Human peripheral and tissue Foxp3-high regulatory T cells in patients with hepatocellular carcinoma [CITE-seq]

CD4⁺ regulatory T cells (Tregs) are pivotal in hepatocellular carcinoma (HCC) progression, but systemic depletion risks autoimmunity. Using single-cell CITE-seq on tumor, non-tumor, and peripheral blood samples, we identified a peripheral Foxp3^high Treg subset that preferentially migrates to tumors via the CCR5–CCL5 axis, where they acquire a terminally differentiated, activated phenotype with elevated LAYN and TRM signatures, and further enhance Foxp3 expression and immunosuppressive activity in response to pro-inflammatory cytokines. The proportion of Foxp3^high Tregs in peripheral blood strongly correlated with their tumor infiltration, and a >3.5% Foxp3^high Tregs/CD4⁺ T cells ratio predicted poor survival and early recurrence (AUROC >0.75). These findings establish peripheral Foxp3^high Tregs as both key mediators of immunosuppression and a promising non-invasive biomarker for HCC prognosis.