## 3 Results
### Cell Cycle Regulatory Genes Are Involved in the Proliferation of Tregs  
Since a seven-day expansion of Tregs is necessary for their ex vivo maturation, we aimed to determine the possible proteins involved in cell cycle regulation. The expression levels of 28 genes in responder cells were analyzed using the Human Cell Cycle RT\({}^{2}\) Profiler PCR Array. The expression levels of the investigated genes were measured at the time of cultivation against the expression of those in freshly isolated CD4\({}^{+}\) T cells (Figure 6). Significant changes in expression levels were found in cyclin genes that promote transition through S and G2/M phases. CCNA1, CCNA2, CCNB1, CCNB2 and CCND1 were upregulated within the first 7 days, whereas G1 phase-associated CCNC appeared to be upregulated within 5-9 days of cultivation. The expression of CCNE, whose activity is required for cell cycle G1/S transition, and the F-box family protein CCNF, remained unchanged [52]. We found that the expression of two cyclin-dependent kinases (CDKs), CDK2 and CDK4, was upregulated in responder cells, and the inhibitors of CDK2 and CDK4, namely, CDKN1A, CDKN1B and CDKN2A, were subsequently reduced. The expression of the CDK/cyclin inhibitor proteins P16 INK4A and P21 was downregulated as expected, since it was previously shown in proliferating lymphocytes [53]. Cell proliferation is associated with the inhibition of P53 and RB tumor suppressor pathways [54]. We found that P53 and RB genes were expectedly downregulated during the entire time of cultivation. Activation of p38 can induce p21-dependent cell cycle arrest [55]. Despite reduced P21 and P38 activator GADD45, the expression of _P38_ was downregulated only at the 3-5-day time points. Along with the reduced RB gene, we found the downregulation of RB transcription factors E2F1 and E2F2. A component of the cell cycle checkpoint complex that causes cell cycle arrest in response to bulky DNA lesions and DNA replication blockage of _HUS1_[56] was also downregulated in proliferating cells. The expression of a nuclear protein associated with cellular proliferation of _KI67_[57] was significantly upregulated. The expression of the serine/threonine-protein kinase genes NEK and CHEK1 [58,59], which are involved in mitotic regulation, and the MTBP protein, which regulates progression through the cell cycle, remained unchanged. Our results indicate that the maturation of Tregs during 9 days of ex vivo cultivation is associated with the modulation of cell cycle regulatory gene expression. Our results indicate that the expression profile of cell cycle-regulating genes was not significantly changed between days 7 and 9 of maturation. Matured eTregs Demonstrate High Suppressive Activity against Autologous CD4\({}^{+}\)CD25\({}^{-}\) Target Cells  
A mixed lymphocyte reaction was performed to compare the functional suppressive activity of 7-day ex vivo expanded eTregs and freshly isolated pTregs from peripheral blood. The proliferative activity of respond CD4\({}^{+}\)CD25\({}^{-}\) T cells that were cocultured with pTregs or eTregs was measured. pTregs were able to inhibit the proliferation of target cells by almost 100% at ratios from 1:1 to 1:4 (Figure 7 A,B). PI in ratio of 1:8 was 55.2 \(\pm\) 4.0. Higher dilutions of pTregs did not demonstrate suppressive activity. High suppressive activity was shown by eTregs, even at a ratio of 1:16 (Figure 7 C,D). PI in ratio of 1:32 was 27.1 \(\pm\) 8.3. Higher dilutions of eTregs did not show statistically significant suppression. These results indicate an approximately two times higher activity of eTregs in comparison to pTregs. Figure 6: Cell cycle-associated gene expression in cells during ex vivo expansion. Total mRNA was isolated from cells, and mRNA levels were determined by real-time RT-PCR using the Human Cell Cycle PCR Array. The figure depicts a heat map diagram of gene expression in cells within the time of cultivation. Gene expression levels were normalized relative to the mean expression of four reference genes: 18S, GAPDH, HPRT1 and GUSB.