The splicing isoform Foxp3Δ2 releases the autoinhibitory conformation and differentially regulates tTregs and pTregs homeostasis

Foxp3 is the master transcription factor for the development and function of regulatory T cells (Tregs). So far, little is known about whether the conformation change in Foxp3 could impact the Tregs biology. Alternative splicing of human Foxp3 results in the expression of two major isoforms: the full-length protein or an exon 2-deleted protein (Foxp3Δ2). Here, AlphaFold2 structure predictions and in vitro experiments demonstrated 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 generated exon 2 deficient mice and found Foxp3Δ2-bearing Tregs in the peripheral lymphoid organ were less sensitive to TCR due to the enhanced binding of Foxp3Δ2 to the Batf promoter and were unsusceptible to IL-2. In contrast, among RORγt+ Tregs in the large intestine, Foxp3Δ2 Tregs expressed much more RORγt-related genes, and more strikingly, the deletion of exon 2 of Foxp3 conferred a competitive advantage over WT RORγt+ Tregs. Together, our results reveal that alternative splicing of exon 2 generates a constitutively active form of Foxp3, which plays a differential role in regulating tTregs and pTregs homeostasis. Highlights 1. Foxp3Δ2 broke the inhibitory loop and generated constitutive DNA-binding activity. 2. Foxp3 isoforms differentially regulate tTregs and pTregs homeostasis 3. Foxp3Δ2-bearing Tregs in the peripheral lymphoid organ were less sensitive to TCR and were unsusceptible to IL-2 4. Foxp3Δ2 RORγt+ pTregs benefited them for better adapting to the gut environmental conditions ### Competing Interest Statement The authors have declared no competing interest.