Molecular heterogeneity and clonal origin of CCR8⁺ effector regulatory T cells in human cancer

CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) are highly activated in tumors and promote disease progression. Specific, universal targeting of these effector Treg cells is limited by the lack of a conserved signature across human cancers and information on their origin. Here we combine analysis of single-cell RNA-sequencing datasets with spectral flow cytometry and identify a core signature of 88 genes consistently upregulated in intratumoral Treg cells among 9 epithelial cancers. We describe 4 Treg cell subsets - CCR7⁺ quiescent, CCR8⁺ effector, CD161⁺ and intermediate, with distinct tissue distribution, function, differentiation trajectories and molecular drivers. By single-cell T cell receptor sequencing, we observe that protumoral, effector CCR8⁺ Treg cells exhibit little clonal relationship with other Treg cell subsets inside tumors, but are clonally related to Treg cells in tumor-draining lymph nodes, as well as conventional T cells in tumors. This resource provides insights for development and fine-tuning of CCR8⁺ Treg cell-targeting therapies in cancer.