Intratumoral stem-like CCR4+ regulatory T cells orchestrate the immunosuppressive microenvironment in HCC associated with hepatitis B

ABSTRACT Background & Aims Regulatory T (Treg) cell depletion increases antitumor immunity. However, severe autoimmunity can occur following systemic Treg cell loss, which could be avoided by selectively depleting intratumoral Treg cells (Tregs). Here, we aimed to investigate the tumor-infiltrating CCR4+ Tregs and provide a potential target strategy for immunotherapy in hepatocellular carcinoma (HCC). Methods CCR4+ Tregs were analyzed by flow cytometry in murine models and clinical samples. Tumor-infiltrating and induced CCR4+ Treg function were interrogated by genetic and epigenetic approaches. To block CCR4+ Tregs chemotaxis, we developed a N-terminus recombinant protein of CCR4 (N-CCR4-Fc) as a neutralizing pseudo-receptor that effectively bounds to its ligand CCL22. Efficacy of CCR4 antagonism as an immunotherapeutic agent was evaluated by tumor weights, growth kinetics and survival curves. Results CCR4+ Tregs were the predominant type of Tregs recruited in the hepatitis B-associated HCC (HBV+ HCC), correlating with sorafenib resistance and HBV load titers. Compared with CCR4- Tregs, CCR4+ Tregs highly expressed IL-10 and IL-35, and enhanced functionality in suppressing the CD8+ T cells. Particularly, CCR4+ Tregs displayed PD-1+TCF1+ stem-like properties. ATAC-seq data revealed substantial chromatin remodeling between tumor-infiltrating Tregs (TIL-Tregs) and induced Tregs (iTregs), suggesting that long-term chromatin reprogramming accounted for CCR4+ TIL-Tregs acquisition of enhanced immunosuppressive stem-like specificity. Treatment with CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade therapy. Conclusions Intratumoral stem-like CCR4+ Tregs orchestrated resource cells of immunosuppression in tumor microenviroment (TME). CCR4 could be a potential precision target for enhancing the antitumor immunity by specifically blocking infiltration of Tregs into the TME and inhibiting TIL-Treg pool maintenance. Lay summary Severe autoimmunity can occur following systemic Treg loss, which could be avoided by selectively depleting intratumoral Tregs. Herein, we showed that intratumoral stem-like CCR4+ Tregs orchestrated resource cells of immunosuppression in the TME. Treatment with CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade therapy, indicative of a potential target strategy for immunotherapy in HCC.