Figure 5

Indoleamine 2,3-dioxygenase-expressing CD11c⁺ dendritic cells essential for antigen-specific CD4⁺CD25⁺ regulatory T-cell induction in tolerized mice. (a) Increased CD4⁺CD25⁺ T-cell proportion through an indoleamine 2,3-dioxygenase (IDO)-dependent mechanism. For regulatory T-cell induction, isolated CD4⁺CD25^- T cells (1 × 10⁵/well) were cultured with CD11c⁺ dendritic cells (DCs) (2 × 10⁴/well) from tolerized mice or collagen-induced arthritis (CIA) mice in the absence or presence of type II collagen (CII) (40 μg/ml) for 3 days. 1-Methyl tryptophan (1-MT) was added to selected cultures. The proportion of CD4⁺CD25⁺ T cells was determined using flow cytometry. Numbers represent the percentage of double-positive cells. (b) Summary of the percentages of CD4⁺CD25⁺ T cells from the coculture experiments in (a). Values are the mean from four independent experiments; individual symbols are the mean in individual animals, and bars show the group means. *P < 0.02. (c) Analysis of Foxp3 expression by converted CD4⁺CD25⁺ T cells. Plots were gated on CD4⁺CD25⁺ DCs. Dotted histogram lines represent cells stained with isotype-matched control monoclonal antibodies. Data represent the mean ± standard deviation and are representative of four independent experiments. (d) Foxp3 mRNA expression in the same conditions as (a). β -Actin was used as an internal control. Results are representative of four independent experiments. (e) Regulatory function of the CII-induced CD4⁺CD25⁺ T cells. CD4⁺CD25⁺ T cells were expanded by exposure to CD11c⁺ DCs from Peyer's patches from tolerized mice in the presence of CII antigen stimulation. Varying numbers of CD4⁺CD25⁺ T cells were cultured for 3 days with CII-reactive CD4⁺ T cells (1 × 10⁵) and irradiated antigen-presenting cells (1 × 10⁵) from mice with CIA in the presence of CII (40 μg/ml). Values are the mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.001. cpm, counts per minute.