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Abstract
BackgroundRegulatory T-cells (Tregs), characterized as CD4+CD25hi T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4+CD25hi, FOXP3 mRNA, protein expression, and suppressive Treg function.MethodologyCord blood mononuclear cells (CBMC) were isolated from 70 healthy neonates, stimulated for 3 days with the microbial stimulus lipid A (LpA), and allergen Dermatophagoides pteronyssinus (Derp1). Tregs (CD4+CD25hi, intracellular, mRNA FOXP3 expression, isolated cells), DNA methylation of the FOXP3-locus and suppressive Treg function were assessed.Principal FindingsDemethylation of FOXP3 in whole blood was specific for isolated CD4+CD25hi Tregs. Demethylation of FOXP3 was positively correlated with unstimulated and LpA-stimulated FOXP3 mRNA-expression (p≤0.05), and CD4+CD25hi T-cells (p≤0.03). Importantly, increased FOXP3 demethylation correlated with more efficient suppressive capacity of Tregs (r = 0.72, p = 0.005). Furthermore, FOXP3 demethylation was positively correlated with Th2 cytokines (IL-5, IL-13) following LpA-stimulation (p = 0.006/0.04), with Th2 and IL-17 following Derp1+LpA-stimulations (p≤0.009), but not Th1 cytokines (IFN-γ).ConclusionsFOXP3 demethylation reliable quantifies Tregs in cord blood. FOXP3 demethylation corresponds well with the suppressive potential of Tregs. The resulting strict correlation with functionally suppressive Tregs and the relative ease of measurement render it into a valuable novel marker for large field studies assessing Tregs as qualitative marker indicative of functional activity.
Highlights
Regulatory T-cells (Tregs) are essential for the downregulation of T cell responses to both foreign and self antigens and play an important role in several immune-mediated diseases, such as allergic diseases [1]
We and others have demonstrated that Tregs were present and functional in cord blood mononuclear cells [3,4,9], though less suppressive compared to adult peripheral blood mononuclear cells (PBMC) [3]
Specificity of FOXP3 demethylation for isolated Tregs in cord blood In FACS-sorted isolated CD4+CD252 T-cells and CD4+CD25hi T-cells from cord blood, FOXP3 demethylation was hardly detectable in CD4+CD252 cells and well detectable in a mean of 81% in the CD4+CD25high T cells (Fig. 1A,B)
Summary
Regulatory T-cells (Tregs) are essential for the downregulation of T cell responses to both foreign and self antigens and play an important role in several immune-mediated diseases, such as allergic diseases [1]. Various studies including ours suggested that Tregs may play a crucial role in early allergy protection by keeping the immune system in balance and counter-regulating potential default pathways [2,3,4]. Tregs were best characterized as CD4+CD25hi T-cells expressing the forkhead box transcription factor (FOXP3), which suppress T cell activation and regulate various immune reactions in vitro and in vivo [6,7,8]. Regulatory T-cells (Tregs), characterized as CD4+CD25hi T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4+CD25hi, FOXP3 mRNA, protein expression, and suppressive Treg function
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