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Dissection of the trans-endocytosis and signal inhibition functions of CTLA-4 through the study of a disease-associated Y139C mutation

Cellular & Molecular Immunology volume 22, pages 1506–1518 (2025)Cite this article

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Abstract

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) plays a crucial role in maintaining peripheral immune tolerance, but its mechanisms of action are highly complicated. Here, through the generation of a gene knock-in (KI) mouse carrying a CTLA-4 Y139C human patient-derived pathogenic mutation, we phenocopied the lethal autoimmune diseases in Ctla4 KO mice due to the impairment of Treg functions. Interestingly, although both KO and KI Treg cells lost the ability to endocytose B7 molecules, the KO and KI mice differed in terms of T-cell proliferation since the KI mutation retained its ability to transmit inhibitory signals. Therefore, this study not only dissected the two distinct immunoregulatory mechanisms of CTLA-4 but also provided genetic evidence highlighting the importance of ligand trans-endocytosis in the function of CTLA-4. Our findings enhance our understanding of CTLA-4 function and CTLA-4 insufficiency disease, providing valuable insights for advancing improved immunotherapy strategies targeting CTLA-4.

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Data availability

The data will be made available upon request. All sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject accession numbers PRJNA1309579 (for Treg Bulk-seq) and PRJNA 1309745 (for scRNA-seq).

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Acknowledgements

We thank Yang Yao from the group and Yanwei Li, Jiajia Wang, and Yingying Huang from the core facilities (Zhejiang University School of Medicine) for their technical assistance in histology analysis and cell sorting. We thank Zejin Cui from the Shanghai Immune Therapy Institute for assisting with the FACS analysis. This work was supported by National Natural Science Foundation of China Grants 31930038, U21A20199, 32141004, and 32350007 (to LL); an innovative research team of high-level local universities in Shanghai SHSMU-ZLCX 20211600 (to LL); the Provincial Natural Science Foundation of Zhejiang Province LQ22H030012; and China Postdoctoral Science Foundation 2022M720087 (to KXZ).

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  1. These authors contributed equally: Zhishan Zhao, Peng Wu, Qingman Li.

Authors and Affiliations

  1. Institute of Immunology and Department of Rheumatology in Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Zhishan Zhao, Qingman Li, Lize Wu, Fan Zhao, Ziwei Chen, Pengkun Yuan, Kaixiang Zhu, Xuexiao Jin, Pengfei Wang & Linrong Lu

  2. Shanghai Immune Therapy Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zhishan Zhao, Di Sun, Kaixiang Zhu & Linrong Lu

  3. Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China

    Zhishan Zhao & Linrong Lu

  4. Department of Cell Biology, School of Basic Medical Science, Department of Cardiology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Peng Wu, Chenyi An & Wei Chen

  5. Department of Rheumatology Immunology and Allergy, Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China

    Yiping Xu & Meiping Lu

  6. Metabolic Medicine Center, International Institutes of Medicine, the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, China

    Dandan Wu & Dante Neculai

  7. Liangzhu Laboratory, Zhejiang University, Hangzhou, China

    Wei Chen & Linrong Lu

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Contributions

ZZ designed, performed, and interpreted the in vitro and in vivo experiments, analyzed the data, and wrote the manuscript. PWu developed and performed the BFP experiments and analyzed the data. QL participated in the design and execution of a segment of in vivo experiments. DS and LW assisted in the RNA-seq and scRNA-seq data analysis. YX assisted in human PBMC sample collection and processing. FZ, DW, ZC, PY, KZ, XJ, PWang, and CA assisted in the cellular experiments. DN and WC assisted in the experimental design and manuscript preparation. ML initiated the project and led the clinical analysis. LL conceptually designed and interpreted the experimental work and prepared the manuscript.

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Correspondence to Meiping Lu or Linrong Lu.

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The authors declare no competing interests. L.L. is an editorial board member of Cellular & Molecular Immunology, but he has not been involved in the peer review or the decision-making of the article.

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Zhao, Z., Wu, P., Li, Q. et al. Dissection of the trans-endocytosis and signal inhibition functions of CTLA-4 through the study of a disease-associated Y139C mutation. Cell Mol Immunol 22, 1506–1518 (2025). https://doi.org/10.1038/s41423-025-01348-w

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