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Background <p>Insufficient T cell infiltration into solid tumors represents a major barrier to effective immunotherapy, particularly in the context of CAR-T therapy. Identifying key molecules capable of promoting T cell migration to tumor sites is therefore critical. GPR183 (EBI2), a receptor for oxidized sterols, has not yet been fully characterized in terms of its role in antitumor T cell immunity or its potential for application in CAR-T cell engineering.</p> Methods <p>Public single-cell transcriptomic data from breast cancer and ovarian cancer were analyzed to identify distinct expression patterns of GPR183 across T cell subsets, which were validated using flow cytometry. Functional assays, including in vitro Transwell migration experiments, as well as GPR183 knockout and overexpression models, demonstrated that GPR183 regulates T cell migration. To investigate the underlying molecular mechanisms, we employed ligand stimulation, co-culture systems, and transcriptome sequencing. Furthermore, we generated HER2-targeted CAR-T cell models with either GPR183 knockdown or overexpression, and systematically evaluated the impact of GPR183 on CAR-T cell function through in vitro cytotoxicity assays, IFN-γ secretion measurements, and in vivo tumor xenograft models in mice.</p> Results <p>Single-cell analysis revealed that GPR183<sup>high</sup> T cells exhibit a central memory phenotype and are enriched in migration-related signaling pathways. Functional experiments confirmed that GPR183 acts as a key positive regulator of T cell migration. Mechanistically, direct contact between T cells and tumor cells induced upregulation of CH25H and CYP7B1 in tumor cells, leading to increased production of 7α,25-OHC, which activated GPR183 and further enhanced its expression, establishing a positive feedback loop. In the HER2-CAR-T model, GPR183 overexpression significantly enhanced tumor infiltration, IFN-γ secretion, and tumor cell killing, and resulted in an additively improved antitumor efficacy in vivo.</p> Conclusion <p>This study reveals, for the first time, that the GPR183-mediated positive feedback loop is a critical novel mechanism governing T cell tumor infiltration. Enhancing GPR183 expression through genetic engineering represents a promising strategy that significantly improves the migratory capacity and antitumor functionality of CAR-T cells, thereby providing a new therapeutic target and theoretical foundation for overcoming current limitations in the treatment of solid tumors.</p>

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GPR183 potentiates CAR-T cell infiltration and antitumor immunity through a positive feedback loop involving the oxysterol 7α,25-OHC

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月17日

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  • Rui Guo,
  • Chang Guo,
  • Sen Qin,
  • Zhiwen Lin,
  • Jinlian Tong,
  • Yiran Wang,
  • Zixuan Zhao,
  • Wen Zuo,
  • Qing Gao,
  • Qin Tan,
  • Jie Ma

摘要

Background

Insufficient T cell infiltration into solid tumors represents a major barrier to effective immunotherapy, particularly in the context of CAR-T therapy. Identifying key molecules capable of promoting T cell migration to tumor sites is therefore critical. GPR183 (EBI2), a receptor for oxidized sterols, has not yet been fully characterized in terms of its role in antitumor T cell immunity or its potential for application in CAR-T cell engineering.

Methods

Public single-cell transcriptomic data from breast cancer and ovarian cancer were analyzed to identify distinct expression patterns of GPR183 across T cell subsets, which were validated using flow cytometry. Functional assays, including in vitro Transwell migration experiments, as well as GPR183 knockout and overexpression models, demonstrated that GPR183 regulates T cell migration. To investigate the underlying molecular mechanisms, we employed ligand stimulation, co-culture systems, and transcriptome sequencing. Furthermore, we generated HER2-targeted CAR-T cell models with either GPR183 knockdown or overexpression, and systematically evaluated the impact of GPR183 on CAR-T cell function through in vitro cytotoxicity assays, IFN-γ secretion measurements, and in vivo tumor xenograft models in mice.

Results

Single-cell analysis revealed that GPR183high T cells exhibit a central memory phenotype and are enriched in migration-related signaling pathways. Functional experiments confirmed that GPR183 acts as a key positive regulator of T cell migration. Mechanistically, direct contact between T cells and tumor cells induced upregulation of CH25H and CYP7B1 in tumor cells, leading to increased production of 7α,25-OHC, which activated GPR183 and further enhanced its expression, establishing a positive feedback loop. In the HER2-CAR-T model, GPR183 overexpression significantly enhanced tumor infiltration, IFN-γ secretion, and tumor cell killing, and resulted in an additively improved antitumor efficacy in vivo.

Conclusion

This study reveals, for the first time, that the GPR183-mediated positive feedback loop is a critical novel mechanism governing T cell tumor infiltration. Enhancing GPR183 expression through genetic engineering represents a promising strategy that significantly improves the migratory capacity and antitumor functionality of CAR-T cells, thereby providing a new therapeutic target and theoretical foundation for overcoming current limitations in the treatment of solid tumors.