In vivo inhibition of IDO prevents tumor-mediated expansion of CD4⁺CD25⁺ T cells by blocking the conversion of CD4⁺CD25⁻ cells. (A) Immunocytochemistry analysis for IDO protein in A20 cells. Image was obtained on an Olympus B×41 microscope (Olympus, Tokyo, Japan) equipped with a 40×/0.75 NA objective lens and an Olympus Camedia camera. No imaging medium or solution was used. Olympus Camedia software was used for image acquisition. (B) Functional enzymatic activity. Depletion of tryptophan from the culture medium (expressed as the percentage of the starting concentration in fresh medium) by human MSCs with or without IFN-γ (positive control sample) and A20 cells. Results are representative of 3 independent experiments. (C) BALB/c mice were injected intrasplenically with 10⁵ A20 cells (TB indicates tumor-bearing mice), and, from the day of tumor injection, they were treated or not with 1-D and L-MT (NT indicates not treated, n = 21; 1-MT, 1-MT–treated, n = 21). Two groups of mice (naive, n = 15) were not injected with the tumor but received 1-MT. Percentage of CD4⁺CD25⁺ T cells among CD4⁺ T cells in the spleen was assessed by flow cytometry analysis. Averages of data collected from experiments independently performed are reported. The data report the mean ± SD of independent experiments. (D) Intracellular Foxp3 expression in purified CD4⁺CD25⁺ T cells obtained from splenocytes of tumor-bearing mice. Results are representative of 4 independent experiments. (E) Anti-CD3 mediated proliferation of naive CD4⁺CD25⁻ T cells in the presence of increased numbers of CD4⁺CD25⁺ cells obtained from splenocytes of tumor-bearing mice (▪). As a positive control, CD4⁺CD25⁻ cells were stimulated in the absence of CD4⁺CD25⁺ T cells (□). Proliferation was evaluated after 3 days by thymidine incorporation assay. Results are expressed as cpm and represent the mean ± SD of 4 independent experiments. *P = .03, experimental versus control sample. (F) In vivo conversion experiments. Purified Thy1.1⁺ CD4⁺CD25⁻ T cells (10 × 10⁶) were transferred into BALB/c mice bearing A20 or CT26 tumors. After 10 days, spleens were collected and labeled with Thy1.1, CD4, and CD25; Thy1.1⁺ CD4⁺ cells were gated and analyzed for CD25 and CFSE expression. Cumulative data of the CFSE^highCD25⁺Thy1.1⁺ cells conversion in spleens cells are reported and represent the mean ± SD of 4 experiments.