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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. (

Organ transplantation is the treatment of choice for patients with advanced chronic or end-stage organ failure. The introduction and continued advances in immunosuppressive regimens during the last decades have led to a dramatic improvement in allotransplant survival rates. However, long-term use of powerful nonspecific immunosuppressants such as calcineurin inhibitors (CNIs) may cause adverse effects often linked with systemic toxicity, in particular nephrotoxicity, and may not properly control chronic immune-mediated allograft rejection.1 , 2 The CNIs tacrolimus and cyclosporine A (CsA) are currently used as the first line of therapeutic regimens in solid organ transplantation. Everolimus (EVR) is an immunosuppressive agent derivative of rapamycin (RAPA). RAPA works as a specific inhibitor of the mammalian target of RAPA (mTOR) when it is a member of the mTOR complex 1 (mTORC1), but not when it is part of the complex 2.3 Everolimus has been approved by the FDA for heart, renal, and liver transplantation,4-6 and it can be used alone or in combination with CNIs to reduce CNI-induced toxicity.7 , 8 In either case, the administration of EVR has been associated with a significant improvement in renal function and overall toxicity after transplant.9-12

Aside from pharmacologic drugs, biologic immunosuppressants have been gaining interest for their enormous clinical potential in sustaining graft acceptance. CD4+CD25hi forkhead box P3 (FoxP3)+ regulatory T (Treg) cells constitute a small fraction of T cells (approximately 1%-5% of circulating CD4+ T cells) that harbor immunosuppressive function and play a critical role in the induction and maintenance of peripheral tolerance and immune homeostasis.13 Tolerant transplant recipients show increased frequencies of Treg cells,14 whereas acute rejection with low levels of circulating Treg cells.15 Indeed, increasing evidence supports the balance between graft-reactive effector cells and graft-protective suppressor Treg cells as a determining factor in long-term allograft survival.16-18 The ability of Treg cells to inhibit the effector immune reactions that trigger graft rejection have been demonstrated in numerous preclinical studies, and Treg cells have become attractive candidates for the development of therapeutic strategies aimed at tolerance induction.19 However, the low frequency of peripheral Treg cells has become a major obstacle for their clinical application. The production of large numbers of clinical grade cells entails the ex vivo expansion of Treg cells in GMP-compatible conditions. Addition of RAPA is of standard use in these protocols because the targeting of the mTOR pathway leads to a preferential or selective expansion of Treg cells over conventional T (Tconv) cells20-23 and the stability of FoxP3 expression after in vivo transfer.24 In a recent study, we reported that the dual targeting of phosphoinositide 3-kinase (PI3K) and mTOR is also inducing the preferential expansion of mouse and human Treg cells, and the resulting cells are able to promote in vivo tolerance.23 In the same study, we showed that dual PI3K/mTOR inhibitors and RAPA induced similar changes in the phenotype and function of cultured Treg cells, although both families of drugs exert their effects through distinct alterations in the signaling and metabolic profiles. Our group has just initiated a phase I/II clinical trial with autologous Treg cell immunotherapy in kidney transplant patients receiving EVR in the conditioning regimen (NCT03284242). Consequently, we wanted to address the option of integrating a common immunosuppressive EVR-based regimen, which includes the expansion of autologous Treg cells ex vivo and the administration of the same mTOR inhibitor to the patient. However, in contrast to RAPA, little information is available regarding the effects promoted by EVR in Treg cells. Earlier studies by our group and others demonstrated the critical sensitivity of Treg cell differentiation, expansion, and function to mTOR pathway perturbations.20-32 In such terms, the aim of this study was to determine the mechanisms of action and efficacy of EVR for the clinical grade expansion of functional Treg cells.

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Ethical Approval of Studies and Informed Consent

All experimental protocols were approved by an Institutional Review Board Committee at the University of Kentucky (16-0779-F6A). Peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis from healthy donors (n = 2) after obtaining written informed consent or isolated from buffy coats of anonymous healthy donors (n = 4) provided by the Kentucky Blood Center (Lexington, KY).

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Purification and Activation of Primary Human T Cells

PBMCs were conjugated for 10 minutes at 4°C with the CliniMACS CD25 MicroBeads reagent (Miltenyi). CD25+ cells were isolated in the CliniMACS system (Miltenyi, program Enrichment 3.2) according to the manufacturer instructions. The sequential gating strategy to identify the viability and purity of Treg cells resulting from the CD25+-enrichment process is depicted in Figure S1 (SDC, Tconv cells were isolated from PBMCs by negative selection with the use of the human CD4+ T cell isolation kit (StemCell Technologies). For initial stimulation of cells, the enriched CD25+ Treg cell fraction was placed in culture in closed, gas permeable GMP cell expansion bags (Miltenyi) at a concentration of 106 cells/mL in TexMACS GMP medium (Miltenyi) supplemented with 2% autologous serum, MACS GMP ExpAct Treg cell kit (Miltenyi) at a bead-to-cell ratio of 2:1, and either RAPA (MACS GMP Rapamycin, 100 nM) or EVR (Selleckchem Chemicals, 100 nM). After 48 hours, recombinant human IL-2 (MACS GMP hrIL2, 500 IU/mL) was added. At day 5, a sample from each condition was collected for short-term analysis, and the remaining cells were restimulated with ExpAct Treg cell beads, IL-2, and RAPA or EVR at a concentration of 106/mL. Cell growth was monitored throughout the remaining of the expansion process to maintain the cell density below 2 × 106 cells/mL.

Cell surface and intracellular staining, western blot analysis, metabolic characterization, mitochondrial and autophagy changes, and methylation of the TSDR region of the FOXP3 locus were assessed as previously described23 , 25 and detailed in the Supplemental Materials and Methods (SDC,

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Data are reported as mean ± SD except where indicated. Experiments were performed at least in triplicate. Statistical significance of differences was analyzed using Kruskal-Wallis analysis of variance followed by Wilcoxon rank sum test for pairwise comparisons. Statistical analyses were performed using GraphPad PRISM 7.0. In all analyses, P <0.05 was considered a statistically significant difference.

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Isolation and Culture of CD4+CD25+ Treg Cell Population

The phenotypic analysis of the CD25+-enriched fraction showed a mean CD4+ T cell purity (±SEM) of 96.7% (±2.8%) and a CD25+/FoxP3+/CD127Low/− purity of 82.3% (±4.6%) with cell viability consistently above 98% (Figure S1, SDC, Because the mTOR inhibitor EVR was not previously described as used for the culture of Treg cells, we first assessed the optimal concentration of EVR needed for limiting the expansion of Tconv cells and increasing the population of CD25+-enriched Treg cells. After 5-day culture, the optimal concentration of EVR that resulted in the highest Treg cell and lowest Tconv cell expansion rates was determined to be 100 nM (Figure S2, SDC, Accordingly, we set this as the default concentration of EVR to culture CD4+CD25+ Treg cell-enriched cells throughout the study. All parameters were compared with the effects induced by RAPA (100 nM), the most widely used mTOR inhibitor to selectively prevent the outgrowth of contaminating Tconv cell in the in vitro culture of Treg cells.19-24

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Regulation of mTOR Signaling Pathway

Western blot analyses were performed on lysates of Treg cells cultured in expansion medium during 5 days alone or with the addition of EVR or RAPA. Figure 1 shows the significant decrease in the expression of phosphorylated 70 kDa ribosomal protein S6 kinase (p70S6k) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), 2 of the major downstream substrates of mTORC1, the canonical target of rapalogs, in EVR- and in RAPA-treated cells compared with the 100% baseline expression of untreated control cells. The mTORC1 signaling attenuation occurred with the simultaneous increase of mTORC2 activity, as measured by the phosphorylation of protein kinase B (AKT) on Ser-473. This overactivation was less prominent in EVR (217.7 ± 16.1%) than in RAPA-treated cells (372.1 ± 33.8%). In addition, a substantial increase in phospho-extracellular signal-regulated kinase (ERK) levels was also noted in response to both drug treatments (151.2 ± 4.6% and 156.0 ± 4.1%, respectively). Although the total expression of ERK was also reduced in drug-treated cells, AKT expression persisted in RAPA-treated cells. In contrast, EVR treatment promoted a significant reduction in AKT expression (75.8 ± 5.5%). The (phosphorylated/total) protein expression indexes (Figure S3, SDC, revealed that, in addition to the regulation of total protein expression, RAPA and EVR differently regulate the phosphorylation of AKT (higher in RAPA-treated cells) and 4EBP1 (higher in EVR-treated cells). In contrast, most of the differences observed in the expression levels of phospho-p70S6k derived from the regulation of total protein expression. Overall, our findings revealed distinct mTORC1/mTORC2 balances induced by RAPA and EVR in 5-day cultured Treg cells.



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Treg Cell Bioenergetics

Treg cells rely preferentially in the mitochondrial oxidation of lipids or glucose to meet cell energy demands, although the glycolytic pathway seems to be also active during in vitro expansion.32-35 In an early study, we reported the distinct bioenergetic profiles induced by RAPA and dual PI3K-mTOR inhibitors in Treg cells.23 After the same experimental approach, we evaluated whether the presence of EVR or RAPA may alter the cellular metabolism in expanding CD25+-enriched Treg cells. The oxygen consumption rate (OCR) (an indirect measurement of oxidative phosphorylation [OxPhos]) and extracellular acidification rate (ECAR) (an indicator of glycolytic flux) bioenergetic profiles were measured in 5-day cultured Treg cells and compared with Tconv cells also grown for 5 days in the same expansion medium (Figures 2 and 3 and Figure S4, SDC, EVR-treated cells exhibited reduced OxPhos across all data points relative to RAPA-treated cells, with lower baseline levels (22.2 ± 1.8 pmol/min and 30.9 ± 3.5 pmol/min) and, after trifluorocarbonylcyanide phenylhydrazone (FCCP) injection, attenuated maximal OCR (128.2 ± 9.1 pmol/min and 211.4 ± 11.6 pmol/min) and the corresponding spare respiratory capacity levels (106.0 ± 8.7 pmol/min and 180.8 ± 11.2 pmol/min), although no differences were noted on the ATP-linked OCRs (Figure 2A–D). Addition of the irreversible inhibitor of carnitine palmitoyltransferase-1, etomoxir enables the evaluation of the relative contribution of endogenous fatty acid (FA) oxidation (FAO) to OxPhos. We found lower FA-dependent (70.4 ± 5.3 pmol/min) and FA-independent OCR (57.9 ± 4.4) in EVR relative to RAPA-treated cells (123.6 ± 9.8 and 87.8 ± 2.7 pmol/min, respectively). Unlike RAPA-treated cells, only FAO (but not FA-independent oxidation) was significantly augmented in EVR-treated cells compared with Tconv cells (37.7 ± 8.2 pmol/min for FAO, and 55.1 ± 4.5 for FA-independent respiration). The FAO index (FA-dependent/FA-independent) greater than 1 in all Treg cell conditions (untreated and EVR- or RAPA-treated) illustrates the preferential use of FA as OxPhos substrate. In contrast, the low OxPhos rates in Tconv cells were mostly driven by non-FA (etomoxir-resistant) substrates. In separate ECAR experiments, addition of RAPA or EVR reduced the glycolytic profile, which may account for the observed lower expansion rates of treated compared with untreated Treg cells. When compared both drugs, EVR treatment resulted in lower ECAR baseline levels (5.9 ± 0.6 mpH/min) and in response to glucose (26.1 ± 3.0 mpH/min) than RAPA (7.5 ± 0.7 mpH/min and 30.70 ± 3.2 mpH/min) (Figue 3A–C). However, the glycolytic capacity (as measured upon injection of the OxPhos inhibitor oligomycin) and the glycolytic reserve (that represents the difference between glycolytic capacity and glycolysis rate) did not differ between both treatments (42.8 ± 7.2 mpH/min vs 50.3 ± 4.7 mpH/min and 16.7 ± 4.3 vs 19.7 ± 1.7 mpH/min, respectively) (Figure 3D and E). As expected by their strong dependency on glycolysis, cultured Tconv cell displayed a sharp increase in the glycolytic rate after the addition of exogenous glucose (54.8 ± 3.0 mpH/min) but a limited capacity to respond to oligomycin (7.3 ± 3.1 mpH/min). Similarly, addition of oligomycin promoted a significant increase in the ECAR values recorded on the OCR experiments in RAPA- and EVR-treated cells (61.9 ± 4.5% and 48.4 ± 5.5%, respectively) (Figure S5A, SDC, In contrast, ECAR levels in Tconv cells remain largely unresponsive to oligomycin. The plot of OCR versus ECAR under basal conditions and under maximal OCR and glycolytic flux (Figure S4A, SDC, illustrates the different bioenergetic profiles and relative utilization of OxPhos and glycolysis of expanding Treg cells and Tconv cells and revealed the reduced cell metabolism of 5-day-EVR-treated cells relative to RAPA. The OCR/ECAR rates (Figure S4B and C, SDC, confirmed the high glycolytic dependency of proliferating Tconv cells and the predominant OxPhos energy profiles of Treg cells. Worth noting also is that both RAPA and EVR treatments promoted the reliance of expanding Treg cells on mitochondrial respiration.





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Mitochondrial Morphology and Function

The different OxPhos activity promoted by EVR and RAPA in Treg cells may associate with changes in mitochondrial membrane potential (ΔΨm), a critical parameter of mitochondrial functional integrity. To address this possibility, we measured ΔΨm with the TMRE fluorescent dye in the 5-day expanded untreated (control) and drug-treated cells. The reliability of the TMRE uptake to measure ΔΨm was confirmed for each condition by the collapse of the ΔΨm upon preincubation with the decoupler proton ionophore FCCP. To rule out any biased results as a consequence of changes in mitochondrial mass, the TMRE loading results were normalized for mitochondrial content and were expressed as the ratios between TMRE and MitoTracker Green FM fluorescences as reported elsewhere.35 The MitoTracker Green FM results revealed the increase in mitochondrial mass in RAPA-treated cells (447 ± 45 mean fluorescence intensity [MFI]) compared with EVR (385 ± 29 MFI,) and untreated cells (367 ± 42 MFI). Once normalized, the ΔΨm results were not significantly different among different conditions (Figure 4A). Because the activation of the PI3K/mTOR pathway is known to negatively influence autophagy and RAPA is reportedly promoting autophagy in different cell settings,36 , 37 we next asked whether EVR induced similar effects in Treg cells. The analyses with the Cyto-ID Green detection reagent demonstrated that EVR also increased the formation of autophagosomal vacuoles (6288 ± 427 MFI) compared with untreated control cells (5518 ± 345). However, the vacuole formation in EVR samples was significantly lower than in RAPA samples (7245 ± 222). Addition of CLQ to measure autophagic flux produced similar accumulation of autophagosomes in response to both drugs (2143 ± 134 MFI for EVR and 2214 ± 68 for RAPA), both significantly higher than in the untreated group (1442 ± 48) (Figure 4B).



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Treg Cell Phenotype and Regulatory Activity

The presence of RAPA or EVR did not induce any significant phenotypic difference in the 5-day expanded cells. The mean fluorescent intensity (MFI) of CD25 and FoxP3 expression was similar under the 2 drug conditions, as well as the expression of other Treg cell-related cell markers including CTLA-4, CD49d (depicted in Figure S6, SDC,, PD1, PDL-1, OX40, GITR, and CD86 (data not shown). The functional properties of expanded Treg cells in the presence of EVR or RAPA were assessed by their ability to suppress the proliferation of Tconv cell. The data demonstrated the equivalent suppressive function elicited by EVR- and RAPA-Treg cells (Figure S7, SDC, The functional properties of expanded Treg cells in the presence of EVR or RAPA were assessed by their ability to suppress the proliferation of Tconv cells. The data demonstrated the equivalent suppressive function elicited by EVR- and RAPA-Treg cells (Figure S7, SDC,

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Generation of Clinical Grade Treg Cells

Manufacturing clinical grade cellular products for application in adoptive immunotherapy requires the ex vivo expansion of the original pool of cells. To validate the use of EVR to generate sufficient cell numbers of high-quality, clinical-grade Treg cells, freshly isolated CD4+CD25+ cells were expanded in the absence or in the- presence of EVR (100 nM) or RAPA (100 nM). Addition of EVR or RAPA promoted similar cell growth rates (Figure 5A and B), although the cells in EVR-medium experienced a temporary delay in early stages of culture (Figure S8, SDC, In the absence of rapalogs, the long-term expansion produced significantly larger cell yields (Figure 5B). However, these cells displayed a reduced suppressor activity when compared with drug-treated cells, whereas no significant differences in suppressive capacity were noted between both rapalog treatments (Figure 5C). The demethylation of the CpG dinucleotides at the highly conserved TSDR region of the FOXP3 locus is necessary for Treg cell lineage stability.38 Both RAPA- and EVR-treated cells show low expression of methylation levels across the 9 CpG sites of the TSDR (ranges between 5.9% and 12.1% and 6.2% to 13.1%, respectively, in 6 samples), whereas the untreated cells displayed a broader range of methylation (between 12.3% and 43.6%, n = 6) (Figure 5D). The same treatments in conventional T cells produce methylation levels at the TSDR CpG sites ranging from 81% to 97% (n = 8). The high purity of the initial CD4+CD25+ Treg cells was sustained along the 21-day ex vivo expansion period in both RAPA- and EVR-treated cells. The absence of rapalogs in the expansion cell culture produced a final population with inconsistent contamination of CD8+ T cells (ranging from less than 3% to 27% of total T cells in 6 experiments) and with a population of CD4+CD25+ displaying lower expression of FoxP3 and CD25 markers compared with EVR- or RAPA-treated cells (Figure 6A). Further phenotype analysis of these cells showed also a different profile of several phenotype markers expressed distinctly in Treg cells and Tconv cells (Figure S9, SDC,, including higher intracellular IL-10, Helios, CCR4, CTLA4, and CD36, and lower expression of TIGIT and PD1 in rapalog-treated cells (Figure 6B), whereas no substantial differences were noted between RAPA and EVR cell culture phenotypes. The functional and phenotypic similarities between long-term expanded drug-treated cells also concur with the convergence of the oxidative metabolism (Figure 7) and glycolytic rates (Figure S5B, SDC, in EVR- and RAPA-treated Treg cells, which, coincidently, displayed equivalent measurements in mitochondrial mass, mΔψ, and autophagy (not shown).







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The allosteric mTOR inhibitors RAPA and EVR are increasingly used in transplantation to minimize the dosage of CNIs in an attempt to reduce the risk of nephrotoxicity and incidence of malignancy.39-41 Comparative pharmacokinetics suggest that EVR exhibits greater intestinal absorption compared with RAPA.42 The relative hydrophobicity of RAPA makes it readily absorbed through the skin and is used in custom topical preparations.43 In contrast, RAPA systemic clearance is half that of EVR,44 , 45 which allows for EVR to reach faster steady-state levels after the initiation of treatment and faster elimination after withdrawal. To date, there are no clinical trials directly comparing EVR and RAPA in cancer therapy or transplant, and there is limited literature on the characterization of the effects induced by EVR on T cells. A comparative study by Roat et al46 among liver transplant patients under CsA or EVR revealed that patients taking EVR had a higher percentage of total and naïve CD4+ T cells than those treated with CsA, a lower percentage and functional response of CD8+ T cells, and a higher percentage of Treg cells. Levistky et al47 reported a significant amplification of newly generated and natural Treg cells in a mixed lymphocyte culture with EVR compared with mycophelonate, RAPA, and tacrolimus. Huijts et al26 showed that mTOR inhibition by RAPA or EVR increased the immunosuppressive capacity of the total Treg cell enriched population caused by the increased frequency of Treg cells but not by the alteration of the suppressive activity per cell.

Here we confirmed the advantage of adding rapalogs for the ex vivo expansion of functional, clinical grade Treg cells and performed a comparative assessment of mechanisms of action and efficacy of EVR and RAPA. Our results demonstrated a similar efficacy of EVR and RAPA to expand functional Treg cells, although EVR treatment showed an early delay in cell growth (Figure S8, SDC, During this early phase, both drugs reduced the glycolytic rates in Treg cells, but only RAPA enhanced the mitochondrial OxPhos activity compared with untreated cells (Figures 2 and 3). The oligomycin-induced inhibition of OxPhos activity resulted in the rapid metabolic shift from OxPhos to aerobic glycolysis (Figure S5, SDC,, which was further corroborated in separate ECAR experiments (Figure 3E). This oligomycin-dependent increase in glycolytic rates illustrates the metabolic plasticity of Treg cells previously suggested by Procaccini et al35 and is consistent with the ability of expanding Treg cells to use glucose as a substrate for both mitochondrial respiration and aerobic glycolysis33 , 34 even in long-term expanded Treg cells (Figure S4B, SDC, In contrast, the glycolytic rates remained steady in Tconv cell upon mitochondrial OxPhos inhibition. The robust spare respiratory capacity and high glycolytic reserve levels in proliferating Treg cells add further evidence of the cells’ metabolic adaptability to sustain their intracellular ATP demand. This bioenergetic plasticity may allow Treg cells to experience temporary metabolic stress without triggering cell death in a similar way as reported in some cancer cells.48 Our results also revealed qualitative differences between Treg cells and Tconv cells in OxPhos substrate utilization, as reflected by the major contribution of FA in Treg cells and non-FA in Tconv cells. The inability of Tconv cells to oxidize glucose suggests the use of alternative non-FA, likely amino acids,49-50 as preferential mitochondrial substrates. In the context of the ex vivo expansion of Treg cells, with the cells growing under conditions of unlimited nutrient availability, RAPA-treated cells appear to exhibit more active metabolism than EVR-treated cells during the early culture phase, which may account for their faster cell expansion growth seen in our study. As suggested for CD8+ memory T cells,49 the increase in mitochondrial mass (Figure 4A) may contribute to the higher oxidative and glycolytic capacity of RAPA-treated Treg cells.

The differences between EVR and RAPA in the regulation of the Treg cell metabolic responses were associated with a different pattern of mTOR signaling activation. Both drugs produced similar attenuation on the phosphorylation of 4EBP1 and p70S6k, 2 main downstream effectors of mTORC1, as well as similar compensatory overactivation of ERK. However, the balance between mTORC1 and mTORC2 activities was differently perturbed; although both treatments increased the expression levels of mTORC2-dependent phosphorylation of AKT in Ser-473 when compared with untreated control cells, the increment was significantly lower in EVR-treated cells. The reduced total AKT expression in EVR-treated cells is likely contributing to the partial overactivation of AKT. However, we cannot discard the participation of different feedback loops and/or compensatory mechanisms within the complex PI3K/AKT/mTOR signaling cluster.27 Because the activation of mTORC2-dependent AKT is a critical marker for increased glycolysis in T cells51 and in agreement with the mTORC2 necessary role in cell growth, proliferation, and survival,28-30 we can rationally speculate a functional link between the weaker activation of AKT, the reduced glycolytic phenotype, and the slower proliferative rates in the early stages of EVR-treated Treg cell culture. In contrast, the suppressive function or the phenotypic profile of expanding Treg cells were independent of this metabolic and signaling fluctuations, which is consistent with the different molecular circuitries that regulate expansion and suppressor activity in de novo differentiated FoxP3+ Treg cells described in a previous study.25 This possibility is also in line with the direct regulation of the suppressive function of Treg cells by mTORC1.31 , 32

The increased activity of mTORC1 is generally perceived as a potent inhibitor of autophagy52 and, consequently, the mTORC1-inhibitor RAPA is widely used to induce autophagy.37 Consistent with the same mTORC1 inhibitory capacity of RAPA and EVR in Treg cells, both drugs induced similar increase of autophagic flux. A potential cause for the increased autophagosome formation in RAPA-treated Treg cells may rely on their high mitochondrial mass.53 , 54 The combination of high autophagosome formation and mitochondrial mass raised the possibility of autophagic stress in RAPA-treated cells. However, long-term cell viability, expansion, phenotype, and function did not differ between EVR and RAPA treatments, suggesting that the differences in autophagy processes did not exceed a threshold value to elicit any measurable damaging effect in the cell. In this context, the metabolic plasticity of Treg cells and their ability to redirect the energy metabolism toward glycolysis may also contribute to minimizing the potential damage associated with high mitochondrial OxPhos activity.55 Additional evidence against the likelihood of autophagy stress induction in RAPA-treated Treg cells was generated. First, we previously reported that autophagy-deficient Treg cells exhibit a significant decrease in ΔΨm as well as metabolic and functional deficits25; in contrast, none of these parameters were similarly altered in the current study after the exposure of Treg cells to RAPA. Importantly, the suppressor activity was consistently equivalent between both treatments throughout the ex vivo expansion process. Second, our findings suggest that the differences induced by EVR and RAPA in day 5 of the ex vivo culture are temporary, as evidenced by the subsequent expansion rates as well as the phenotypic, functional, and metabolic profile progressions of both cell treatments.

In the absence of EVR or RAPA, the expansion of Treg cells, even in our conditions of low activation, may produce a significant degree of contaminant CD8+ non-Treg cells. In addition, the fact that the expression levels of standard Treg cell markers such as CD25 and FoxP3 are low in untreated CD4+ T cells brings into question the degree of purity of these Treg cells, further unsettled by the higher methylation status of the TSDR-FOXP3 region. In the absence of Treg cell-specific membrane markers, the discrimination between effector and Treg cells for clinical use may be challenging. From the results generated in this study, we are currently analyzing the link between the different expression of TIGIT and CD36 in untreated and treated Treg cells and their functional capacities. On the other hand, the genomic location of FOXP3 on X chromosome should caution from a sex-biased expression. X chromosome inactivation in female mammals generates a transcriptionally silent inactive X chromosome (Xi) that, in case of FOXP3, remains highly methylated.56-58 However, sex differences remain on the methylation status of the FOXP3 gene even after corrected with a factor of 2. Although there is no evidence to date that FOXP3 is among the immune-related genes that escape X chromosome inactivation in humans,56 , 59 other sex-specific differences are arising with respect to the increased expression of FOXP3 in females, including androgen-dependent sensitivity of FOXP3 expression60 and the potential role of some functional FOXP3 variants.61-66 These sex-specific epigenetic states and regulatory cues are likely to have important implications for understanding sex dimorphic variability of Treg cells in health and disease and strongly support the stratification of the Treg cell studies based on sex.

Similar to our pilot study (NCT03284242), the polyclonal Treg cell yield required in phase I/II clinical trials is in the range of 1 to 10 × 108 cells. We choose to expand the cells in a rather low activation regimen (relative low dose of IL2 and bead concentration) to reduce the response of potential contaminating effector cells. Addition of RAPA or EVR will further support this purpose while allowing an expansion rate of 80 times the original cell yield. From the initial leukapheresis product, we obtain a number of CD25+ Treg cells ranging from 50 to 80 × 106, which allow us to reach (and exceed) the intended Treg cell number. In agreement with others,20-22 our results suggest that the absence of rapalogs in the expansion cell culture may represent a significant risk of contamination with unwanted non-Treg cells.

Our results also revealed the efficacy of ex vivo EVR Treg cell expansion and support EVR as a potential alternative to RAPA in the generation of clinical grade Treg cells. We reported several novel key findings regarding the distinct mechanisms of action of EVR in short-term cultured Treg cells, including the lower mTORC2 activity associated with an overall reduced metabolism and slow early expansion rates compared with RAPA. This initial EVR-Treg cell expansion delay, however, was overcome at later stages, and both RAPA and EVR treatments produced a similar number of competent Treg cells with equivalent phenotype and functional suppressive activity. Overall, our findings support the implementation of a common immunosuppressive EVR-based regimen in the transplant patient that includes the adoptive infusion of ex vivo EVR-expanded autologous Treg cells.

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