AbstractThe CD4⁺ regulatory T (Treg) cell lineage, defined by FOXP3 expression, comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. As a model for Treg cells, studies employ TGF-β-induced (i)Treg cells generated from CD4⁺ conventional T (Tconv) cells in vitro. Here, we describe how human iTreg cells relate to human blood-derived tTreg and Tconv cells according to proteomic analysis. Each of these cell populations had a unique protein expression pattern. iTreg cells had very limited overlap in protein expression with tTreg cells, regardless of cell activation status and instead shared signaling and metabolic proteins with Tconv cells. tTreg cells had a uniquely modest response to CD3/CD28-mediated stimulation. As a benchmark, we used a previously defined proteomic signature that discerns ex vivo naïve and effector Treg cells from Tconv cells and includes conserved Treg cell properties. iTreg cells largely lacked this Treg cell core signature and highly expressed e.g. STAT4 and NFATC2, which may contribute to inflammatory responses. We also used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naïve Treg cells. iTreg cells contained part of this effector Treg cell signature, suggesting acquisition of pTreg cell features. In conclusion, iTreg cells are distinct from tTreg cells and share limited features with ex vivo Treg cells at the proteomic level.