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. 2022 Sep 26;13:5644. doi: 10.1038/s41467-022-33285-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a MC38-Vec./Scr., MC38-USP14/Scr., and MC38-USP14/shIDO1 cells were cultured for 24 h with or without KYNU treatment. KYN and TRP levels in cell supernatants were determined by ELISA (n = 3 biological replicates). Vec., vector; Scr., scramble. b MC38-Vec./Scr., MC38-USP14/Scr., and MC38-USP14/shIDO1 cells were injected subcutaneously into C57BL/6 J mice at day 0. HPLC-MS/MS analysis was used to generate the tryptophan metabolomic profiles of mouse plasma at day 21, and the KYN/TRP ratio in the mouse plasma was calculated (n = 3 biological replicates). c Representative immunofluorescence images of the indicated mouse tumors stained with anti-KYN. Scale bars, 100 µm. d Quantification of the proliferation of splenic CD8⁺ T cells. Unstimulated T cells were used as a negative control (n = 3 biological replicates). e IFN-γ secretion by splenic CD8⁺ T cells measured by ELISA (n = 3 biological replicates). f, g The percentage of GzB⁺ cells in CD8⁺ T cells and CD25⁺FOXP3⁺ cells in CD4⁺ T cells isolated from the indicated tumors was measured by flow cytometry and analyzed by FlowJo software. h The percentage of PD-1⁺ cells in CD8⁺ T cells isolated from the indicated tumors was measured by flow cytometry and analyzed by FlowJo software. i, j Representative images and quantification of GzB⁺CD8⁺ cells and FOXP3⁺CD4⁺ cells analyzed by IF staining in tumors (n = 3 biological replicates). The small red boxed areas were amplified images of cells. Multiple GzB⁺CD8⁺ cells and FOXP3⁺CD4⁺ cells were marked with red arrowheads. The far-right images in each panel were close-ups of the boxed region. Scale bars, 50 µm. k The percentage of GzB⁺CD8⁺ T cells and CD4⁺CD25⁺FOXP3⁺ T cells per milligram of the indicated tumors was measured by flow cytometry and analyzed by FlowJo software (n = 3 biological replicates). In a, b, e–h, j, and k, error bars represent the mean ± SD of three independent experiments, two-sided Student’s t-test. GzB Granzyme B. Source data are provided as a Source Data file.