Radiotherapy Reprograms CCR8⁺ Regulatory T Cells via REL-NF-κB to Enforce Spatial Immune Exclusion in Hepatocellular Carcinoma.

Radiotherapy is increasingly incorporated into the treatment of hepatocellular carcinoma (HCC), but adaptive immunosuppressive remodeling of the tumor microenvironment can limit efficacy. We integrated public bulk, single-cell, and spatial transcriptomic cohorts with a murine single-cell atlas, paired human radiotherapy specimens, and functional murine studies. High intratumoral regulatory T cell (Treg) infiltration was associated with poor prognosis across two cohorts. Analyses of single cells identified a CCR8⁺ Treg state that was enriched in advanced tumors, exhibited enhanced suppressive features, and was conserved across species. Spatial analyses showed that CCR8⁺ Tregs accumulated in peritumoral regions enriched in myeloid cells and fibrotic stroma, where CD8⁺ T cell infiltration was limited. Hypofractionated radiotherapy expanded suppressive CCR8⁺ Tregs, while radiation enhanced CCL1 secretion by M2-like tumor-associated macrophages (TAMs), providing a recruitment signal. Mechanistically, REL knockdown, chromatin immunoprecipitation, and promoter reporter assays demonstrated that radiation activates REL, which directly binds to and transactivates the CCR8 promoter. CCR8 blockade enhanced radiotherapy efficacy, reduced suppressive Treg features, and increased cytotoxic CD8⁺ T cell activity. These findings identify a REL-NF-κB-CCR8 axis as a mediator of immune adaptation following radiotherapy and support combination therapies targeting CCR8 in HCC.