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

Abstract

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.

Trial registration: ClinicalTrials.gov NCT03284242.

Conflict of interest statement

Disclosure: The authors declare no commercial or financial conflicts of interest

Figures

Figure 1.. Effect of rapamycin and everolimus on the expression of AKT/mTOR signaling proteins in Treg cells.

Cells were expanded in Treg expansion medium either alone or with the addition of 100nM of either RAPA (R) or EVR (E). After 5 days, lysates from 106 Treg cells were subjected to western blot analysis to determine the levels of phosphorylated (TOP) and total (BOTTOM) expression of the mTORC2 substrate AKT, the mTORC1 substrates 4EBP1 and p70S6k, and ERK. β-actin expression was used to normalize the loaded protein levels in each line (indexes indicated as numerical values at the corresponding line in the bottom panel) of both phosphorylated and total protein blots. Right panels illustrate the average ± SD of band relative densities pooled from three different experiments (n=3) and shown as percentage of individual protein expression (phosphorylated -TOP- and total –BOTTOM-) relative to untreated-control cells (leveled at 100 and depicted as a dashed line). *p<0.05 and **p<0.001 indicate significant difference between RAPA and EVR-treated cells as determined by Wilcoxon’s rank sum test. NS indicate no significant differences between both treatments.

Figure 2.. Effect of rapamycin and everolimus on the oxygen consumption rates of Treg cells.

(A) Oxygen Consumption Rates (OCR, pMoles/min) were measured in 5 day-expanded Treg cells in the presence of 100nM of rapamycin (closed circles) or everolimus (open circles). The results were compared with the profile generated with untreated Treg cells (Control, closed squares) and conventional T cells (Tconv, closed triangles) cultured in the same Treg cell expansion medium for 5 days. (B-F) The OCR parameters were generated from a modification of the Seahorse XF Mito Stress Test Report Generator detailed in SDC, Material and Methods, and are shown as follows: (B) Baseline OCR, (C) ATP-linked oxygen consumption, (D) Maximal respiratory capacity -Max. Resp.-, (E) (F) non fatty acid oxidation-driven capacity -FA-independent-, and (E) endogenous fatty acid oxidation-driven capacity -FA-dependent.-, ().-, Data represents the mean ± SD pooled from at least three independent (n=3) experiments and five replicates per condition in each experiment. *p<0.05 and **p<0.001 indicate significant differences as measured by Kruskal–Wallis ANOVA followed by post-hoc Wilcoxon test to assess treatment-specific differences. NS indicate no significant differences.

Figure 3.. Effect of rapamycin and everolimus on the extracellular acidification rates of Treg cells.

(A) The extracellular acidification rates (ECAR, mpH/min) were measured in Treg cells expanded 5 days in the presence of 100nM of rapamycin (closed circles) or everolimus (open circles), and compared with 5 day-expanded untreated Treg cells (Control, closed squares) and conventional T cells (Tconv, closed triangles) cultured in the same Treg cell expansion medium. (B-E) The ECAR parameters were generated with the Seahorse XF Glycolysis Stress Test Report Generator and are shown as follows: (B) Baseline ECAR, (C) Glycolysis, (D) Maximal glycolytic capacity -Glycol. Capacity-, and (E) Glycolytic reserve -Glycol. Reserve-. Data is showing as the mean ± SD pooled from at least three independent (n=3) experiments and five replicates per condition in each experiment. *p<0.05 and **p<0.001 indicate significant pairwise differences in Wilcoxon test following Kruskal–Wallis ANOVA test. NS indicate no significant differences among treatments.

Figure 4.

Effect of rapamycin and everolimus on the mitochondrial integrity and autophagy in Treg cells were measured by flow cytometry analysis. Histograms (left) are representative single individual experiments of the corresponding bar-graphs (right) depicting the pooled (n=6) average mean fluorescence intensity (MFI)± SD from 5-day untreated (CTRL), 100nM rapamycin-treated (RAPA), or 100nM everolimus-treated (EVR) expanded Treg cells. (A) Mitochondrial mass (TOP) was measured with MitoTracker Green, which is independent from the mitochondrial membrane potential (mΔΨ). (BOTTOM) Changes in mΔΨ were analyzed by flow cytometry with the fluorescent probe TMRE. The resulting MFI within cells treated only with TMRE was corrected from the corresponding MFI of cells pre-treated with FCCP. Results for each condition are shown as the rate between TMRE increments (±FCCP) and the corresponding MitoTracker Green value, which normalize mΔΨ per equal mitochondrial mass. (B) The measurements of autophagic vacuole formation (TOP) were performed with the Cyto-ID autophagy detection kit. The accumulation of autophagosomes after the addition of the autophagic inhibitor Chloroquine (CLQ) for 6 hours before the addition of Cyto-ID Green solution was used to determine the autophagic flux (BOTTOM). The autophagosome flux is shown as the average MFI mean fluorescence intensity increase in cells pre-treated with CLQ compared to the cells in the absence of CLQ. Statistical differences among treatments were tested by Kruskal–Wallis ANOVA followed by post-hoc Wilcoxon test to assess treatment-specific differences. *p<0.05 and **p<0. 001 indicate significant pairwise differences; NS, no significant differences.

Figure 5.. Long-term effect of rapamycin and everolimus on the expansion, FOXP3 TSDR methylation, suppressor function and phenotype of Treg cells.

CD4+CD25+ cells were expanded in Treg expansion medium in the presence of 100nM RAPA or 100 nM EVR for 21 days. (A) Cell numbers for RAPA (closed circles) or EVR (empty circles)-treated cells were quantified every 2–4 days. The plot illustrates the temporal fold increase in cell numbers with respect to the initial seeding. (B) Cell yield corresponding to the 21-day expansion in untreated (C), RAPA-treated (R), or EVR-treated (E) cells shown as interquartile (25/75) range box-and-whiskers plots with median and individual values of n=4 measurements. (C) Suppressive activity induced in allogeneic CFSE-labeled responder T cells in co-culture for 72 hours with different of Treg:Target labeled cell ratios. Suppressor activity was measured by the decrease of division indexes in the CFSE-labeled target cells. (D) Methylation status (as percentage) of the TSDR FOXP3 in untreated (C), RAPA-treated (R), or EVR-treated (E) cells is shown as interquartile range box-and-whiskers plots with median and individual values of n=6 measurements.

Figure 6.. Long-term effect of rapamycin and everolimus on the phenotype of Treg cells.

Representative phenotype analysis of 21-day expanded CD4+/FoxP3+/CD25+ population (A) Top dot-plots represent the expression of FoxP3 and CD25 in cell cultures in the absence (Control) or in the presence of RAPA or EVR. Bottom panels depict CD25/FoxP3 countour plots of untreated (grey lines) overlaid with treated (black) cells. The corresponding GeoMean Fluorescence Intensities of FoxP3 and CD25 are indicated inside the panels (grey: untreated / black: treated). (B) Histogram plots comparing the expression profiles of different proteins in untreated (shaded), RAPA-treated (dotted line) and EVR-treated (solid line) cells. These proteins are differentially expressed in Tregs and Tconv cells (see Figure S7 in SDC). The expression levels of the different proteins are indicated inside the panels as GeoMean Fluorescence Intensities (Gm) or as percentages of positive cells for untreated, RAPA-treated and EVR-treated cells (first / second / third value, respectively). FMO controls are depicted as dashed lines. This is a representative of 3 different experiments.

Figure 7.. Long-term effect of rapamycin and everolimus on oxygen consumption rates of Treg cells.

(A) The same CD4+CD25+ cells expanded in Treg expansion medium in the presence of 100nM RAPA (closed circles) or 100 nM EVR (empty circles) for 21 days depicted in Figure 5A were used to analyze the oxygen consumption rate (OCR), as in Figure 2. (B) Baseline OCR and maximal respiratory capacity (Max. Resp.) levels for RAPA- and EVR-treated cells were generated as in Figure 2. Fatty acid-dependent (FAd) and fatty acid-independent (FAi) rates are shown as stacked columns in Max. Resp. (*) indicates a significant (p<0.05) difference between RAPA and EVR treatments as measured by Wilcoxon’s rank sum test out of 4 independent experiments and five replicates per condition in each experiment; NS, not significant difference