Abstract 5243: Peripheral Foxp3high regulatory T cells develop into tumor-associated counterparts by proinflammatory cytokines and act as prognostic biomarker in patients with hepatoma

CD4+ regulatory T cells (Tregs) play a critical role in the progression of hepatocellular carcinoma (HCC). However, indiscriminate depletion of Tregs risks triggering autoimmunity. While previous research highlights the phenotypic and functional heterogeneity of Treg subpopulations, a unified classification system remains elusive. This study aims to characterize Treg subtypes in HCC patients using single-cell CITE-seq and validate their clinical relevance and prognostic potential. CITE-seq profiling of 51,067 CD4+ T cells from eight HCC patients identified distinct Treg populations in tumor, non-tumor, and peripheral blood samples. Validation studies conducted on 96 HCC patients and 53 healthy donors using flow cytometry, functional assays, and clinical data revealed a peripheral Foxp3high Treg subset that preferentially migrates to tumor sites. Upon infiltration, these Tregs undergo terminal differentiation, exhibiting heightened activation, immunosuppressive functions, and increased expression of LAYN and TRM-associated genes. Proinflammatory cytokines within the tumor microenvironment further amplify Foxp3 expression and the suppressive capacities of these cells. Mechanistic analysis implicated the CCL5/CCR5 signaling axis as essential for the recruitment of Foxp3high Tregs from peripheral blood to the tumor microenvironment. Peripheral Foxp3high Tregs strongly correlated with their tumor-infiltrating counterparts, suggesting their potential as a non-invasive biomarker. A threshold of peripheral Foxp3high Tregs/CD4+ T cells > 3.5% was found to predict overall survival and early recurrence, achieving AUROCs exceeding 0.75. In conclusion, peripheral Foxp3high Tregs transition into tumor-associated counterparts via the CCR5-CCL5 axis and proinflammatory cytokines, contributing to immunosuppression in HCC. Their abundance in peripheral blood correlates with tumor infiltration and clinical outcomes, underscoring their utility as a prognostic biomarker for HCC. Citation Format: Wei-Ting Ku, Chien-Hao Huang, Jayashri Mahalingam, Cheng-Heng Wu, Tsung-Han Wu, Jian-He Fan, Chung-Wei Su, Po-Ting Lin, Chien-Wei Peng, Wei Teng, Wen-Juei Jeng, Wen-Juei Jeng, Wei-Ting Chen, Chen-Chun Lin, Shi-Ming Lin, I-Shyan Sheen, Yung-Chang Lin, Chun-Yen Lin. Peripheral Foxp3 regulatory T cells develop into tumor-associated counterparts by proinflammatory cytokines and act as prognostic biomarker in patients with hepatoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5243.