PD-1 expressing CD8⁺ T cells in the liver of AML-bearing mice displayed impaired function. B6 mice were injected intravenously with 10⁶ C1498FFDsR cells and killed 14, 20, or 25 days after tumor injection. Flow cytometric analysis was performed on liver leukocytes and splenocytes (A-C). (A) Flow dot plot of PD-1 expression on liver CD8⁺ T cells 25 days after AML injection. (B) PD-1 was up-regulated on liver CD8⁺ T cells at late phase of AML progression. (C) PD-1 expression was not found on the spleen CD8⁺ T cells of AML-bearing mice. (D) PD-L1 expression on C1498FFDsR left untreated or treated with IFN-γ for 48 hours was assessed by flow cytometric analysis. PD-L1 expression was found on C1498FFDsR, and mean fluorescent intensity was increased by IFN-γ treatment. (E) Percentage of IFN-γ–producing PD-1⁺/PD-1⁻ CD8⁺ T cells was determined by flow cytometry analysis. Percentage of IFN-γ–secreting cells. PD-1⁺ fraction was much lower than the PD-1⁻ fraction. (F) Liver samples from naive mice (left side) or AML-bearing mice 25 days after C1498FFDsR injection (right side) were evaluated by immunofluorescence staining. Slides were mounted with VECTASHIELD (Vector Laboratories) and images were taken at 40×/1.30 oil objective through Olympus UPlanApo oil lens and an Olympus FV500 camera, compiled with Fluoview software (Version 4.3), then cropped in Adobe Photoshop CS3. (Top panels) Colocalization of tumor cells (DsR⁺, blue), CD8⁺ T cells (CD8⁺, green), and Tregs (Foxp3⁺, red) is designated by white arrows. (Bottom panels) Colocalization of tumor cells, CD8⁺ T cells, and PD-1 (red) is designated by white arrows. Results from one of 5 representative experiments are shown. Bar graphs represent mean ± SD.