Dynamic demethylation of the IL2RA promoter during in vitro CD4+ T cell activation in association with IL2RA expression

Abstract

IL2RA, a subunit of the high affinity receptor for interleukin-2 (IL2), plays a crucial role in immune homeostasis. Notably, IL2RA expression is induced in CD4+ T cells in response to various stimuli and is constitutive in regulatory T cells (Tregs). We selected for our study 18 CpGs located within cognate regulatory regions of the IL2RA locus and characterized their methylation in naive, regulatory, and memory CD4+ T cells. We found that 5/18 CpGs (notably CpG + 3502) show dynamic, active demethylation during the in vitro activation of naive CD4+ T cells. Demethylation of these CpGs correlates with appearance of IL2RA protein at the cell surface. We found no influence of cis located SNP alleles upon CpG methylation. Treg cells show constitutive demethylation at all studied CpGs. Methylation of 9/18 CpGs, including CpG +3502, decreases with age. Our data thus identify CpG +3502 and a few other CpGs at the IL2RA locus as coordinated epigenetic regulators of IL2RA expression in CD4+ T cells. This may contribute to unravel how the IL2RA locus can be involved in immune physiology and pathology.

Keywords: CD4+ T cells; CpG methylation; IL2RA; Tregs; age.

Figure 1.

(A) Levels of methylation of the 18 studied CpGs in three subpopulations of CD4+ T lymphocytes: naive CD4+ T cells (n = 76, black dots), memory T cells (n = 5, blue dots) and T regulatory cells (n = 7, red dots). The x-axis shows the studied CpGs (black lollipops), the positive regulatory regions (PRR), the negative regulatory region (NRE), and the TSS (black arrow). (B) Table showing the comparison of methylation levels for the 18 CpGs in the studied groups of T1D patients and controls.

Figure 2.

Lack of correlation between CpG +3502 methylation and genotypes for the 7 studied SNPs at the IL2RA locus. The asterisk (*) indicates the two closest SNPs flanking CpG +3502.

Figure 3.

The four PRRVI CpGs (−8452 to −8564 bp) and the PRRIV CpG +3502 show significant demethylation (P <3.10⁻¹⁶) following in vitro stimulation of naive CD4+ T cells (T1D ○, n = 36; children controls ●, n = 8; Adult controls ■, n = 32).

Figure 4.

Time course of CpG +3502 demethylation during in vitro activation of naive CD4+ T cells. Each point represents the mean ± SD of methylation at CpG +3502 in 3 independent in vitro experiments.

Figure 5.

The demethylation occurring at CpG +3502 correlates with (a) CD4+ T cell expression of IL2RA at the cell surface of CD4+ T cells activated in vitro (R = 0.52, P = 3.10⁻³, n = 28) and (b) the concentration of IL2RA in cell supernatant (R = 0.51, P = 4.10⁻⁵, n = 52).

Figure 6.

The decrease in CpG +3502 methylation correlates with the concentration of IL2 in supernatant of CD4+ T cells activated in vitro (R = 0.46, P = 3.10⁻⁴, n = 59).

Figure 7.

Parallel time course of the demethylation of CpG +3502 at the IL2RA promoter (solid line) and demethylation of CpG −252 at the IL2 promoter (dotted line).