## 3 Results
### Stability of FoxP3 Expression Depends on the Demethyllation Levels of TSDR in Its Promoter
A gradual increase in FoxP3 expression corresponded to an increase in the rate of TSDR demethylation (Figure 3) and the ratio of FoxP3-expressing cells measured by flow cytometry (Figure 2C,D). The proportions of FoxP3 splice variants demonstrated rather complex expression profiles (Figure 4E). The full-length variant was repressed 3-5 days after initial cell stimulation and demonstrated a significant increase at days 7 and 9. The splice variant with the deletion of exon 3 (\(\Delta\)3) remained unchanged  
Figure 4: Upregulation of telomerase and FoxP3 is associated with the induction of alternative splicing of their mRNA: (**A**) Induction of total hTERT expression during the time of cell cultivation. (**B**) Changes in the proportion of hTERT splice variants. (**C**) Representative TRAP gel electrophoresis demonstrating elevated telomerase activity in Tregs upon maturation. (**D**) Gradual increase in FoxP3 gene expression during the time course of Treg maturation. (**E**) Changes in the proportion of FoxP3 splice variants. \(n\) = 4. * \(p\)\(\leq\) 0.05 vs. initial cells at 0-day time-point. FL, full-length variant. \(\alpha\) or \(\beta\) indicates corresponding splice variants (see main text). \(\Delta\) indicates the deletion of the relevant exon. in the first five days after the beginning of expansion and was detected at minor levels in mature eTregs at days 7 and 9. The expression of the splice variant with the deletion of exon 8 (\(\Delta\)8) demonstrated a significant increase of up to 21.6% in total FoxP3 mRNA at days 3 and 5 while, in mature eTregs, it was almost undetectable. Similar expression was observed for the variant with deletions of both exons 3 and 8 (\(\Delta\)3\(\Delta\)8). This splice variant demonstrated a small increase in expression at days 3 and 5 of up to 12.4% in total FoxP3 mRNA, and it was almost undetectable in mature eTregs. Ex Vivo eTreg Maturation Is Associated with Changes in the Expression of Ikaros Family Zinc Finger Gene Members and in the Levels of Their Splice Variants  
Ikaros family zinc finger members (IKZF1-IKZF5) act as transcription factors and can cooperate with FoxP3 to support Treg development [49]. Multiple splice variants have been described for these five genes [50,51]; however, their involvement in Treg biology has not been investigated. Using real-time RT-PCR, we examined the expression levels of their splice variants during the entire time of eTreg maturation ex vivo. The expression of total IKZF1 (IKAROS) mRNA increased 3 days after initial cell stimulation and remained at a high level up to day 9 of expansion (Figure 5A). The full-length splice variant in initial CD4\({}^{+}\) T cells was expressed at a level no more than 17.3% of total IKZF1 mRNA; however, its expression increased after cell stimulation by up to 64.7% in mature eTregs (Figure 5B). This result demonstrated that the increase in the full-length variant contributed the most to the increase in total mRNA. The opposite effect was observed for the IKZF1 splice variant with a deletion of exon 4 (\(\Delta\)4). In initial cells, it comprised the majority (up to 68.2%) of total mRNA and was significantly reduced in mature eTregs (19.8%). The variant with a deleted exon 3 (\(\Delta\)3) was expressed at a minimal rate (2.1%) in initial cells but increased up to 9.2% in mature cells. The splice variant with a deletion of exon 8 (\(\Delta\)8) comprised up to 10.0% of CD4\({}^{+}\) T cells and unexpectedly failed to be detected in expanding cells after initial stimulation. Figure 5: Expression profiles of IKAROS family zinc finger gene members and their splice variants during the time course of eTreg maturation ex vivo. The expression levels of total (**A**) IKZF1 (IKAROS), (**C**) IKZF2 (HELIOS), (**E**) IKZF3 (AIOLOS), (**G**) IKZF4 (EOS) and (**I**) IKZF5 (PEGASUS) in proliferating cells. (**B**,**D**,**F**,**H**,**J**) Proportions of corresponding splice variants. \(n=4\). * \(p\leq 0.05\) vs. initial cells at 0-day time-point. \(\Delta\) indicates the deletion of the relevant exon.