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mTOR Inhibitor Everolimus in Regulatory T Cell Expansion for Clinical Application in Transplantation

Gedaly, Roberto MD1; De Stefano, Felice MD1; Turcios, Lilia PhD1; Hill, Marita2; Hidalgo, Giovanna2; Mitov, Mihail I. PhD3,4; Alstott, Michael C.3; Butterfield, D. Allan PhD3,5; Mitchell, Hunter C.1,6; Hart, Jeremy MD2; Al-Attar, Ahmad MD2; Jennings, Chester D. MD2; Marti, Francesc PhD1

doi: 10.1097/TP.0000000000002495
Original Basic Science—General

Background. Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells 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 Treg cells 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. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin 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 protein kinase B compared with 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.

1 Transplant Division, Department of Surgery, University of Kentucky, College of Medicine, Lexington, KY.

2 Department of Pathology and Laboratory Medicine, University of Kentucky, College of Medicine, Lexington, KY.

3 Redox Metabolism (RM) Shared Resource Facility (SRF), Markey Cancer Center, University of Kentucky, College of Medicine, Lexington, KY.

4 Idaho College of Osteopathic Medicine (ICOM), Meridian, ID.

5 Department of Chemistry, University of Kentucky, College of Medicine, Lexington, KY.

6 Asbury University, Wilmore, KY.

Received 9 May 2018. Revision received 9 October 2018.

Accepted 11 October 2018.

R.G. and F.M. contributed equally to this article.

The authors declare no conflicts of interest.

This research was supported by the National Institute of Allergy and Infectious Diseases (NIAID) NIH grant R03-AI135592 to F.M., and by the National Center for Research Resources and the National Center for Advancing Translational Sciences, NIH grant UL1TR001998 to R.G. and F.M. 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.

R.G. and F.M. designed research. F.D.S., L.T., G.H., M.I.M., M.C.A., H.C.M., and F.M. collected data. M.H., D.A.B., J.H., C.D.J. contributed analytic tools. R.G., A.A.T., and F.M. analyzed data. R.G. and F.M. wrote the article. All authors participated in the Critical editing of content. All authors approved the final version.

Clinical Trial Notation: National Cancer Trial Registry; NCT03284242.

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (

Correspondence: Roberto Gedaly, MD, University of Kentucky Transplant Center 740 South Limestone, K301 Lexington, KY 40536-0284. (

Francesc Marti, PhD, University of Kentucky Transplant Center 740 South Limestone, K301 Lexington, KY 40536-0284. (

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