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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

From: Carboxyamidotriazole combined with IDO1-Kyn-AhR pathway inhibitors profoundly enhances cancer immunotherapy

Fig. 3

| Combining CAI with 1-MT or DMF synergistically disrupts PD-1 expression and promotes IFN-γ production in CD8+ T cells. Sorted CD8+ T cells were activated by CD3/CD28 beads for 48 h and treated with CAI (10 μM), DMF (20 μM) or a combination of CAI and DMF for 24 h. In another experiment, the sorted CD8+ T cells were treated with CAI (10 μM), 1-MT (0.2 mM) or a combination of CAI and 1-MT for 24 h. a-b Flow cytometry plots showing the number of PD-1+ CD8+ T cells after various treatments. Representative histogram plots (left) and the statistical histogram plots (right) are shown. c The percentage of IFN-γ-producing T cells in each group was measured by flow cytometry (n = 3). d and e Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 10 healthy volunteers, and PBMC-derived CD8+ T cells were incubated with the indicated single drug or a combination of drugs for 48 h. The percentages of PD-1-positive cells and IFN-γ-producing cells in each group were measured by flow cytometry. f and g Activated CD8+ T cells from the spleens of C57BL/6 mice were cocultured with B16 cells, or PBMC-derived CD8+ T cells were cocultured with HCT116 cells at a ratio of 20:1 for 24 h. Tumor cell apoptosis was analyzed by flow cytometry. h ChIP-qPCR analysis of AhR-dependent PD-1 expression after various treatments. The ChIP enrichment ratio relative to the control is shown. i CD8+ T cells isolated from mouse spleens were activated with anti-CD3/CD28 beads for 48 h. At the same time, activated T cells were treated with CAI (10 μM), DMF (20 μM), 1-MT (0.2 mM) or a combination of CAI and DMF/1-MT for 24 h. Then, the CD8+ T cells were fixed and strained with an anti-AhR antibody and imaged by confocal microscopy. Bar, 2 μm. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001 by one-way ANOVA (A, B, E-H)

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