Fibrocytes mediate immune suppression via IDO. (A) Fibrocytes do not co-stimulate T cells treated with submitogenic concentrations of anti-CD3. Elutriated lymphocytes (5 × 104 cells/well) and designated electronically sorted cell subsets (2.5 × 104 cells/well) were added to a 96-well plate precoated with the designated concentration of anti-CD3, and thymidine incorporation was measured on day 3. This is representative of 5 experiments using cells from 5 separate subjects. (B) Suppression of anti–CD3-mediated T-cell proliferation by fibrocytes. T cells were plated as in (A), but the designated electronically sorted cell subsets (purity >90%) were added (2.5 × 104 cells/well) at a ratio of 2 lymphocytes:1 suppressor, and thymidine incorporation was measured on day 3. This is representative of 6 experiments using cells from 6 separate subjects. (C) Semiquantitative real-time PCR was used to measure IDO, Arginase I, and INOS2. Relative amounts compared with normal peripheral blood mononuclear cells are shown. The mean ± SEM was derived from 6 experiments. (D) Fibrocyte-mediated suppression can be reversed by the addition of an IDO inhibitor, 1MT. Cultures were set up as described in (A) and, where designated, 1MT was added at a concentration of 20 μM at the beginning on the co-culture. Results are representative of 5 experiments using fibrocytes from 5 separate subjects. All experiments in Figure 3 used electronically sorted fibrocytes with purity >90%.
