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 Tregs CD4+CD25+FoxP3+CD127low 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 FOXP3, 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.

The aim of this work was to identify genes whose mRNAs were subjected to alternative splicing by apoptotic endonuclease EndoG in CD4+ T lymphocytes from healthy humans, mice, and rats. In order to induce EndoG, lymphocytes were transfected with an EndoG-containing plasmid, or a control pGFP plasmid, or were incubated with cisplatin. Efficiency of transfection, number of cells with DNA damages and the level of EndoG expression have been monitored. Total cell mRNA has been sequenced and the changes in proportion of splice variants of genes were analyzed. The changes in the proportion of 28 mRNA splice variants have been identified in human and murine lymphocytes in both transfected with EndoG gene or incubated with cisplatin. Thus, EndoG can be considered as a potent modulator of alternative splicing of mRNA of identified genes.