## 3 Results
### Stability of FoxP3 Expression Depends on the Demethyllation Levels of TSDR in Its Promoter  
Demethylation events in the FoxP3 TSDR promoter region are responsible for the stability of FoxP3 expression in mature cells. We studied changes in TSDR methylation/demethylation levels during the time of Treg expansion using methyl sensitive PCR. We found a gradual increase in demethylation during seven days of ex vivo expansion (Figure 3A,B). The demethylation level was 41.7 \(\pm\) 8.3% (28.5; 49.5) in pTregs while, in seven-day expanded eTregs, it increased to 59.5 \(\pm\) 5.4% (50.7; 65.6). There was no difference in the TSDR demethylation levels between cells at days seven and nine of cultivation. We analyzed the methylation of each CpG site at TSDR. Cells differed in the percentage of demethylation but we found no consistency in demethylation level differences at single positions (Figure 3C). These results demonstrate that mature Tregs have a higher degree of demethylation in the TSDR than initial cells, which is associated with increased expression of FoxP3. Induction of Telomerase and FoxP3 during Treg Expansion Is Associated with Changes in Their Splice Variants  
High proliferation of T cells is supported by active telomerase, which is activated upon immune stimulation [43; 44]. Telomerase activity in cells depends on the expression level of the telomerase main catalytic subunit hTERT [19; 45], as well as on the predominance of the  
Figure 3: The level of TSDR demethylation increases during ex vivo expansion of Tregs. TSDR methylation analysis was performed during the time of cell expansion: (**A**) Levels of TSDR methylation and demethylation during Treg ex vivo maturation. (**B**) Mean level of TSDR methylation during Treg expansion. (**C**) Representative result of demethylation percentage of each CpG site in the TSDR color-coded according to the legend. Each column corresponds to a single CpG site. Absolute coordinates according to the GRCh37/hg19 human genome assembly are indicated. \(n\) = 5. * \(p\)\(\leq\) 0.05 vs. initial cells at the 0-day time-point. full-length \(\alpha\)+\(\beta\)+ active splice variant [46; 47]. We investigated the rate of hTERT expression in proliferating cells. In the first five days, hTERT expression was not different from that of the initial CD4\({}^{+}\) cells; however, after day seven, a significant increase in hTERT expression was observed (Figure 4A). In freshly isolated initial CD4\({}^{+}\) T cells (0-day time-point), the \(\alpha\)+\(\beta\)- truncated splice variant comprised approximately 80% of total hTERT, while the ratio of the active full-length variant was only 10% (Figure 4B). Three days after cell stimulation with IL-2, TGF-beta and antibodies, the rate of the full-length variant increased up to 60%. No significant changes in the expression of the minor splice variants \(\alpha\)-\(\beta\)+ and \(\alpha\)-\(\beta\)- were observed within the entire time of cultivation. The increase in the active full-length variant was associated with an increase in telomerase activity, which was measured using a TRAP assay (Figure 4C). After day five of cultivation, no significant changes in the rates of hTERT splice variants were observed. However, telomerase activity gradually increased until day seven. This observation leads to the conclusion that, in the first five days of maturation, telomerase activity is induced by the shift in hTERT splicing patterns toward the full-length active variant while, after day five, the increase in total hTERT expression has a major impact on enhancing telomerase activity. FoxP3 is considered to be a master regulator protein for Tregs that determines their differentiation and proliferation and their supports suppressive activity [48]. Splice variants of FoxP3 are involved in different biological processes [12]. We observed the gradual induction of total FoxP3 expression throughout eTreg ex vivo expansion (Figure 4D). However, a significant increase was achieved only on days seven and nine.