Tumor-migrating peripheral Foxp3-high regulatory T cells drive poor prognosis in HCC

Background and Aims: CD4 + regulatory T cells (Tregs) are pivotal in HCC progression. However, systemic depletion of all Tregs risks autoimmunity. Existing subgroup classifications highlight Tregs’ phenotypic and functional heterogeneity but lack coherent consensus. This study aimed to classify Treg subtypes using single-cell CITE-seq, integrating data from tumor, non-tumor, and blood samples in HCC patients. We also validated the clinical relevance and prognostic value of this classification. Approach and Results: CITE-seq analysis was performed on 51,067 CD4 + T cells from 8 HCC patients. Validation involved 96 HCC patients and 53 healthy donors using flow cytometry, functional assays, and clinical data. Trajectory and TCR analyses identified a peripheral Foxp3 high Treg subset that preferentially migrates to tumor sites, acquiring a terminally differentiated, activated phenotype. These tumor-infiltrating Foxp3 high Tregs exhibit elevated LAYN and TRM signatures and further increase their Foxp3 expression and immunosuppressive functions in response to pro-inflammatory cytokines in tumor tissue. The CCL5/CCR5 axis is crucial for recruiting Foxp3 high Tregs from peripheral blood into the tumor microenvironment. A strong correlation was observed between the percentage of Foxp3 high Tregs in peripheral blood and their counterparts in tumor regions, suggesting their potential as peripheral biomarkers. Notably, a peripheral Foxp3 high Tregs/CD4 + T cells percentage >3.5% predicted overall survival and early recurrence, with AUROCs exceeding 0.75. Conclusions: Peripheral Foxp3 high Tregs migrate to tumor sites via the CCR5–CCL5 axis and mature in response to pro-inflammatory cytokines. The proportion of these Tregs in peripheral blood correlates with their presence in tumors, making them a potential biomarker for predicting HCC outcomes.