Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on InVitro Expanded Regulatory T Cells

摘要

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promisingoption for reducing graft rejection in allogeneic transplantation. To gain therapeuticlevels of Tregs there is a need to expand obtained cells ex vivo, usually in the presenceof the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg Tcells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide withmTOR inhibitory activity that has been shown to exert immunomodulatory effects on severaltypes of immune cells. In this study we investigated the effects of Azithromycin, comparedwith Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, andmemory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells aswell as in the culture medium was measured for up to 48 h after supplemented. Tregphenotype was assessed by flow cytometry and Treg function was measured as inhibition ofresponder T-cell expansion in a suppression assay. The concentration of Rapamycin wasquantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs,while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memoryTregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increasedtheir suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retainedintracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro,further studies elucidating the effects of Azithromycin treatment on Tregs are needed todetermine its potential use.