The splicing isoform Foxp3A2 differentially regulates tTreg and pTreg homeostasis

Foxp3 is the master transcription factor for regulatory T cells (Tregs). Alternative splicing of human Foxp3 re-sults in the expression of two isoforms: the full length and an exon 2-deleted protein. Here, AlphaFold2 pre-dictions and in vitro experiments demonstrate that the N-terminal domain of Foxp3 inhibits DNA binding by moving toward the C terminus and that this movement is mediated by exon 2. Consequently, we find that Foxp3A2-bearing thymus-derived Tregs (tTregs) in the peripheral lymphoid organ are less sensitive to T cell receptor (TCR) stimulation due to the enhanced binding of Foxp3A2 to the Batf promoter and are hy-poresponsive to interleukin-2 (IL-2). In contrast, among RORyt+ peripherally induced Tregs (pTregs) in the large intestine, Foxp3A2 pTregs express many more RORyt-related genes, conferring a competitive advan-tage. Together, our results reveal that alternative splicing of exon 2 generates an active form of Foxp3, which plays a differential role in regulating tTreg and pTreg homeostasis.