T regulatory (Treg) cells help maintain immunologic tolerance and control inflammation in many contexts. Their
dysfunction leads to multi-organ autoimmunity in FoxP3-deficient mice and human IPEX patients, with variable
clinical manifestations. Treg function and homeostasis are dependent on the transcription factor FoxP3,
encoded on ChrX, which determines a substantial portion of their specific transcriptome. Our prior work on
FoxP3 structure/function explored how FoxP3’s interactions with different cofactors within multimolecular
complexes that reside in different nuclear compartments, and condition its transactivating potential on different
transcriptional targets. Further, single-cell cytometry and transcriptomics of T cells from FOXP3-deficient
patients and mice revealed a narrow cell-intrinsic signature of the deficiency, directly controlled by FoxP3, and
a larger disease signature, conferred in a cell-extrinsic manner, that affects both Treg and conventional CD4+
T cells. We hypothesize that these components vary according to the actual FOXP3 lesion, mutations in
different interaction facets leading to different pathology, and that environmental and activation triggers
promote the unfolding of the full cell-extrinsic IPEX signature. In Aim1, to better understand the components of
Treg dysfunction in IPEX patients, we will analyze the impact of missense FOXP3 mutations by single-cell
RNAseq in CD4+ T cells from paired IPEX patients and carrier female relatives (typically mothers), in which we
distinguish Treg-like cells expressing wild-type or mutant FOXP3 through random ChrX-inactivation and SNP-
based cell identification. Effects on chromatin will also be mapped (ATACseq), and related back to
symptomatology in these patients. To enable further analysis in a genetically and environmentally controlled
environment, where sample access is not limiting, Aim2 uses CRISPR editing to introduce eight selected
missense FOXP3 mutations from IPEX patients into B6 mice, where lymphoid and tissue-resident Tregs will be
examined and profiled. Super-resolution microscopy and HiChIPseq will assess the effect of the FOXP3
mutations on localization of the mutant proteins in the nucleus, in terms of nuclear structure and enhancer-
promoter loops. We will also analyze the timing and determinants of acquisition of the dominant Treg-extrinsic
signature. Aim3 will use this FoxP3 mutant mouse panel to ask how mutation-specific transcriptional features
relate to the IPEX-relevant clinical characteristics in mice: appearance of spontaneous autoimmunity (scurfy-
like disease), susceptibility to induced autoimmunity (colitis, dermatitis). Challenges with defined microbes and
microbial molecules will test the hypothesis that clinical variability in IPEX results, at least in part, from
environmental and infectious triggers. These interconnected Aims, with complementary explorations on mice
and humans, will bring unique information on how variation in a transcription factor modulates its ability to
influence gene expression through chromatin and genomic 3D architecture, and how these relate to a
monogenic loss of tolerance in human patients.