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Fig. 2 | Journal for ImmunoTherapy of Cancer

Fig. 2

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

Fig. 2

| CAI stimulation of the IDO-Kyn metabolic circuitry and the effects of the metabolite Kyn on CD8+ T cells. After CAI treatment (10 μM, 48 h) (a), the production of Kyn in the B16/T cell coculture system (left) and B16 tumor tissues (right) were determined. b and c The mRNA and protein expression of IDO1 determined by RT-PCR and Western blotting. d CTLs were treated with 200 mM Kyn for 2 days. The transfer of AhR from the cytosol to the nucleus determined by immunostaining assay. Bar, 2 μm. e ChIP-qPCR analysis of AhR-dependent PD-1 expression after Kyn treatment. The ChIP enrichment ratio relative to the control is shown. f CTLs were incubated with vehicle (DMSO), Kyn (200 mM) or DMF (20 μM) alone or a combination of Kyn and DMF for the indicated time spans, and the PD-1+ CD8+ T cells were analyzed by flow cytometry. Representative histograms (left) and the overall results (right) are shown. g B16 tumor-bearing mice received an intratumoral injection of Kyn with or without DMF treatment (10 mg/kg). Tumor-infiltrating lymphocytes (TILs) were then isolated from the tumor tissues, and the PD-1+ CD8+ T cells were analyzed by flow cytometry. Representative histogram (left) and the statistical histogram (right) are shown. h Intratumoral injection of Kyn reduced the proportion of IFN-γ-positive T cells in TILs isolated from B16 tumor tissues, and DMF treatment (10 mg/kg) rescued this inhibition. Representative histograms (left) and the statistical histograms (right) are shown. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA (a, g, f and h) or Student’s t test (b and e)

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