Fludarabine and cyclophosphamide downregulate IDO expression. (A) Jeko-1 cells were treated with mafosfamide (2 μg/mL), fludarabine (20 μM), or both for 24 hours. Cells were then washed and cultured with or without IFN-γ. Protein extracts were prepared for IDO immunoblot analysis. Results are representative of 3 independent experiments. (B) PBMCs from 2 CLL patients were treated with mafosfamide (2 μg/mL), fludarabine (20 μM), or both for 24 hours. Cells were then washed and incubated with IFN-γ. Twenty-four hours later, proteins were extracted, and IDO expression was revealed by immunoblot. (C) Schematic representation of the experiments in SCID/Beige mice comparing the antitumor effect of chemotherapy, CD19-CARTs, or their combination on Jeko-1 tumors. Mice were subcutaneously injected with luciferase-transduced Jeko-1 cells (3 × 10⁶). Five days later, mice were treated with 0.75 mg fludarabine (Flu) and 0.75 mg cyclophosphamide (Cy) intraperitoneally. Two days after chemotherapy, CD19-CARTs (10 × 10⁶) were infused intravenously. (D) Time course of Jeko-1 tumor bioluminescence. Data represent mean ± SD of 7 mice per group from 2 independent experiments (*P < .05).