Deconvolution of regulatory T cell heterogeneity identifies cycling precursors as conductors of immune escape during breast carcinoma progression

Tumor-immune microenvironment is instrumental in shaping breast cancer tumor evolution. We previously identified an evolutionary bottleneck demarcating ductal carcinoma in situ (DCIS) from invasive ductal carcinoma (IDC). How the microenvironment evolves during such transition remains elusive. We dissected, in parallel, single-cell composition of DCIS and IDC patients and rat tumors that faithfully recapitulate human breast cancer. We discovered T cell diversity distinguishing DCIS and IDC and a novel cycling Treg state occupying the pseudotime bifurcation between DCIS and IDC, preceding more activated Treg subtypes. Multiscale analysis of patient cohorts as part of HTAN Consortium established that cycling Treg predicts recurrence in low-grade DCIS. We proved cycling Treg as Treg progenitors critical for immune escape and demonstrated that OX40 agonism restored immunotherapeutic sensitivity, where responders displayed increased inflammatory CAF subtype heterogeneity. Our study thus established cycling Treg’s importance in preserving immune escape and underscored its prognostic value to stratify future recurrence.