Wiskott-Aldrich syndrome protein is required for regulatory T cell homeostasis.

Wiskott-Aldrich syndrome protein (WASp) is essential for optimal T cell activation. Patients with WAS exhibit both immunodeficiency and a marked susceptibility to systemic autoimmunity. We investigated whether alterations in Treg function might explain these paradoxical observations. While WASp-deficient (WASp(-/-)) mice exhibited normal thymic Treg generation, the competitive fitness of peripheral Tregs was severely compromised. The total percentage of forkhead box P3-positive (Foxp3(+)) Tregs among CD4(+) T cells was reduced, and WASp(-/-) Tregs were rapidly outcompeted by WASp(+) Tregs in vivo. These findings correlated with reduced expression of markers associated with self-antigen-driven peripheral Treg activation and homing to inflamed tissue. Consistent with these findings, WASp(-/-) Tregs showed a reduced ability to control aberrant T cell activation and autoimmune pathology in Foxp3(-/-)Scurfy (sf) mice. Finally, WASp(+) Tregs exhibited a marked selective advantage in vivo in a WAS patient with a spontaneous revertant mutation, indicating that altered Treg fitness likely explains the autoimmune features in human WAS.

Severe food allergy as a variant of IPEX syndrome caused by a deletion in a noncoding region of the FOXP3 gene.

BACKGROUND & AIMS: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX; OMIM 304930) syndrome is a congenital syndrome characterized by autoimmune enteropathy, endocrinopathy, dermatitis, and other autoimmune phenomena. In the present work, we aimed to uncover the molecular basis of a distinct form of IPEX syndrome presenting at the edge of autoimmunity and severe allergy. METHODS: The FOXP3 gene was sequenced, FOXP3 messenger RNA (mRNA) was quantified by real-time polymerase chain reaction (PCR), and protein expression in peripheral blood lymphocytes was analyzed by flow cytometry after intracellular staining. In coculture experiments (CD4(+)CD25(-) and CD4(+)CD25(+) cells), the functions of regulatory T cells were analyzed. Expression of interferon gamma and interleukin 2 and 4 mRNA within the inflamed intestinal mucosa was quantified by real-time PCR. RESULTS: Here, we describe a distinct familial form of IPEX syndrome that combines autoimmune and allergic manifestations including severe enteropathy, food allergies, atopic dermatitis, hyper-IgE, and eosinophilia. We have identified a 1388-base pair deletion (g.del-6247_-4859) of the FOXP3 gene encompassing a portion of an upstream noncoding exon (exon -1) and the adjacent intron (intron -1). This deletion impairs mRNA splicing, resulting in accumulation of unspliced pre-mRNA and alternatively spliced mRNA. This causes low FOXP3 mRNA levels and markedly decreased protein expression in peripheral blood lymphocytes of affected patients. Numbers of CD4(+)CD25(+)FOXP3(+) regulatory T cells are extremely low, and the CD4(+)CD25(+) T cells that are present exhibit little regulatory function. CONCLUSIONS: A new mutation within an upstream noncoding region of FOXP3 results in a variant of IPEX syndrome associating autoimmune and severe immunoallergic symptoms.

Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor.

Foxp3 has been shown to be both necessary and sufficient for the development and function of naturally arising CD4+ CD25+ regulatory T cells in mice. Mutation of Foxp3 in Scurfy mice and FOXP3 in humans with IPEX results in fatal, early onset autoimmune disease and demonstrates the critical role of FOXP3 in maintaining immune homeostasis. The FOXP3 protein encodes several functional domains, including a C2H2 zinc finger, a leucine zipper, and a winged-helix/forkhead (FKH) domain. We have shown previously that FOXP3 functions as a transcriptional repressor and inhibits activation-induced IL-2 gene transcription. To characterize the role of each predicted functional domain on the in vivo activity of FOXP3, we have evaluated the location of point mutations identified in a large cohort of patients with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) and found them to cluster primarily within the FKH domain and the leucine zipper, but also present within the poorly defined N-terminal portion of the protein. The molecular functions of each of the IPEX-targeted domains were investigated. We show that FOXP3 is constitutively localized to the nucleus and this localization requires sequences at both the amino and C-terminal ends of its FKH domain. Moreover, FOXP3 was found to homodimerize through its leucine zipper. We also identify a novel functional domain within the N-terminal half of FOXP3, which is required for FOXP3-mediated repression of transcription from both a constitutively active and a NF-AT-inducible promoter. Furthermore, we demonstrate that IPEX mutations in these domains correlate with deficiencies in FOXP3 repressor function, corroborating their in vivo relevance.