Generation of Functional Regulatory T Cells From Umbilical Cord Blood Naïve T Cells: Potential Role of Hydroxymethylation in the Epigenetic Reprograming and Transcriptional Induction of FoxP3

• Published in: Blood 2012-11, Vol.120 (21), p.4835-4835
• Main Authors: Haddad, Rodrigo, dos Santos Schiavinato, Josiane Lilian, Saldanha-Araújo, Felipe Saldanha, Scheucher, Priscila S, Araújo, Amélia G, Rego, Eduardo M., Covas, Dimas T, Zago, Marco A., Panepucci, Rodrigo Alexandre
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.4835.4835

Abstract 4835 The development and functionality of CD4+CD25hi regulatory T cells (Tregs) depends on stable FoxP3 expression, a central regulator of Treg differentiation. It is believed that this is accomplished by regulatory regions in the promoter and 3 evolutionarily conserved noncoding sequences, termed CNS1, CNS2 (or TSDR) and CNS3. The activation of TCR (with anti-CD2/3/28) in CD4+CD25− naïve T cells from PBMCs, in the presence of IL-2, TGF-β and atRA, induces the generation of Foxp3+ induced regulatory T cells (iTreg). While demethylation of 5mC residues in the CNS2 is associated with stable FoxP3 expression in nTregs, the epigenetic events involved in the regulation of FoxP3 in iTregs remains unexplored. Recently, the oxidation of 5-mC, originating hidroxymethylated 5-hmC residues, have been described as a key mechanism of active demethylation, with roles in biological processes, such as regulation of pluripotency and differentiation of hematopoietic stem cells. In contrast to PBMCs, in umbilical cord blood (UCB) T cells are mainly naïve making UCB an attractive source for the development of protocols for generation of iTregs. Here, we evaluated the iTregs generation from UCB naïve T cells. In addition, we compared the expression of FoxP3 on iTregs and on naturally occurring Tregs (nTregs) obtained from PBMCs. Also, we evaluated the methylation pattern of promoter and CNS2 and CNS3 in nTregs, fleshly isolated naïve T cells, activated naïve T cells (Teff), and iTregs. Finally, we evaluated the ability of iTregs, to suppress the proliferation of activated T cells, as compared to nTregs. For this, CD4+CD25-CD45RA+ naïve T cells were immunomagnetically isolated from UCB and activated with anti-human CD2/CD3/CD28 beads (1:2 beads:cell ratio) in the presence of IL-2 (50 U/ml) with (iTregs) or without (Teff) TGF-β (5 ng/ml) and atRA (100 nM) for 5 days. In parallel, PBMCs from 5 individuals were obtained for nTregs phenotypic characterization. CD4+ gated cells from iTregs and from PBMC were analyzed by flow cytometry for FoxP3 expression in the CD25+, CD25hi and CD25−population. nTregs (CD4+CD25+CD127−) were immunomagnetically isolated from PBMCs and CD4+CD25hi and CD4+CD25− populations were FACS-sorted from iTreg to observe the potential in regulate the proliferation of CD3+ T cells (CFSE staining). Finally, methylation pattern analysis of FoxP3 locus, including CNS2 and CNS3, were performed in naïve T cells, nTregs, iTreg and Teff. The mean percen