Figure 2

Oral tolerance induction in indoleamine 2,3-dioxygenase-expressing CD11c⁺ dendritic cells of tolerized mice. The induction of oral tolerance increases the proportion of indoleamine 2,3-dioxygenase (IDO)-expressing CD11c⁺ dendritic cells (DCs) in Peyer's patches of tolerized mice. (a) Flow cytometric analysis of IDO in CD11c⁺ DCs isolated from Peyer's patches. Mononuclear cells obtained from Peyer's patches of tolerized mice and of CIA mice were probed with Fluorescein isothiocyanate-labeled anti-CD11c mAb and were fixed with CytoPerm/CytoFix for 20 minutes. Cells were probed for intracellular IDO using anti-mouse IDO antibody and were analyzed by flow cytometry. The histograms were gated on CD11c⁺ DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Results are the mean ± standard deviation of replicate samples from seven independent experiments. SSC. (b) Analysis of IDO transcription in tolerized mice and CIA mice. CD11c⁺ DCs were isolated from Peyer's patch mononuclear cells using the magnetic-activated cell sorting system. The expression of IDO mRNA was analyzed using RT-PCR. β₂-Actin was used as an internal control. Each value is the mean ± standard deviation of replicate determinations in one of four experiments. *P < 0.05. (c) Immunofluorescent confocal microscopic examination of IDO expression by CD11c⁺ DCs. Mononuclear cells obtained from Peyer's patches of tolerized and CIA mice were stained with Fluorescein isothiocyanate-labeled anti-CD11c (green) and anti-IDO (red), fixed, and were examined using confocal microscopy. Isotype-matched control antibody staining was negative (data not shown). Data are representative of three independent experiments.