Data availabilityAll data are available in the main text or the supplementary materials. The raw DNA sequencing data of the CRISPR screen have been deposited under SRA PRJNA1183302. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD057240. The scRNA-seq FASTQ files are available at GEO under accession GSE281314. All the materials generated in this study are available upon reasonable request to the lead contact.ReferencesFreeman, G. J. et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192, 1027–1034 (2000).CAS PubMed PubMed Central Google Scholar Pauken, K. E., Torchia, J. A., Chaudhri, A., Sharpe, A. H. & Freeman, G. J. Emerging concepts in PD-1 checkpoint biology. Semin Immunol. 52, 101480 (2021).CAS PubMed PubMed Central Google Scholar Leach, D. R., Krummel, M. F. & Allison, J. 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This work was supported by grants from the National Key R&D Program of China (2022YFC3401500 and 2023YFC3402100 to J.Z.), the National Natural Science Foundation of China (82273062 to J.Z., 82103149 to H.Y., 82503802 to B.C.), the Fundamental Research Funds for the Central Universities (2042022dx0003 to J.Z., 2042021kf0075 to H.Y.), the Postdoctoral Fellowship Program of CPSF (GZC20241276 to W.X., GZC20250976 to C.H.), the China Postdoctoral Science Foundation (2022M722462 to H.L., 2024M762491 to C.H., 2025M772321 to W.X.), the Postdoctoral Project of Hubei Province (2004HBBHJD057 to C.H.), Natural Science Foundation of Wuhan (2024040701010031 to J.Z.), the Open Projects of Hubei Key Laboratory of Tumor Biological Behavior (220172107 to J.Z.), the Natural Science Foundation of Hubei Province of China (2022CFA008 to J.Z.), the Open Research Fund of the National Center for Protein Sciences at Peking University in Beijing (KF-202504 to J.Z.), the Natural Science Foundation Youth Category A of Hubei Province (JCZRJQ202500008 to H.Z.), the Natural Science Foundation of Chongqing, China (CSTB2024NSCQ-MSX0066 to H.Z.), the translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University (ZNJC202312 to J.Z.), and USA National Institutes of Health P50 CA101942 (G.J.F.).Author informationAuthor notesThese authors contributed equally: Yishuang Sun, Zijian Zhang, Haiou Li, Xia Bu, Li Chen.Authors and AffiliationsDepartment of Hepatobiliary and Pancreatic Surgery, State Key Laboratory of Metabolism and Regulation in Complex Organisms, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei, ChinaYishuang Sun, Xiyong Wang, Lijun Kong, Wenjing Song, Xiangling Xiao, Jie Shi, Bolin Xiang, Chuan He, Yingmeng Yao, Wenjun Xiong, Haisheng Yu, Hudan Liu, Guoliang Qing, Zhiyong Yang & Jinfang ZhangDepartment of Pathology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, ChinaYishuang Sun, Lifang Fan & Sufang TianTaikang Center for Life and Medical Sciences, Wuhan University, Wuhan, Hubei, ChinaYishuang Sun, Xiyong Wang, Lijun Kong, Xiangling Xiao, Jie Shi, Bolin Xiang, Chuan He, Yingmeng Yao, Wenjun Xiong, Haisheng Yu, Hudan Liu, Guoliang Qing & Jinfang ZhangKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Hainan University, Haikou, Hainan, ChinaZijian ZhangNational “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan, Hubei, ChinaZijian Zhang & Haichuan ZhuDepartment of Dermatology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, ChinaHaiou LiDepartment of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USAXia Bu & Gordon J. FreemanDepartment of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USALi Chen & Wenyi WeiDepartment of Colorectal and Anal Surgery, Low Rectal Cancer Diagnosis and Treatment Center, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, ChinaBaoxiang Chen, Congqing Jiang & Qun QianDepartment of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Provincial Clinical Research Center for Cancer, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, ChinaPanpan Dai & Jinfang ZhangAuthorsYishuang SunView author publicationsSearch author on:PubMed Google ScholarZijian ZhangView author publicationsSearch author on:PubMed Google ScholarHaiou LiView author publicationsSearch author on:PubMed Google ScholarXia BuView author publicationsSearch author on:PubMed Google ScholarLi ChenView author publicationsSearch author on:PubMed Google ScholarXiyong WangView author publicationsSearch author on:PubMed Google ScholarLifang FanView author publicationsSearch author on:PubMed Google ScholarBaoxiang ChenView author publicationsSearch author on:PubMed Google ScholarLijun KongView author publicationsSearch author on:PubMed Google ScholarPanpan DaiView author publicationsSearch author on:PubMed Google ScholarWenjing SongView author publicationsSearch author on:PubMed Google ScholarXiangling XiaoView author publicationsSearch author on:PubMed Google ScholarJie ShiView author publicationsSearch author on:PubMed Google ScholarBolin XiangView author publicationsSearch author on:PubMed Google ScholarChuan HeView author publicationsSearch author on:PubMed Google ScholarYingmeng YaoView author publicationsSearch author on:PubMed Google ScholarWenjun XiongView author publicationsSearch author on:PubMed Google ScholarHaisheng YuView author publicationsSearch author on:PubMed Google ScholarCongqing JiangView author publicationsSearch author on:PubMed Google ScholarQun QianView author publicationsSearch author on:PubMed Google ScholarHudan LiuView author publicationsSearch author on:PubMed Google ScholarSufang TianView author publicationsSearch author on:PubMed Google ScholarGuoliang QingView author publicationsSearch author on:PubMed Google ScholarZhiyong YangView author publicationsSearch author on:PubMed Google ScholarWenyi WeiView author publicationsSearch author on:PubMed Google ScholarGordon J. FreemanView author publicationsSearch author on:PubMed Google ScholarHaichuan ZhuView author publicationsSearch author on:PubMed Google ScholarJinfang ZhangView author publicationsSearch author on:PubMed Google ScholarContributionsY.S. performed most of the experiments with help from Z.Z., Haiou L., X.B., L.C., X.W., L.K., P.D., W.S., X.X., J.S., B.X., C.H., Y.Y., W.X., and H.Y.; J.Z., Y.S., G.J.F., H.Z., and W.W. designed experiments; L.F., B.C., C.J., and Q.Q. collected human colorectal cancer tissues; X.B. and G.J.F. provided essential reagents; J.Z., H.Z., G.J.F., and W.W. guided and supervised the project. J.Z. and Y.S. wrote the manuscript. X.B., Z.Y., G.Q., S.T., Hudan L., W.W., and G.J.F. provided discussion and edited the manuscript. All authors commented on the manuscript.Corresponding authorsCorrespondence to Wenyi Wei, Gordon J. Freeman, Haichuan Zhu or Jinfang Zhang.Ethics declarationsCompeting interestsG.J.F. has patents/royalties on the PD-L1/PD-1 pathway from Bristol-Myers-Squibb, Roche, Eli Lilly, and Novartis. G.J.F. has served on advisory boards for iTeos, NextPoint, IgM, GV20, IOME, Bioentre, Santa Ana Bio, Simcere of America, and Geode. G.J.F. has equity in Nextpoint, Triursus, Xios, iTeos, IgM, Invaria, GV20, Bioentre, and Geode. W.W. is a co-founder and consultant for the ReKindle Therapeutics. The other authors declare no competing interests.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary informationSupplementary information, Fig. S1Supplementary information, Fig. S2Supplementary information, Fig. S3Supplementary information, Fig. S4Supplementary information, Fig. S5Supplementary information, Fig. S6Supplementary information, Fig. S7Supplementary information, Fig. S8Supplementary information, Fig. S9Supplementary Information, Table S1Supplementary Information, Table S2Supplementary Information, Table S3Supplementary Information, Table S4Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article