Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome

Abstract

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this “IPEX-like” syndrome presently remains unclear.To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3+ T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively.TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3+ and CD25+FOXP3+ T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Autoimmune and other cytopenias in primary immunodeficiencies: pathomechanisms, novel differential diagnoses, and treatment

“…[57][58][59] In addition to the mutations in FOXP3 that cause IPEX, 60,61 other components of the Treg activation pathway may be defective and may thus reduce the function and/or number of Tregs and lead to an IPEX-like syndrome (eg, deficiency of CD25 or STAT5b and gain-of-function mutations in STAT1). [62][63][64][65] Interestingly, the classical central T-cell tolerance defect, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome ([APECED] due to a defect of the autoimmune regulator transcription factor) is not typically associated with cytopenias. 66 Furthermore, immune dysregulatory processes such as hemophagocytosis or lymphoproliferation (and subsequent splenic sequestration of blood cells) may cause secondary cytopenia in critically ill patients.…”

Forkhead box P3: The Peacekeeper of the Immune System

“…Except for rare patients with mutations in CD25 [42] or STAT5b [43], in most of the cases the cause of the IPEX-like syndromes remains unknown. We have recently reported that in at least a subset of these patients autoimmunity may be caused by a quantitative defect of tTreg cells [44], thus indicating Treg cells as the key contributors to disease development in both IPEX and IPEX-like syndromes.…”

“…Furthermore, a population of genetically imprinted Treg cells, i.e. T cells carrying specific epigenetic modifications in the regulatory regions of the FOXP3 locus, are present in peripheral blood of IPEX patients, regardless of the type or site of the mutation [44,55]. 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].…”

“…T cells carrying specific epigenetic modifications in the regulatory regions of the FOXP3 locus, are present in peripheral blood of IPEX patients, regardless of the type or site of the mutation [44,55]. 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.…”

The Role of HTLV-1 in Alteration of Immune Regulation Through Regulatory T Cell Dysfunction and Exosomal Manipulation

“…Recently, it has been demonstrated that FoxP3 TSDR demethylation can be characterized from whole blood PBMCs if used in conjunction with normalization to a specific T cell population (Barzaghi et al 2012). We took a similar approach by enumerating demethylation of the FoxP3 TSDR in i) PBMCs; ii) CD4 + T cells; iii) CD4 + CD25 + T cells of HAM/TSP patients normalized to the activated CD4 + T cell population as determined by flow cytometry.…”