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. *Both authors contributed equally to this manuscript #Corresponding Authors: Roberto Gedaly, MD and Francesc Marti, PhD, University of Kentucky Transplant Center, 740 South Limestone, K301, Lexington, KY, 40536-0284, USA, Email: rgeda2@uky.edu & fmart3@uky.edu. Tel: 1-859-323-4661, FAX: 1-859-257-3644 Clinical Trial Notation: National Cancer Trial Registry; # NCT03284242 Authorship: Designed research: RG and FM Collected data: FDS, LT, GH, MIM, MCA, HCM and FM Contributed analytic tools: MH, DAB, JH, CDJ Analyzed data: RG, AAT and FM Wrote the manuscript: RG and FM Critical editing of content: all authors Approval of final version: all authors Disclosure: The authors declare no commercial or financial conflicts of interest Funding sources: This research was supported by the National Institute of Allergy and Infectious Diseases (NIAID) NIH grant R03-AI135592 to FM, and by the National Center for Research Resources and the National Center for Advancing Translational Sciences, NIH grant UL1TR001998 to RG and FM. The Redox Metabolism Shared Resource (RMSR) and the University of Kentucky Flow Cytometry and Immune Monitoring (FCIM) core facilities received support from the National Cancer Institute (NCI) NIH Cancer Center Support Grant P30CA177558 awarded to the University of Kentucky Markey Cancer Center. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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