mTOR Inhibitor Everolimus in Regulatory T cell Expansion for Clinical Application in Transplantation.

BACKGROUND: Experimental and pre-clinical evidence suggest that adoptive transfer of regulatory T cells (Tregs) could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel Phase I/II clinical trial with ex vivo expanded autologous Tregs as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen.

METHODS: The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux and suppressor function of the cells. PI3K/AKT/mTOR signaling pathway profiles of treated cells were analyzed by western blot and cell bioenergetic parameters by extracellular flux analysis.

RESULTS: EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of AKT compared to RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA.

CONCLUSIONS: Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.