## 4 Discussion  
Despite the good reproducibility of Treg ex vivo induction protocols [60; 61], these methods have certain limitations, including long cultivation times and relatively low yields of Tregs. Therefore, we developed a simple and rapid method for the ex vivo expansion of regulatory T cells based on selecting a starting cell population of CD4\({}^{+}\) T cells. The resulting eTregs expressed all typical markers of Tregs CD4\({}^{+}\)CD25\({}^{+}\)FoxP3\({}^{+}\)CD127 low. The starting population of CD4\({}^{+}\) T cells contained approximately 7-12% Tregs; after seven days of ex vivo cultivation, approximately 98-99% of cells expressed typical markers of Tregs. According to our findings, almost all CD4\({}^{+}\) cells undergo division, demonstrating that the proliferation of Tregs in the CD4\({}^{+}\) population and the transformation of conventional CD4\({}^{+}\)CD25\({}^{-}\) T cells occur simultaneously. Restimulation performed after 96 hours supports cell proliferation until day 7. Longer cultivation does not increase cell numbers; rather, it decreases cell viability, probably because of the lack of nutrients in growth media. Our protocol involves stimulation of initial cells with IL-2 and TGF-beta combined with TCR stimulation with anti-CD3 and anti-CD28 antibodies. IL-2-dependent conversion of the nonregulatory T cells into eTregs involves TGF-beta, which induces FoxP3 expression [62]. The costimulatory receptor CD28, as well as TCR and the IL-2 receptor, have been shown to participate in the transcriptional regulation of the FoxP3 gene [63]. Anti-CD3 and anti-CD28 antibodies and IL-2 have been used initially to stimulate the proliferation of precursor cells [64], and then TGF-beta has been used to induce the expression of the transcription factor FoxP3, which follows the  
Figure 7: Enhanced suppressive activity of eTregs: Representative proliferative diagram of **(A)** pTregs and **(C)** eTregs. CFSE-labeled CD4\({}^{+}\)CD25\({}^{-}\) responder cells were cocultured with accessory cells and pTregs or eTregs at various ratios. Results are shown as the percentage of proliferated target cells measured by reduction of the CFSE signal using flow cytometry. Neg. ctrl—negative control, CD4\({}^{+}\)CD25\({}^{-}\) cells only. Pos. ctrl—positive control, CD4\({}^{+}\)CD25\({}^{-}\) cells cocultured with accessory cells. Proliferation index of CD4\({}^{+}\)CD25\({}^{-}\) responder cells cocultured with accessory cells and **(B)** pTregs and **(D)** eTregs at various ratios. Ctrl—control, CD4\({}^{+}\)CD25\({}^{-}\) cells cocultured with accessory cells. \(n=5\). * \(p\leq 0.05\) vs. control cells. emergence of both specific Treg markers and their potent suppressive function [65]. The engagement of TCRs (at least in some circumstances) is required to ensure the phenotypical stability of eTregs by inducing epigenetic changes, e.g., the establishment of T cell-specific CpG hypomethyllation patterns [66]. In our experiments, the number of Tregs increased from 5 \(\times\) 10\({}^{6}\) to 150 \(\times\) 10\({}^{6}\) during seven days of cultivation. The total number of cells was elevated 40-50 times, and the number of Tregs increased 400-450 times from the initial CD4\({}^{+}\) T cells. It is expected that such rapid cell growth should be accompanied by the activation of a number of cell proliferation genes. The investigation of gene expression within the time of ex vivo Treg proliferation and expansion revealed increased expression of genes responsible for cell cycle regulation. FoxP3 is the master gene for Tregs. It is responsible for the suppressive activity of Tregs [3]. FoxP3 stabilizes and supports the growth and differentiation of Tregs. We compared the expression of FoxP3 in eTregs and in initial cells freshly isolated from peripheral blood. Expression was assessed by real-time RT-PCR and flow cytometry. The expression of FoxP3 in eTregs increased as much as two times in comparison with that in initial cells.