## 4 Discussion
FoxP3 gene expression is under the epigenetic control of the highly conserved CpG-rich TSDR region in the FoxP3 gene promoter, which is selectively demethyllated in Tregs [7]. Molecular characterization of the TSDR revealed that this element has transcriptional enhancer activity and determines the stability of FoxP3 expression [67]. Our study confirms that ex vivo expansion of Tregs is characterized by the increased demethylation of FoxP3. Our data are not in accordance with the results obtained by [PERSON] et al. [7], where it was shown that TGF-beta-induced Tregs in vitro demonstrate a much lower degree of demethylation compared to naturally occurring peripheral CD25\({}^{+}\)CD4\({}^{+}\) Tregs. The difference in the results relies on the use of naive CD4\({}^{+}\) T cells for initial cells, which have a different initial status of TSDR demethylation. In addition to FoxP3, T cell activation and proliferation are accompanied/caused by the expression of Ikaros family transcription factors [68]. Members of this family are DNA binding proteins containing two highly conserved zinc finger N-terminal domains and a protein binding domain (C-terminal) [69]. IKZF1 (IKAROS) may have dual effects and act as an enhancer or a silencer of gene expression [70]. Our results demonstrate that IKZF1 is significantly upregulated in mature eTregs. It is known that IKZF2 (HELIOS) upregulates FoxP3 by binding to the FoxP3 promoter [71]. Changes in IKZF2 expression determine changes in FoxP3 in Tregs [72]. We found upregulated expression of IKZF2 in eTregs which, together with upregulated demethylation, supports elevated FoxP3 expression. The activity of IKZF3 (AIOLOS) has the same effect in immune cells as IKZF1 [73]. In our experiments, the expression of IKZF3 in mature eTregs was not different from that in initial cells; however, its expression was reduced within the first 5 days of expansion. IKZF4 (EOS) has the opposite effect and can mediate Foxp3-dependent gene silencing in Tregs [74;75]. These data are in accordance with our results, where we demonstrated significant downregulation of IKZF4 in mature eTregs. The results of our study also demonstrate the changes in the expression profile of splice variants of FoxP3 and IKZF family members during eTreg maturation from initial CD4\({}^{+}\) T cells. These results are very difficult to interpret, as the biological activities of such splice variants and their involvement in different processes are still poorly understood. Rapid proliferation of activated lymphocytes is supported by active telomerase [43;44], and elevated activity was found in cells upon stimulation. Telomerase activity is known to be regulated by hTERT expression [76] and by alternative splicing of its mRNA [77;78]. The regulation of hTERT gene expression has been rather well investigated [79], whereas very little is known about telomerase regulation by hTERT. To date, 22 splice variants of hTERT have been described; however, only full-length hTERT is catalytically active [80]. Two splice variants comprise the major forms of total hTERT mRNA. Deletion of 36 nucleotides from exon 6 (\(\alpha\)-variant) leads to the loss of a portion of the reverse transcriptase domain in the hTERT protein, and results in the loss of catalytic activity. Deletion of 182 nucleotides from exons 7 and 8 (\(\beta\)-variant) leads to a reading frame shift, and results in the formation of a premature stop codon in exon 10 and the synthesis of a truncated hTERT protein [81]. This hTERT splice variant acts as a dominant negative protein [82]. Our results demonstrated that, in the first five days of maturation, telomerase activity was induced by the shift in hTERT splicing patterns toward the full-length active variant while, after day five, the increase in total hTERT expression had a major impact on the enhancement of telomerase activity.