Functional IDO Is Expressed on CD34⁺- and Monocyte-Derived Dendritic Cells According to Differentiation Status.

Indoleamine 2,3-dioxigenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expression by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs). Although human monocyte-derived dendritic cells (Mo-DCs) have been shown to express IDO, little is known about the expression of IDO in other subsets of human DCs, including those generated from CD34⁺ hematopoietic progenitors (CD34⁺-derived DCs) In the present study, we performed a full characterization of IDO expression and function by human dendritic cells including Mo-DCs and CD34⁺-derived DCs. Mo-DCs were generated from purified CD14⁺ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-gamma and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6, PGE2). CD34+-derived DCs were generated from purified CD34⁺ cells after 7 days of culture with GM-CSF and TNF-alpha, followed by 7 day-treatment with GM-CSF and IL-4 and then matured with the same stimuli used for MoDCs. After culture, DCs were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogenic proliferation and Tregs induction. Monocytes lack IDO expression. Immature Mo-DCs have little if any expression of IDO. During maturation, the cytokine cocktail is the most effective in up-regulating IDO, both at mRNA and protein level, which is paralleled with higher kynurenine production and inhibition of allogeneic T cell proliferation. PGE2 has a crucial role in inducing IDO expression, while IL6 has an opposite effect. Mature Mo-DCs are shown to generate a population of CD4⁺CD25⁺FOXP3⁺ Tregs which are capable of suppressing allogeneic T-cell proliferation. This inhibitory effect is abrogated by the addition of the IDO inhibitor 1-methyl tryptophan (1-MT). CD34⁺ cells lack IDO mRNA expression. During DC differentiation, IDO expression is observed at day 7, but not at day 14. Flow cytometry analysis of day 7 cells reveal a population of CD34⁻, CD14⁺, CD1a^+/− cells. Similarly to Mo-DCs, maturation stimuli induce a marked up-regulation of IDO mRNA. Similar results are observed when IDO protein is measured. Production of kynurenine, inhibition of allogeneic T cell proliferation and generation of Tregs from normal CD3⁺ T-cells are also paralleled with IDO expression. In conclusion, in human DCs the maturation status is associated with a differential expression of IDO both in Mo-DCs and CD34⁺-derived DCs. Differentiation of CD34⁺ cells into DCs results in a transient and early expression of IDO at precursor level. Given its functional capacity to inhibit T-cell-mediated immune response, the expression of IDO along DC differentiation may indicate a protective role of the pool of precursor cells which are committed to DC lineage.