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

F Barzaghi, L Passerini, E Gambineri, S Ciullini Mannurita, T Cornu, E S Kang, Y H Choe, C Cancrini, S Corrente, R Ciccocioppo, M Cecconi, G Zuin, V Discepolo, C Sartirana, J Schmidtko, A Ikinciogullari, A Ambrosi, M G Roncarolo, S Olek, R Bacchetta, F Barzaghi, L Passerini, E Gambineri, S Ciullini Mannurita, T Cornu, E S Kang, Y H Choe, C Cancrini, S Corrente, R Ciccocioppo, M Cecconi, G Zuin, V Discepolo, C Sartirana, J Schmidtko, A Ikinciogullari, A Ambrosi, M G Roncarolo, S Olek, R Bacchetta

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.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Figures

Fig. 1
Fig. 1
TSDR demethylation analysis in the peripheral blood of IPEX-like patients. (A) Comparison between median Treg percentages (measured by TSDR demethylation assay), in the peripheral blood of HS (white bar, n = 40) and IPEX-like patients (grey bar, n = 28). Middle line indicates the median. The box represents 50% of all events and the whiskers extend to 95%. Statistical analysis was performed with Mann–Whitney U-test. (B) ROC curve describing the performance of TSDR assay in discriminating patients and HS. AUC value reflecting the discrimination between groups is indicated. (C, D) Scatter plot and linear regression between the percentage of Treg cells and age in healthy paediatric controls (n = 27), and IPEX-like paediatric patients (n = 22), all under 16 years.
Fig. 2
Fig. 2
Flow cytometric analysis of Treg cells in the peripheral blood of IPEX-like patients. FOXP3+ cells were detected by flow cytometry in the peripheral blood of IPEX-like patients (n = 13) and HS (n = 25). Percentage of FOXP3+ T cells in the (A) CD4+ and (B) CD4+CD25+ T cell gates are plotted in the graphs. Mean fluorescence intensity of marker expression in (C) CD4+FOXP3+ and (D) CD4+CD25+ T cells is also shown. Differences between groups were verified by means of Mann–Whitney U-test.
Fig. 3
Fig. 3
Treg cell normalization to CD3+ T cell counts. (A) Robust regression plots of the % of Treg cells vs the % of CD3-expressing cells (as measured by TSDR and TLSDR assays) in the peripheral blood of HS (left panel) and IPEX-like patients (right panel) are shown. Pearson correlation values were 0.617 (p < 0.001) for HS and 0.476 (p = 0.010) for patients. (B) The percentage of Treg cells normalized to TLSDR was plotted versus the CD3+ cell counts, as measured by TLSDR assay. Region (I) comprises lymphopenic patients, in whom the % of CD3-expressing cells in whole blood was below 14.9%; region (II) identifies patients with lymphocyte cell counts within normal ranges. (C) The box-plots show the % of Treg normalized to CD3+ T cell amount (as measured by TSDR and TLSDR combined analysis) in HS (white bar) and non-lymphopenic IPEX-like (light grey) and control patients (dark grey). Control pathologies include: IPEX syndrome (n = 5), T1DM (n = 10), Celiac (n = 9) and Crohn (n = 8) diseases. Middle lines represent the median. Statistical analysis was performed with Mann–Whitney U-test, p < 0.05 was considered significant. (D) The ROC curve describes the performance of the combined TSDR/TLSDR assays in discriminating HS and IPEX-like patients. AUC value reflecting the discrimination between groups is indicated.
Fig. 4
Fig. 4
Suppressive function of Treg cells isolated from the peripheral blood of IPEX-like patients. CD4+CD25+ T cells isolated from the peripheral blood of IPEX-like patients were used as suppressor cells in a suppression assay, using allogeneic and/or autologous PBMC as responders. CD4+CD25+ T cells isolated from the peripheral blood of one HS were tested in parallel and used as control. (A) Histograms of CFSE dilution of one representative patient (#20) are shown. Analysis was performed by gating either on CD4+ or on CD8+ responder T cells. Numbers in the plots indicate percent inhibition. The relative [suppressor: PBMC responder] cell ratio is also indicated. (B) Average %inhibition of proliferation versus the indicated responder cells is plotted in the graphs. The highest [suppressor: responder] cell ratio tested is plotted for each patient. Black symbols refer to patients, white symbols to HS tested in parallel. ●: Patient #20; ▲: Patient #17; ■: Patient #15; ♦: Patient #16.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3314976/bin/figs1.jpg

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