Insulin signaling establishes a developmental trajectory of adipose regulatory T cells

Systematic characterizations of adipose regulatory T (T_reg) cell subsets and their phenotypes remain uncommon. Using single-cell ATAC-sequencing and paired single-cell RNA and T cell receptor (TCR) sequencing to map mouse adipose T_reg cells, we identified CD73^hiST2^lo and CD73^loST2^hi subsets with distinct clonal expansion patterns. Analysis of TCR-sharing data implied a state transition between CD73^hiST2^lo and CD73^loST2^hi subsets. Mechanistically, we revealed that insulin signaling occurs through a HIF-1α-Med23-PPAR-γ axis to drive the transition of CD73^hiST2^lo into a CD73^loST2^hi adipose T_reg cell subset. T_reg cells deficient in insulin receptor, HIF-1α or Med23 have decreased PPAR-γ expression that in turn promotes accumulation of CD73^hiST2^lo adipose T_reg cells and physiological adenosine production to activate beige fat biogenesis. We therefore unveiled a developmental trajectory of adipose T_reg cells and its dependence on insulin signaling. Our findings have implications for understanding the dynamics of adipose T_reg cell subsets in aged and obese contexts.