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
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 
Published: October 2019

Abstract
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 percentage of FoxP3+ cells in CD4+CD25hi from iTreg was 98.5%, as compared to 82.4% in PBMCs. Interestingly, the percentage of FoxP3+ cells in total CD4+CD25+ was higher in cells from iTreg (97,3%) than on PBMCs (26,8%). Moreover, while the percentage of FoxP3+ cells in the CD4+CD25− population, was very low in PBMCs (2,8%), up to 55% of the cells derived from iTreg were FoxP3+. The immunossupression assay showed that, compared to activated CD3+ T cells cultured alone, nTregs (CD4+CD25+CD127−) decreased the proliferation of CD3+ T cells in 55%, while iTregs (CD4+CD25hi) decreased the proliferation in 46%. Interestingly, the CD4+CD25− population from iTreg (55% of FoxP3+ cells) also decreased the proliferation of CD3+ T cells, but to a lower extent (21%). Additionally, while naïve T cells and Teff presented low level of 5hmC in both segments evaluated of CNS2 (∼1%); upon in vitro induction, iTregs presented 5hmC levels comparable to that of nTregs (5–11% and 5% respectively), in line to FoxP3 expression. Furthermore, CNS3, which was found to be partially demethylated in naïve T cells and nTregs (45 and 50% respectively), presented even higher levels of demethylation upon activation in iTregs and nTregs (77 and 82% respectively). In summary, we show that functional Foxp3+CD4+CD25hi T cells can be generated in vitro from UCB naïve T cells. Additionally, our results indicate that active demethylation of CNS2 occurs in a TGF-β and atRA-dependent manner during iTregs generation. Moreover, the partial demethylation of CNS3 observed in naïve T cells and nTregs, and the increased demethylation promoted by activation (in Teff and iTreg), is consistent with the role of CNS3 as a pioneer element that initiates FoxP3 transcription. Our results contribute to the understanding of the epigenetic mechanisms underlying the differentiation of Tregs and may help in the development of protocols for the generation of functional iTregs for future therapeutic applications. Support: FAPESP, CNPq.

Disclosures:
No relevant conflicts of interest to declare.