The splicing isoform Foxpdelta2 releases the autoinhibitory conformation and differentially regulates tTregs and pTregs homeostasis

Abstract

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 (Foxp3{Delta}2). 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 Foxp3{Delta}2-bearing Tregs in the peripheral lymphoid organ were less sensitive to TCR due to the enhanced binding of Foxp3{Delta}2 to the Batf promoter and were unsusceptible to IL-2. In contrast, among ROR{gamma}t+ Tregs in the large intestine, Foxp3{Delta}2 Tregs expressed much more ROR{gamma}t-related genes, and more strikingly, the deletion of exon 2 of Foxp3 conferred a competitive advantage over WT ROR{gamma}t+ 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. HighlightsO_LIFoxp3{Delta}2 broke the inhibitory loop and generated constitutive DNA-binding activity. C_LIO_LIFoxp3 isoforms differentially regulate tTregs and pTregs homeostasis C_LIO_LIFoxp3{Delta}2-bearing Tregs in the peripheral lymphoid organ were less sensitive to TCR and were unsusceptible to IL-2 C_LIO_LIFoxp3{Delta}2 ROR{gamma}t+ pTregs benefited them for better adapting to the gut environmental conditions C_LI