The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood

Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4⁺ T cells. Methods: CB-CD4⁺ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4⁺ T cells was calculated, and the incidence of CD25⁺Foxp3⁺ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25⁺Foxp3⁺ cell population in CD4⁺ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25⁺Foxp3⁺ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4⁺ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25⁺Foxp3⁺ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x10⁷ iTregs were obtained from the initial input of 5×10⁴ CD4⁺ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4⁺ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4⁺ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting.

No relevant conflicts of interest to declare.