Foxp3 M370I mutation allows the activation of T effector programs in regulatory T cells 3759

Description The transcription factor Foxp3 in mice dictates the development and function of regulatory T cells (Tregs) to maintain self-tolerance. The mutation of amino acid 370 (M to I) of FOXP3 protein was identified in IPEX patients with type-2 autoimmunity. We generated an M370I-knock in (KI) mouse by CRISPR-Cas9. The homozygous female KI (Foxp3M370I/M370I) mice and the hemizygous male KI (Foxp3M370I/Y) mice showed T cell activation with multi-organ inflammation. The KI Tregs were capable to produce effector T cell cytokines (IL-2, IFN-γ, IL-4, and IL-17A). We also generated heterozygous KI female (Foxp3M370I/GFP) mice which were healthy in general without type-2 inflammation. However, the KI Tregs still possessed the ability of producing IL-2/IFN-γ. IP-MS of Foxp3 showed an altered pattern of Foxp3 interacting proteins between WT and KI Tregs, including NFAT and RUNX1. Structural modelling indicated that M370I may disrupt head-to-head dimerization of FOXP3. While another amino acid substitution F374C that sits in the center of H4 helix, may form an intra-domain disulfide to favor swap-domain dimer. We further generated mice carrying FOXP3-F374C mutation and these mice indeed had similar phenotypes. Thus, our results indicate that M370I and F374C KI Tregs displayed effector-phenotypes, possibly resulting from enhanced formation of domain-swap FOXP3 dimerization. This work was supported by the Division of Intramural Research of NIAID, NIH.