Multiple sclerosis (MS) is an autoimmune disease characterized by defect in regulatory function of CD4CD25 T cells. We demonstrated difference in proportion of regulatory T cells CD4CD25FoxP3CD127 (Tregs) within the same patients' relapse and remission. Proportion of peripheral Tregs (pTregs) dropped almost two times in the relapse compare to remission. Levels of pTregs in patients' remission were lower than in healthy donors. Suppressive ability of pTregs was decreased in MS patients compared to healthy donors. Injections of expanded ex vivo autologous Tregs (eTregs) could be helpful in bringing up the level of Tregs in patients' blood. We developed a simple method for ex vivo expansion of autologous Tregs within a short period of time. The final pool of cells consisted of 90-95% eTregs. When we started the culture with 10-20 × 10 CD4 T cells, we yield 300-400 × 10 eTregs in a week. Expression of FoxP3 and Helios was calculated by two methods. Expanded ex vivo patients' and donors' Tregs were characterized by increased from three to five times expression of FoxP3, as well as almost doubled Helios expression. Peripheral Tregs in MS patients have decreased demethylation of FoxP3 gene promoter in comparison with donors. On the contrary, eTregs showed stable up-regulated demethylation without difference between MS patients and donors. MS patients' and donors' eTregs have much more suppressive ability than pTregs. Our data showed that eTregs can be applied as immunotherapy for MS patients and other autoimmune diseases if further investigated.

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 numbers of pTregs in the blood of these patients elevated by 1.5-2 times. No adverse-effects, a decrease of relapses and stabilization of disability index were observed. It has been suggested that ex vivo expanded Tregs can compensate the impaired function of pTregs and can be used for adoptive immunotherapyof multiple sclerosis.

Regulatory T-cells CD4⁺CD25⁺FoxP3⁺CD127low (Tregs) play a key role in the maintenance of tolerance to auto antigens, inhibit function of effector T and B lymphocytes, and provide a balance between effector and regulatory arms of immunity. Patients with autoimmune diseases have decreased Treg numbers and impaired suppressive activity. Transformed ex vivo autologous Tregs could restore destroyed balance of the immune system. We developed a method for Treg precursor cell cultivation. Following the method, we were able to grown up 300-400 million of Tregs cells from 50 ml of peripheral blood during a week. Transformed ex vivo Tregs are 90-95% CD4⁺CD25⁺FoxP3⁺CD127low and have increased expression of transcription genes FoxP3 and Helios. Transformed ex vivo Tregs have increased demethylation of FoxP3 promoter and activated genes of proliferation markers Cycline B1, Ki67 and LGALS 1. Transformed ex vivo Tregs have increased suppressive activity and up to 80-90% these cells secrete cytokines TNFα и IFNγ. Our data suggest transformed ex vivo autologous Tregs have genetic, immunophenotypic and functional characteristics for regulatory T-cells and further can be used for adoptive immunotherapy autoimmune diseases and inhibition of transplantation immunity.