Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4(+)CD25(+)FOXP3(+) T regulatory cell subpopulations

Abstract

Adoptive transfer of ex vivo expanded CD4(+)CD25(+)FOXP3(+) regulatory T cells is a successful therapy for autoimmune diseases and transplant rejection in experimental models. In man, equivalent manipulations in bone marrow transplant recipients appear safe, but questions regarding the stability of the transferred regulatory T cells during inflammation remain unresolved. In this study, protocols for the expansion of clinically useful numbers of functionally suppressive and stable human regulatory T cells were investigated. Regulatory T cells were expanded in vitro with rapamycin and/or all-trans retinoic acid and then characterized under inflammatory conditions in vitro and in vivo in a humanized mouse model of graft-versus-host disease. Addition of rapamycin to regulatory T-cell cultures confirms the generation of high numbers of suppressive regulatory T cells. Their stability was demonstrated in vitro and substantiated in vivo. In contrast, all-trans retinoic acid treatment generates regulatory T cells that retain the capacity to secrete IL-17. However, combined use of rapamycin and all-trans retinoic acid abolishes IL-17 production and confers a specific chemokine receptor homing profile upon regulatory T cells. The use of purified regulatory T-cell subpopulations provided direct evidence that rapamycin can confer an early selective advantage to CD45RA(+) regulatory T cells, while all-trans retinoic acid favors CD45RA(-) regulatory T-cell subset. Expansion of regulatory T cells using rapamycin and all-trans retinoic acid drug combinations provides a new and refined approach for large-scale generation of functionally potent and phenotypically stable human regulatory T cells, rendering them safe for clinical use in settings associated with inflammation.

Figure 1.

Expansion, phenotype and suppressive activity of untreated, RAPA-, ATRA- and RAPA+ATRA-treated human Tregs. (A) CD25 and FOXP3 expression of fresh cells and 28-day untreated or drug-treated Treg lines. Dot plots show a representative example from 7 independent experiments. (B) Population doublings during in vitro expansion from 10 different T-cell lines. Cell counts were performed at weekly intervals (R) throughout culture period. (C) Expansion rates of 10 different 28-day T-cell lines cultured in the presence of only IL-2 (1000 IU/mL) or IL-2 and RAPA (100nM), ATRA (2 μM) or RAPA+ATRA. Mid lines indicate median expansion. (D) Mean fluorescence intensity (MFI) of CD25 and FOXP3 expression in 28-day Treg lines. (E) Suppressive abilities at different ratios of Treg:Teff of 28-day Treg lines. Data, mean ± s.e.m. of 9 independent experiments, are expressed as percentage of inhibition of Teff proliferation. Statistical analysis shown only on significantly different data. *P<0.05, **P<0.01, ***P<0.001.

Figure 2.

Analysis of Treg plasticity. (A) IL-17 and IFN-γ concentrations in 7-day culture supernatant from 28-day Treg cultures; cumulative mean ± s.d. from 5 experiments. (B) Percentage of FOXP3⁺CD161⁺ cells in the presence of RAPA (100 nM), RAPA+ATRA (100 nM and 2 μM, respectively) or different concentrations of ATRA; cumulative mean ± s.d. from 3 independent experiments. (C) Treg stability in the presence of pro-inflammatory milieu. IL-17 concentration in fresh CD4⁺CD25⁺ T-cell and Treg culture supernatants. IL-17 concentration was measured after 1-week culture in the absence of drugs and in the presence of only IL-2 (10 IU/mL) or 2 different cytokine cocktails. Cocktail A (CA): IL-2, IL-1β, IL-6, TGF-β. Cocktail B (CB): IL-2, IL-21, IL-23, TGF-β. (See text for details). Graphs show pooled mean ± s.d. from 3 independent experiments. Statistical analysis shown only on significantly different data. *P<0.05, **P<0.01, ***P<0.001.

Figure 3.

Interleukin 17 and IFN-γ production from in vitro expanded and in vivo infused Tregs. (A) Representative dot plots showing the gating strategy to analyze in vivo infused Tregs. (B and C) IL-17 and IFN-γ production from Treg infused in a humanized mouse model of xeno-GvHD. Cumulative plots of percentages of IL-17⁺ or IFN-γ⁺ Tregs from different number of mice (n ≥ 3). Data are representative of 3 independent experiments. (B) In vitro Treg production of IL-17 and IFN-γ. Cumulative plots of percentages of IL-17⁺ or IFN-γ⁺ T cells from 6 and 5 Treg lines, respectively. Mid lines indicate mean expression. Statistical analysis shown only on significantly different data. *P<0.05.

Figure 4.

Treg subsets expansion in the presence of different treatment. (A) Gating strategy for Treg subset cell sorting. (B and C) Cumulative data from 5 different P1 (B) and P3 (C) Treg lines after 4 rounds of stimulation. (D) Population doublings after the first round of stimulation (R1) in differently treated P1 and P3 Treg lines. Data from 5 different Treg lines. Graphs show pooled mean ± s.d. Statistical analysis shown only on significantly different data. *P<0.05, **P<0.01.

Figure 5.

Treg subset stability and CD161 expression in the presence of different treatment. (A and B) Comparison of IL-17 (A) and IFN-γ (B) production by ELISA in 7-day supernatants of P1 and P3 Treg lines. (C) Representative dot plots showing by intracellular staining the percentage of IL-17⁺ and IFN-γ⁺ cells in CD25⁺FOXP3⁺ P1 and P3 cultured with different treatments. (D) Comparison of CD161 expression in P1 and P3 Treg lines. All data are from Treg lines after 4 rounds of stimulation. Plots are representative of 5 independent experiments. Statistical analysis shown only on significantly different data. *P<0.05, **P<0.01, ***P<0.001.