were not affected by the addition of 1-MT.