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.

Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development.

Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25+CD4+ regulatory T cell (T(R)) development and function in mice. In humans, its role in mediating T(R) development has been controversial. Furthermore, the fate of T(R) precursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human T(R) development. Unlike murine Foxp3- T cells, a small subset of human CD4+ and CD8+ T cells transiently up-regulated FOXP3 upon in vitro stimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, only ex vivo FOXP3+ T(R) cells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a T(r) developmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or without FOXP3 mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing a FOXP3 mutation enabling expression of stable FOXP3(mut) protein exhibited FOXP3(mut)-expressing cells among a subset of highly activated CD4+ T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from T(R) precursors.