Ex vivo expanded regulatory T cells CD4⁺CD25⁺FoxP3⁺CD127^Low develop strong immunosuppressive activity in patients...

Multiple sclerosis (MS) is an autoimmune disease characterized by defect in regulatory function of CD4⁺CD25⁺ T cells. We demonstrated difference in proportion of regulatory T cells CD4⁺CD25⁺FoxP3⁺CD127^low (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 × 10⁶ CD4⁺ T cells, we yield 300-400 × 10⁶ 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.