*Purpose: 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 pharmacological inhibition of mTOR has become a critical component to obtain high numbers of functional Treg cells for clinical use. We have shown that PI3K/mTOR signaling pathway controls the metabolic switch that determines the balance between graft-reactive effector T cells and graft-protective Tregs. For the University of Kentucky Transplant Center Phase I/II clinical we have assessed the efficacy of dual mTOR/PI3K inhibitors with the standard mTOR inhibitors in the expansion and function of ex vivo expanded Tregs. The aim of this study is to compare the effect of mTOR inhibitors Everolimus and Rapamycin with dual PI3K/mTOR inhibitors PI-103 and PKI-587 in the development of functional, clinically competent primary human Treg cells for their therapeutic use in transplantation.

*Methods: CD25⁺ Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in culture with Everolimus, Rapamycin, PI-103 or PKI-587. Multi-parametric FACS analyses were used to monitor the expansion rates, viability, phenotype and suppressor function of the cells. Cellular bioenergetic parameters, including mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic rates, were assessed on an extracellular flux analyzer.

*Results: The expansion rates, viability, phenotype and suppressor function of the Treg cells in culture with Everolimus were similar to those in culture with Rapamycin and substantially better than the results obtained with dual inhibitors. In spite of the resemblances between the results with Rapamycin and Everolimus, the cells showed some differences on their cell energy metabolism profile, with lower OXPHOS rates in Rapamycin-treated cells.

*Conclusions: Our results demonstrate that pharmacological mTOR inhibition with Everolimus or Rapamycin produce better expansion of functionally competent Treg cells for clinical use when compared to dual PI3K/mTOR inhibition.

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