Wild-type FOXP3 is selectively active in CD4+CD25hi regulatory T cells of healthy female carriers of different FOXP3 mutations

Abstract

Forkhead box P3 (FOXP3) is constitutively expressed by CD4 ؉ CD25 hi regulatory T cells (nTregs). IntroductionNaturally occurring CD4 ϩ CD25 hi regulatory T cells (nTregs) play a central role in immune tolerance. nTregs are generated in the thymus, comprise 5% to 10% of peripheral CD4 ϩ T cells, and constitutively express forkhead box P3 (FOXP3), a transcription factor essential for their regulatory activity. 1 Indeed, knockdown of FOXP3 in nTregs reduces suppressive capacity and overexpression of FOXP3 confers suppressive activity to conventional CD4 ϩ T cells. [2][3][4] However, an essential role of FOXP3 in the development and maintenance of nTregs in humans has not yet been shown. Moreover, FOXP3 can be expressed in activated effector T cells, suggesting that it may also be important for cells other than nTregs. 5,6 In male infants, mutations of FOXP3 cause a severe autoimmune disease known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). 7-9 Depending on the mutation, nTregs may be present in IPEX patients, but display an impaired function. 10 The FOXP3 gene (Xp11.23) is subject to X-chromosome inactivation (XCI), 11 a random process by which 1 of the 2 X-chromosomes is methylated during embryogenesis and remains stably inactive in daughter cells. Accordingly, female carriers of FOXP3 mutations are expected to be cell mosaics in which either the wild-type (WT) or the mutated (mut)-FOXP3 allele is active. Therefore, they represent a unique system to investigate the role of WT versus mut-FOXP3 in nTregs and other cell subsets in humans. Because in heterozygous Foxp3 wt/null female mice, only nTregs expressing WT-Foxp3 have the nTreg phenotype and are maintained in the periphery, 12 we investigated whether expression of human WT-FOXP3 is necessary for the development and/or homeostasis of nTregs, by analyzing the pattern of XCI in T-cell subsets from women carrying FOXP3 mutations. Methods SubjectsCarriers of FOXP3 mutations are healthy adults with no autoimmune symptoms. Peripheral blood was obtained upon informed consent from healthy females and carriers of FOXP3 mutations in accordance with approval from the Internal Ethical Committee of the San Raffaele Scientific Institute (protocol TIGET02) and with the Declaration of Helsinki.For complete materials and methods information, see supplemental methods (available on the Blood website; see the Supplemental Materials link at the top of the online article). Results and discussion Healthy carriers of FOXP3 mutations are mosaics of cells expressing either the WT or the mut-FOXP3 alleleNine healthy female carriers of 5 different FOXP3 mutations, relatives of severely affected IPEX patients, were studied (Figure 1A For personal use only. on April 11, 2019. by guest www.bloodjournal.org From and supplemental methods). The distribution of active WT and mut-FOXP3 alleles in peripheral blood mononuclear cells (PBMCs; n ϭ 9), CD4 ϩ T (n ϭ 8), CD8 ϩ T (n ϭ 3), CD56 ϩ natural killer (n ϭ 2), and CD19 ϩ B (n ϭ 3) cells was evaluated by de...

Glycoantigens Induce Human Peripheral Tr1 Cell Differentiation with Gut-homing Specialization

“…For example, Tr1 cells isolated from tolerized human transplant patients do not express Foxp3 but produce high levels of IL-10 (13). In addition, IPEX patients with loss of function mutations in the Foxp3 gene were shown to have suppressive T cell activity through their Tr1 cells, demonstrating that Foxp3 is not required for suppressive Treg cell function in humans (14). The present results are consistent with these human studies, which collectively suggest that the mouse studies showing PSA-specific Foxp3 ϩ iTregs may have limited applicability to human immunology.…”

“…Human Tr1 cells, isolated from tolerant transplant patients, were shown to produce large amounts of IL-10, are typically defined by their high IL-10:IL-4 ratio, and do not express constitutive levels of Foxp3 (13). Because suppressive Tr1 cells can be differentiated from IPEX patients who genetically lack functional Foxp3, it is known that Foxp3 is not required for all suppressive T cell activity in humans (14). Work done by Papadakis et al (15) has demonstrated that mucosal T cells that express the CC chemokine receptor 9 (CCR9) have a suppressive Tr1 cell phenotype.…”

Concurrent FOXP3- and CTLA4-associated genetic predisposition and skewed X chromosome inactivation in an autoimmune disease-prone family

“…1). Because the mutation was found in two of the three females with AITD, we further explored the females' XCI (see legend) (12,13,14,15,16). Skewing was found in the peripheral blood of the mother and the sister of the patient (both FOXP3 C/K ), but not in the aunt with AITD without a FOXP3 mutation.…”

“…FOXP3 is an X-linked gene whose mutated copy will be selectively silenced in the relevant Treg cell population of female carriers (14,16). Thus, no adverse effects ensue from this specific genetic defect, and any risk of autoimmunity in these individuals is therefore most likely due to the respective CTLA4 variant.…”

Forkhead box P3:The Peacekeeper of the Immune System

“…Therefore, while the functional impairment of FOXP3-mutated Treg cells is undisputed, the identification of circulating cells with figure. specific demethylation of the Treg-cell-Specific-Demethylated-Region (TSDR) (see below), demonstrated that wild type FOXP3 is dispensable for thymic development of Treg cell precursors in humans [44,55]. The lack of functional FOXP3 impairs Treg peripheral maintenance, as demonstrated by the observation that in healthy female carriers of FOXP3 mutations [102] and in transplanted IPEX patients with low peripheral donor chimerism [48] only Treg cells expressing a wild type FOXP3 are detectable in peripheral blood.…”