Foxp3 Processing by Proprotein Convertases and Control of Regulatory T Cell Function

Foxp3 is a 47-kDa transcription factor central to regulatory T cell (Treg) function. The importance of Foxp3+ Tregs in controlling self-reactive T cells and preventing autoimmunity is well established. Our analysis of Foxp3 expression in natural Tregs led to identification of a shorter 41-kDa Foxp3 species in activated Tregs, indicating that Foxp3 may be processed by proteolytic cleavage upon cell activation. Searches of murine and human Foxp3 sequences for potential cleavage sites responsible for the generation of the short Foxp3 species revealed the presence of two RXXR proprotein convertase (PC) motifs, 48RDLR51 and 414RKKR417, located near the N- and C-terminal ends, respectively. We show, using retroviral expression of Foxp3 in CD4+ T cells, that Foxp3 is cleaved at both the N- and C-terminal RXXR sites and that mutagenesis of the RXXR motif prevents cleavage. The cleaved forms of Foxp3 are found in the chromatin fraction but not in nuclear or cytoplasmic extracts. CD4+ T cells expressing Foxp3 species engineered to mimic N-terminally, C-terminally, or N- and C-terminally cleaved Foxp3 forms are functionally distinct, as indicated by differences in expression of key Treg genes, such as interleukin-10 and cytotoxic T-lymphocyte antigen 4 (CTLA-4). In addition, CD4+ cells expressing C-cleaved Foxp3 are superior to those that express WT Foxp3 in preventing experimental colitis. Coexpression of Foxp3 with PC1 or PC7 results in cleavage of the Foxp3 C terminus. The mechanism by which Foxp3 is processed likely extends to other members of the FoxP subfamily, because Foxp1 and Foxp2 also have N-terminal RXXR proteolytic cleavage motifs at similar locations to Foxp3. Our results indicate that the generation of fully functionally competent Tregs is complex and dependent on the generation of multiple forms of Foxp3 that have differing effects on Treg cytokine production and suppressive function.