Abstract
FoxP3+ regulatory T cells (Tregs) are a central element of immunological tolerance. FoxP3 is the key determining transcription factor of the Treg lineage, interacting with numerous cofactors and transcriptional targets to determine the many facets of Treg function. Its absence leads to devastating lymphoproliferation and autoimmunity in scurfy mutant mice and immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. To finely map transcriptionally active regions of the protein, with respect to disease-causing variation, we performed a systematic alanine-scan mutagenesis of FoxP3, assessing mutational impacts on DNA binding and transcriptional activation or repression. The mutations affected transcriptional activation and repression in a variegated manner involving multiple regions of the protein and varying between different transcriptional targets of FoxP3. There appeared to be different modalities for target genes related to classic immunosuppressive function vs. those related to atypical or tissue-Treg functions. Relevance to in vivo Treg biology was established by introducing some of the subtle Foxp3 mutations into the mouse germline by CRISPR-based genome editing. The resulting mice showed Treg populations in normal numbers and exhibited no overt autoimmune manifestations. However, Treg functional defects were revealed upon competition or by system stress, manifest as a strikingly heightened susceptibility to provoked colitis, and conversely by greater resistance to tumors. These observations suggest that some of the missense mutations that segregate in human populations, but do not induce IPEX manifestations, may have unappreciated consequences in other diseases.