Deciphering the mechanisms by which FoxP3 regulates treg identity : an analysis of naturally occurring IPEX mutations

Overview & Synopsis

The transcription factor (TF) FoxP3 plays a crucial role in the immune system by directing the development and function of regulatory T cells (Tregs). These cells maintain immune homeostasis and prevent autoimmunity by suppressing aberrant immune responses. Mutations in FoxP3 can disrupt Treg function, leading to the fatal multisystemic autoimmune disorder IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked). The phenotype is especially severe when the FoxP3 DNA-binding domain (FKHD) is disturbed. However, the exact mechanisms by which FoxP3 maintains Treg identity remain elusive. Significant inter-individual variability is observed in the disease manifestation, even among family members carrying the same mutation. This discrepancy between genotype-and phenotype adds a level of complexity to our understanding of FoxP3's operation. With this in mind, our study aims to unravel the structure-function relationship of FoxP3, by characterizing the molecular effects of naturally occurring FoxP3 mutations associated with IPEX, both in humans and under controlled conditions in mice. First, the flow cytometric and transcriptomic profiles of blood CD4+ T cells from a cohort of 12 IPEX patients, were analyzed at both the population and the single-cell levels. A strong monomorphic signature affecting the whole CD4+ compartment complicated the examination of mutation-specific transcriptional effects in Treg-like cells. This signature appeared to be cell-extrinsic, disappearing in CD4+ from two heterozygous mothers of IPEX patients, where normal Treg cells exerted dominant suppression. Then, as a second strategy, 6 missense mutations from this cohort, spread across the FoxP3 protein, were engineered into B6 mice. This approach allowed us to study these mutations under both the hemizygous (male) and the heterozygous (female) conditions, with a significant number of replicates, and without genetic and environmental confounders. Two classes of mutations emerged from the combined immunologic and genomic analyses. A mutation in the FKHD, R337Q, showed a similar lymphoproliferation and multi-organ infiltration in hemizygous males as complete FoxP3 knockouts (KO), but delayed by months. A deeper structural analyze of the FKHD mutation, R337Q, showed a specific disturbance of FoxP3 dimerization. Conversely, mutations in other domains didn't show clear phenotype at steady state, but immunological challenges or intercrossing with NOD alleles revealed mutation-specific diseases (dermatitis, colitis, diabetes). Our analyses of transcriptomics and chromatin accessibility in a heterozygous setting indicated that these mutations had subtle but distinct intrinsic impacts on the FoxP3-dependent signature and on the chromatin accessibility, in comparison to the strong effect of the FKHD mutation. This suggests that different tissue-specific cofactors might be involved in the function of each mutated version of FoxP3. Finally, using a complete FoxP3 KO heterozygous mouse model, we analyzed single-cell chromatin accessibility in both WT Tregs and Treg-like cells, to understand the overall impact of FoxP3 on Treg TF network. We found that FoxP3 influences Treg chromatin programs through both repressive and activating roles. Interestingly, a subset of Tregs, the Rorg+ Tregs, can function independently of FoxP3. Overall, our research proposes that disease pathogenesis due to specific FoxP3 domain disturbances primarily stems from the mutation's position, illustrated by the fact that mutation directly impacting the DNA-binding domain is sufficient to induce disease. However, non-FKHD mutations, likely interfering with FoxP3-cofactor interactions, also require a combination of genetic and environmental factors to clinically manifest. This provides a clear explanation for the clinical heterogeneity observed in IPEX.