To obtain a molecular definition of regulatory T (Treg) cell identity, we performed proteomics and transcriptomics on various populations of human regulatory and conventional CD4 T (Tconv) cells. A protein expression signature was identified that defines all Treg cells, and another signature that defines effector Treg cells. These signatures could not be extrapolated from transcriptome data. Unique cell-biological and metabolic features in Treg cells were defined, as well as specific adaptations in cytokine, TCR, and costimulatory receptor signaling pathways. One such adaptation-selective STAT4 deficiency-prevented destabilization of Treg cell identity and function by inflammatory cytokines, while these signals could still induce critical transcription factors and homing receptors via other pathways. Furthermore, our study revealed surface markers that identify FOXP3CD4 T cells with distinct functional properties. Our findings suggest that adaptation in signaling pathways protect Treg cell identity and present a resource for further research into Treg cell biology.

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FOXP3-expressing regulatory T (Treg) cells safeguard immunological tolerance. Treg cells can be generated during thymic development (called thymic Treg [tTreg] cells) or derived from mature conventional CD4 + T cells that underwent TGF-bmediated conversion in the periphery (called peripheral Treg [pTreg] cells). Murine studies have shown that tTreg cells exhibit strong lineage fidelity, whereas pTreg cells can revert into conventional CD4 + T cells. Their stronger lineage commitment makes tTreg cells the safest cells to use in adoptive cell therapy, increasingly used to treat autoimmune and inflammatory disorders. Markers to distinguish human tTreg cells from pTreg cells have, however, not been found. Based on combined proteomic and transcriptomic approaches, we report that the Ig superfamily protein GPA33 is expressed on a subset of human Treg cells. GPA33 is acquired late during tTreg cell development but is not expressed on TGF-b-induced Treg cells. GPA33 identifies Treg cells in human blood that lack the ability to produce effector cytokines (IL-2, IFN-g, IL-17), regardless of differentiation stage. GPA33 high Treg cells universally express the transcription factor Helios that preferentially marks tTreg cells and can robustly and stably be expanded in vitro even without rapamycin. Expanded GPA33 high Treg cells are suppressive, unable to produce proinflammatory cytokines, and exhibit the epigenetic modifications of the FOXP3 gene enhancer CNS2, necessary for indelible expression of this critical transcription factor. Our findings thus suggest that GPA33 identifies human tTreg cells and provide a strategy to isolate such cells for safer and more efficacious adoptive cell therapy.