###### Abstract  
Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of TES CD4\({}^{+}\)CD25\({}^{+}\)FoxP3\({}^{+}\)CD27\({}^{\text{low}}\) over 7 days of cultivation from initial CD4\({}^{+}\) T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXPP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells. T 20 May 2021 17 June 2021 22 June 2021 2021 20 May 2021 17 June 2021  
proliferation and differentiation of Tregs [7; 8]. IKAROS family zinc member transcription factors [9], along with reactivation of telomerase [10], also have well-documented roles in the development of immune cell populations, and the process of alternative splicing of their pre-mRNA has a significant impact on cellular biology [11; 12; 13]. A number of studies have examined the various suppressive functions of Tregs and how these mechanisms work to maintain immune tolerance. One of the most promising targets is the inhibitory molecule CTLA-4. CTLA-4 makes contact with the costimulatory molecules CD80 and CD86 and outcompetes the activating receptor CD28 for binding with these two ligands, thereby abrogating the activating signal in effector cells [14; 15]. The CD4-related protein LAG-3 (CD223) has been implicated in suppressive functions of Tregs via direct interactions with Tregs and effector cells and via the modulation of antigen-presenting cells [16; 17]. Another mechanism by which Tregs implement their suppressive activity is the induction of endonuclease G-mediated alternative splicing of the pre-mRNA of the telomerase main catalytic subunit that leads to target cell senescence [18; 19]. The results of the present study provide evidence that human Treg cells suppress telomerase activity and induce target cell senescence in a contact-independent manner. We also show that telomerase activity inhibition in target cells is associated with EndoG-induced hTERT alternative splicing. Tregs are defective in many autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosusous and type 1 diabetes [20; 21; 22; 23; 24; 25]. Tregs are either dysfunctional or unstable, especially in the inflamed peripheral site where self-reactivity occurs. Restoration of Treg function could inhibit the onset and progression of autoimmune diseases [26; 27; 28; 29; 30]. Several approaches have been explored to obtain large amounts of ex vivo expanded Tregs (eTregs). The most widely applied method is T cell receptor (TCR) stimulation in the presence of a large amount of exogenous interleukin-2 (IL-2). Combined alloantigen and polyclonal stimulation with anti-CD3 and anti-CD28 antibodies has been applied to increase antigen specificity [31].