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Differential Effects of Calcineurin and Mammalian Target of Rapamycin Inhibitors on Alloreactive Th1, Th17, and Regulatory T Cells

Gallon, Lorenzo, MD1,2; Traitanon, Opas, MD1,2; Yu, Yuming, MD2,3; Shi, Bo, PhD4; Leventhal, Joseph R., MD, PhD2,3; Miller, Joshua, MD2,3; Mas, Valeria, MD, PhD6; L, Xu, PhD1,2; Mathew, James M., PhD2,3,5

doi: 10.1097/TP.0000000000000717
Original Basic Science—General
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Background Previously, we had reported the role of tacrolimus (TAC) versus sirolimus (SRL) on the generation of regulatory T cells (Tregs) in primary MLR assays with SRL, demonstrating a uniquely supportive effect. However, the mechanisms associated with their actions on alloreactive human T cells are not fully understood. Therefore, we tested whether TAC and SRL differentially affect already alloactivated human CD4+ T-cell subsets.

Methods Alloreactive CD4+CD45RA/CD45RO+ T cells generated in 9-day MLR were cocultured with anti-CD3 and autologous antigen presenting cells plus interleukin (IL)-2 in presence of TAC, SRL, or both, and the Tregs generated after another 5 to 6 days were phenotypically, molecularly, and functionally characterized.

Results Tacrolimus significantly and SRL modestly inhibited interferon (IFN)-γ (Th1) and IL-17 (Th17)–producing cells. At clinical therapeutic concentrations, SRL, however, significantly increased forkhead/winged helix transcription factor P3 (FOXP3+) Tregs, whereas TAC inhibited this T-cell population dose dependently and significantly. When used in combination, TAC and SRL had additive effects on inhibition of IFN-γ– and IL-17–producing cells. This was in contrast to the ability of SRL to reverse TAC-mediated inhibition of FOXP3-expressing cells. Proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) added to cultures caused significant decrease in FOXP3+ Tregs that was again reversed by SRL. Sirolimus-derived Tregs were phenotypically normal, anergic to allostimulation, and suppressed proliferation of allogeneic effector T-cells.

Conclusions Thus, although TAC inhibits all alloreactive T cells, SRL promotes the differentiation and expansion of donor-specific Tregs without secondary reprogramming to IFN-γ+FOXP3+ and IL-17+FOXP3+ Treg subsets. These results, although performed in an artificial in vitro model, add clinically applicable information on how these agents affect T-cell subpopulations.

The authors find that tacrolimus (TAC) vs sirolimus (SRL) differentially affect already alloactivated human CD4+ T IFN-γ+, Th17+ or FOXP3+-cell subsets. While TAC inhibits all alloreactive T cells, SRL promotes donor-specific Treg without secondary reprogramming to IFN-γ+FOXP3+ and IL-17+FOXP3+ Treg subsets.

1 Department of Medicine-Nephrology, Northwestern University Feinberg School of Medicine, Chicago, IL.

2 Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL.

3 Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL.

4 Department of Medicine and Rheumatology Division, Northwestern University Feinberg School of Medicine, Chicago, IL.

5 Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL.

6 Department of Surgery, University of Virginia, Charlottesville, VA.

Received 9 September 2014. Revision requested 10 December 2014.

Accepted 14 December 2014.

The authors declare no funding or conflicts of interest.

L.G. participated in research design, data analysis and the writing of the paper. O.T. participated in research performance, data analysis and the writing of the paper. Y.Y. participated in research performance and data analysis.B.S. participated in research performance and data analysis. J.L. participated in research design and the writing of the paper. J.M. participated in data analysis and review of the manuscript. V.M. participated in research design and the writing of the paper. L.X. participated in research design, research performance, data analysis and the writing of the paper. J.M.M. participated in research design, data analysis and the writing of the paper.

Correspondence: Lorenzo Gallon, MD, Comprehensive Transplant Center, Northwestern University, Feinberg School of Medicine, Arkes Family Pavilion, Suite 19, 676 North St. Clair Street, Chicago, IL 60611-2923. (l-gallon@northwestern.edu) and James M. Mathew, PhD. (james-mathew@northwestern.edu).

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 (www.transplantjournal.com).

CD4+ T cells play an important role in allograft rejection as well as tolerance. They can be classified as naive and into subsets of effector T helper cells (Th1, Th2, and Th17) and regulatory T cells (Tregs). Upon activation and expansion, different effector T helper cell subsets produce distinct cytokines and mediate separate functions.1-3 In the transplant setting, both Th1 and Th17 cells and their cytokines interferon (IFN)-γ and interleukin (IL)-17 are involved in allograft rejection, whereas Tregs favor long-term graft survival, and Th2 cells and their cytokines have differential effects.4-7 Therefore, a shift toward graft-protective Tregs may promote clinical transplant tolerance.8 By contrast, proinflammatory cytokines, such as IL-1β, IL-6, and tumor necrosis factor (TNF)-α, favor the generation of donor-reactive Th1/Th17 cells while preventing the commitment of T cells into Tregs.2,3,9 Given the plasticity of regulatory into inflammatory T cells,2-4,9 treatments targeted at stabilizing Tregs might favor long-term graft acceptance.4,6,8

Calcineurin inhibitors (CNI), such as tacrolimus (TAC), usually nonspecifically reduce the activity of both tissue-destructive Th1 and Th17 and tissue-protective Treg cells,10 whereas mammalian target of rapamycin (mTOR) inhibitors like sirolimus (SRL) augment Treg and memory T cells.11-18 Mechanistically, CNIs inhibit T cell receptor (TCR)-induced calcineurin/nuclear factor of activated T cells-translocation to block IL-2 transcription, or directly interfere with nuclear factor of activated T cells/forkhead/winged helix transcription factor P3 (FOXP3) interaction, leading to a reduced function of both effector T cells and Tregs.19,20 On the other hand, mTOR inhibitors directly bind the TORC1 and inhibit the PI3K/AKT/mTOR signaling, which is part of CD28 costimulatory and IL-2 receptor signaling pathways in generating full effector T-cell activation.21

Previously, we had reported the role of TAC versus SRL on the generation of Tregs in primary MLR assays with SRL, demonstrating a uniquely supportive effect.22 In the present study, we questioned if these agents alone or in combination will have varying effects on the differentiation and expansion of alloreactive effector Th1/Th17 versus protective Tregs by examining associated mechanisms. To do this, already activated alloreactive CD4+CD45RO+ T cells were restimulated nonspecifically by low concentrations of anti-CD3 plus unstimulated autologous antigen presenting cells (APCs).23,24 Because in vitro induction of Tregs with high concentrations of anti-TCR/CD28 plus transforming growth factor (TGF)-β resulted in Tregs with unstable FOXP3 expression,25-27 we also included a high concentration of IL-2 as a stabilization mediator. Such a standardized culture system caused the generation of all 3 alloreactive subsets, that is, IFN-γ+ Th1, IL-17+ Th17, and FOXP3+ Tregs. This provided us with an ideal plastic/variable experimental system to study the mechanism of action by the immunosuppressive drugs on these T-cell subsets in alloimmunity in vitro.

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MATERIALS AND METHODS

Subjects

Human peripheral blood samples were obtained from laboratory volunteers after informed consent under a Northwestern University Institutional Review Board-approved protocol.

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Reagents

Sirolimus and TAC were from Axxora (San Diego, CA). Human IL-1β, IL-6, and TNF-α were from R&D system (Minneapolis, MN). Anti-CD3 (UCHT1) and anti-CD28 (L293) monoclonal antibodies were from BD Biosciences (San Diego, CA). Anti-CD3/CD28-coated microbeads were from Invitrogen (Carlsbad, CA).

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Cell Isolation and Cultures

CD4+CD45RA+ T cells and CD14+ monocytes were positively selected from Ficoll-Hypaque purified peripheral blood mononuclear cells (PBMCs) using magnetic microbeads from Miltenyi Biotech (Carlsbad, CA). The cells were 95% to 99% of the targeted populations as detected by flow cytometric analysis (data not shown). These CD4+CD45RA+ naive T cells (1.5 × 106/well) and allogeneic irradiated CD14+ cells (7.5 × 105/well) were cocultured in 24-well culture plates in complete media consisting of RPMI 1640 with 15% normal human AB serum, 2 mM l-glutamine, 10 mM Hepes and 1× pen/strep/glutamine solution (GIBCO-BRL, Gaithersburg, MD). This culture step was designated as primary MLR.

After 7 to 11 days, CD4+CD45RA/CD45RO+ cells were purified by depleting residual CD45RA+ cells and then positively selecting CD4+ T cells using magnetic microbeads. These alloreactive CD4+CD45RA/CD45RO+ T cells were rested overnight and were cocultured (1 × 105/well) in 96-well U-bottom plates with 100 ng/mL anti-CD3 and autologous CD14+ cells (0.5 × 105/well) or anti-CD28 and with 20 to 100 U/mL IL-2. Tacrolimus, SRL, or both were added at the beginning of these secondary cultures. Interleukin-1β (10 ng/mL), IL-6 (20-100 ng/mL), and TNF-α (50 ng/mL) were also added alone or in combination as indicated. After another 5 to 6 days, the phenotypic, molecular, and functional characterizations of the responding cells were made as described below.

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Cell Surface and Intracellular Staining

The cells were first stained for surface markers with fluorochrome-conjugated anti-CD4, -CD25, -CD127, -CD45RO, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), glucocorticoid-induced TNF receptor–related protein monoclonal antibodies (all from BD Biosciences) using standard procedures. For intracellular staining, the cells were subsequently fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences). Then, phycoerythrin (PE)-Cy5-anti-FOXP3 (PCH101) Regulatory T-cell Staining Kit (eBioscience) or rabbit polyclonal anti–DNA methyltransferase 1 (DNMT1) (H-300) and anti-rabbit immunoglobulin-PE (Santa Cruz, CA) were used for intracellular FOXP3 or DNMT1 staining following the manufacturer’s protocols. When cytokine secreting cells were enumerated, the cultures were restimulated with 20 ng/mL phorbol 12-myristate 13-acetate and 500 ng/mL ionomycin plus GolgiStop for 6 hours before surface staining; fluorescein isothiocyanate–anti-IFN-γ, –––anti-IL-10, and PE-anti-IL-17, −anti-TGF-β (BioLegend, San Diego, CA) were used for intracellular staining. Data were acquired on a FACSCalibur flow cytometer and analyzed by CellQuest software (BD Biosciences).

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Cytokine Assays

Supernatants were collected at the termination of the culture period and stored at −20 °C. Cytokines were quantified with the flowcytomix cytokine assay kit (eBioscience) collecting approximately2000 events gated for the target cytokines on a Beckman Coulter CMP500 flow cytometer, and using FlowCytomix Pro 2.4 software (eBioscience) for data analysis.

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Western Blotting Analysis

Western blot was performed as previously described.28 Anti-pAKT (Ser473) and anti-pFOXO3a (Ser253) were from Cell Signaling Technologies (Boston, MA).

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Quantitative Real-Time Polymerase Chain Reaction Analysis

For transcription factor expression measurements, real-time polymerase chain reactions (PCRs) were performed. Total RNA was extracted from secondary readout cultures using the RNeasy Mini Kit (Qiagen, Valencia, CA). Two micrograms of total RNA was then reverse transcribed using high capacity RNA-to-cDNA kit, and transcripts were quantified by real-time quantitative PCR using TaqMan Fast Universal PCR kit on an ABI 7500 Fast Real-Time PCR System (all RT-PCR reagents from Applied Biosystems, Foster City, CA). The primers used for transcription factors T-bet (Hs00203436_m1), RORγ;t (Hs01076112_m1), GATA-3 (Hs00231122_m1) and FOXP3 (Hs00203958_m1) were also from Applied Biosystems. Reactions were carried out using TaqMan Universal PCR Fast Master Mix and the amplification conditions were: 95 °C for 10 minute, 40 cycles of 95 °C for 3 seconds, and 60 °C for 30 seconds. Specific gene expression was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Expression level of specific messenger RNA (mRNA) was calculated by first determining the average threshold cycle (ΔCt) for each culture, which corresponded to the following: (average specific gene threshold cycle − average GAPDH threshold cycle) from triplicate cultures. The replicate threshold cycle (ΔΔCt) was then calculated with the following formula: (ΔCt with drug − ΔCt without drug). Transcription factors T-bet, RORγt, GATA-3, and FOXP3 mRNA were expressed as fold GAPDH value (2−ΔΔCt).

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Suppression Assays

CD4+CD25highCD127−/low Tregs from the readout cultures treated with 10 ng/mL SRL were purified using Treg isolation kit (Miltenyi Biotech). The capacity of the Tregs to suppress T-cell responses were assessed by their addition at indicated numbers to newly setup MLR cultures. In these MLRs, fresh autologous CD4+CD25 responders (5 × 104) were stimulated with irradiated PBMC (1 × 105) from the original allogeneic donor used in the primary MLR or a third party in presence of graded number (1,000, 5,000, and 25,000) of purified Tregs or additional autologous PBMC as controls. Suppressive capacity was assessed by thymidine incorporation on day 7 as described previously22 and using the formula:

In another approach, carboxyfluorescein succinimidyl ester (CFSE)–labeled CD4+CD25 responder cells were stimulated with irradiated PBMC from the original allogeneic donor used in the primary MLR or a third party in presence of PKH26-labeled Treg cells or PBMC controls and flow cytometric analyses were performed after 5 to 7 days. Indicated surface and intracellular markers and indicated responder subsets were analyzed after gating out the PKH26-labeled Tregs. It was reasoned that since the Tregs did not proliferate even after allogeneic stimulation (please see below), they would not dilute PKH26 and would not contribute to the dye (CFSE) diluted/negative cells analyzed as proliferating responders.

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Statistical Analysis

Significance (P < 0.05) was determined by Student paired and/or 2-tailed t test. Data are presented as mean ± SD.

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RESULTS

Antigen Presenting Cells and IL-2 are Essential for Optimal Induction of Alloreactive Th17 and Treg Cells (Optimization of Cell Cultures)

CD4+CD45RA+ naive T cells (that might also include minor population of terminally differentiated RA+ cells) from laboratory volunteers were stimulated with allogeneic APCs in primary MLR. This resulted in these T cells differentiating into CD45RO+ effector/memory cells peaking on day 9. These cells were IFN-γ+Th1 and FOXP3+Treg subsets, with very few (∼1%) IL-17+Th17 cells (Figure 1).

FIGURE 1

FIGURE 1

The CD4+CD45RA/CD45RO+ T cells isolated from the above primary cultures were then stimulated with submitogenic dose of anti-CD3 (100 ng/mL) in the absence or presence of costimulatory signals provided by anti-CD28 or autologous APCs to further expand the T cell subsets. Anti-CD3 plus autologous APC-treated cultures (hereafter referred as readout cultures) generated significantly higher IL-17+ Th17 cells in these secondary cultures than those of anti-CD3 plus anti-CD28 (Figure 2A). Addition of low concentrations of exogenous IL-2 into the readout cultures expanded the Th1 and Th17 cells; however, high concentrations (100 U/mL IL-2) significantly enhanced the CD4+FOXP3+ cells (n = 4, P < 0.05; Figure 2B). Taken together, these results suggested that in alloreactive CD4 T cells, optimal induction and maintenance of Th1, Th17, and Treg cells required weak TCR stimulation and additional cross-talk with APCs as well as the concomitant presence of IL-2. Using this experimental readout, we analyzed associated mechanisms as follows:

FIGURE 2

FIGURE 2

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TAC and SRL Differentially Inhibit Th1 and Th17 in the Readout Alloreactive CD4 T-Cells

The presence of TAC even at subtherapeutic dose (2 ng/mL) blocked the expansion of IFN-γ– and IL-17–producing cells by more than 90% (Figure 3A) even in the presence of 100 U/mL IL-2 (Figure 3B), whereas at therapeutic concentrations (5 ng/mL) inhibited all cellular growth. Therefore, hereafter the data with 2 ng/mL or less of TAC will only be shown (although other doses were also tested). Sirolimus, by contrast, at therapeutic concentrations (≥5 ng/mL) caused moderate inhibition of Th1 and Th17 cells (30% and 60%, respectively, Figure 3A), and the presence of IL-2 reversed the inhibition of Th1 cells (Figure 3B). However, combination of the 2 drugs at therapeutic levels showed significant inhibition on both IFN-γ– and IL-17–producing cells (Figure 3A and B).

FIGURE 3

FIGURE 3

In parallel experiments, when culture supernatant cytokines were measured, TAC strongly blocked IFN-γ and inhibited IL-17 production, in agreement with intracellular staining described above. However, SRL showed more profound inhibition of IFN-γ and IL-17 secretion into the culture media (Figure 3C and D) in contrast to the relatively lower inhibition of the percentages of Th1 and Th17 cells (Figure 3B). Altogether, these results suggested that TAC completely blocked the differentiation and expansion of preformed alloreactive Th1 and Th17 cells, whereas SRL caused only moderate inhibition, and it was more effective in inhibiting Th17 than Th1.

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TAC and SRL Differentially Affect the Proportion of FOXP3+ Cells in the Readout Alloreactive CD4 T Cells

To determine the effect of TAC and SRL on Treg differentiation and expansion, enriched alloreactive CD4+CD45RA T cells were cocultured with autologous APCs plus anti-CD3 together with these agents. In preliminary experiments without or with low concentrations of IL-2 (≤20 U/mL), both SRL and TAC had no significant effects on the percentage of cells expressing FOXP3 (data not shown). In the presence of high concentration of IL-2 (≥100 U/mL), SRL between 2 and 20 ng/mL augmented the percentages of CD4+FOXP3+ cells approximately 2-fold compared to medium controls (n = 6; P < 0.05) (Figure 4A). This was in contrast to dose-dependent decrease in the proportion of CD4+FOXP3+ cells by TAC. When used in combination, SRL abrogated the CD4+FOXP3+ cell inhibition mediated by TAC in low concentrations. Despite these differential effects of TAC versus SRL on the proportion of Tregs, both drugs at therapeutic concentrations inhibited the expansion when compared to no drug controls (n = 6; P < 0.05) (Figure 4B). Because the starting culture was with 0.5 × 106 cells, this represented an actual cell loss with TAC (0.3 ± 0.2 × 106), but doubling in presence of SRL (1.1 ± 0.4 × 106) and a 6-fold expansion in medium control (3.0 ± 1.2 × 106). Additionally, when the absolute numbers were calculated, it was observed that from the CD4+CD45RA responder T cells (Tresp) containing 2.5 ± 0.8 × 104 CD4+FOXP3+ Tregs at the initiation of the readout cultures, TAC maintained the CD4+FOXP3+ Tregs (3.6 ± 0.7 × 104) whereas SRL amplified them 10-fold to 28 ± 12 × 104 (as opposed to 41 ± 5.2 × 104 Tregs in the medium controls; please see Table S1, SDC,http://links.lww.com/TP/B144, for additional data on these and other subsets).

FIGURE 4

FIGURE 4

In agreement with the flow cytometry results, TAC effectively blocked both T-bet and RORγt mRNA expression, whereas SRL had little effect on T-bet but moderate inhibition on RORγt (Figure 4C). In the presence of 100 U/mL IL-2, SRL augmented, whereas TAC moderately inhibited, FOXP3 mRNA expression. A combination of the two showed a net increase of FOXP3 expression (Figure 4C), indicating that SRL could reverse the transcription inhibition of this molecule mediated by TAC.

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SRL Does Not Increase IFN-γ+ FOXP3+ and IL-17+FOXP3+ Subsets in the Readout Cultures

Murine and human FOXP3+Tregs do not appear to be terminally differentiated2,3,29 and may include IFN-γ+FOXP3+ and IL-17+FOXP3+ subsets. Even though these have been implicated as effector T cells in autoimmune and inflammatory diseases30,31 and in cancer,32 incomplete data on them are available in alloimmunity. Accordingly, in the present system, we detected only low levels of IL-17+FOXP3+ (1.84 ± 0.63%) or IFN-γ+FOXP3+ (3.02 ± 0.87%) cells in these alloreactive readout CD4+CD45RA T cells (Figure 4D). Importantly, in the SRL-treated cultures, there was no increase of IL-17+FOXP3+ or IFN-γ+FOXP3+ cells and SRL together with very low dose of TAC (1 ng/mL) diminished the percentages of these subsets to below 1%.

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Proinflammatory Cytokines Plus SRL Differentially Affect Alloreactive Th1, Th17, and Treg Expansion

It has been reported that TGF-β plus IL-2 are essential for Treg induction,25-27 whereas TGF-β plus IL-6 or TGF-β plus IL-21 or IL-1β are involved in differentiation/expansion of Th17 cells.33,34 We tested the effects of proinflammatory cytokines (10 ng/mL IL-1β, 50 ng/mL TNF-α, and 20-100 ng/mL IL-6) individually or in combination, plus/minus SRL and/or TAC on the amplification of Th1, Th17 and Treg cells in the readout cultures. Notably, when tested individually, IL-1β, IL-6, or TNF-α did not appreciably augment the expansion of IFN-γ+ and IL-17+ cells (Figure 5A and B). However, a combination of these 3 cytokines increased the proportion of IFN-γ+ and IL-17+ cells by 40% and 30%, respectively, when compared to the controls (Figure 5A and B); but SRL at therapeutic concentrations was able to abrogate these inflammatory cytokine-mediated increases, whereas TAC even at subtherapeutic levels totally eliminated these cells.

FIGURE 5

FIGURE 5

Differentiation and expansion of alloreactive CD4+CD45RA T cells into FOXP3-expressing regulatory cells was marginally affected by IL-1β, IL-6, or TNF-α when used individually; but combination of the three significantly downregulated Tregs (n = 4-6, P < 0.05; Figure 5C). Even in this inflammatory condition, SRL at therapeutic concentrations (5 ng/mL) significantly increased CD4+FOXP3+ Tregs although this was lower than that induced by the same concentration of SRL without cytokines.

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SRL-Derived Tregs Suppress T-Cell Proliferation and the Differentiation of Th1 and Th17 Subsets

To test the functional effects of SRL-derived Tregs on the other T-cell subsets, CD127CD4+CD25+ cells isolated from secondary readout cultures were tested as modulator cells. The purified Tregs showed typical lineage markers including high expression of FOXP3, memory marker CD45RO, costimulatory molecule CTLA-4 (and glucocorticoid-induced TNF receptor–related protein, not shown); a minor population also produced some IL-10 and TGF-β; and they were functionally anergic (Figure 6A and B). As expected, based on our previous results,22 the purified Tregs derived from both medium and SRL-spiked cultures had similar phenotypic profiles (Figure 6A).

FIGURE 6

FIGURE 6

When functionally tested, purified CD127CD4+CD25+ cells from SRL-treated secondary readout cultures potently inhibited the 3H-thymidine incorporation by fresh CD4+CD25 autologous Tresp in a dose dependent manner (Figure 6C). This inhibition was quasi-antigen–specific in that at higher Treg-to-Tresp ratios, SRL-Tregs downregulated even the anti–third party responses, but with decreasing ratios, this nonspecific inhibition decreased, whereas the inhibition to specific stimulator was maintained (Figure 6C, right grey bars vs black bars, respectively).

Next, we determined whether there was inhibition of alloreactive Th1 and Th17 subsets by SRL-Tregs using CFSE-labeled CD4+CD25 responders and intracellular staining for IFN-γ or IL-17. As shown in Figure 6D, the SRL-Tregs not only inhibited the proliferation of total CD4+ responders (upper panel) but also inhibited the differentiation and expansion of Th1 and Th17 subsets (middle and lower panels), again in a dose-dependent manner. Finally, this inhibition of Th1 and Th17 cells by SRL-Tregs was reflected in the amount of IFN-γ and IL-17 secreted into the coculture supernatants (Figure 6E).

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DNA Demethylation Plays a Role in the Stability of FOXP3 Expression Modulated by SRL

Recently, everolimus, a rapamycin derivative was found to have a stabilizing effect on FOXP3 expression by partially interfering with the expression of DNA methyltransferase 1 (DNMT1).35 Therefore, we tested whether SRL versus TAC had a similar effect on DNMT1 expression in alloactivated CD4+CD45RA cells. As shown in Figure 7A, SRL-treated cells showed significantly lower levels of DNMT1 compared to untreated or TAC-treated cultures. These Western blot results were further confirmed by intracellular staining for DNMT1 protein using flow cytometry. Compared to the untreated control or TAC, SRL decreased mean fluorescent intensity of DNMT1 expression in the total CD4+, FOXP3+CD127 Treg and FOXP3CD127+ non–Treg cells by approximately 25% (P < 0.05), whereas the positive control PI3K inhibitor LY294002, which has broad inhibition of the upstream of PI3K signaling, decreased DNMT1 expression by 50% (Figure 7B and C). Taken together, these results were consistent with the notion that SRL was stabilizing FOXP3 expression by interfering with DNA demethylation.

FIGURE 7

FIGURE 7

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DISCUSSION

Previously, we had investigated the role of the commonly used transplant immunosuppressive agents TAC and SRL and had observed that SRL had a beneficial effect on the generation of Tregs in primary MLR.22 In the present report, we further investigated the effects of these 2 agents on the next steps of alloreactive responses, differentiation and expansion of primed T-cell subsets, including Th1, Th17, and Tregs, in light of recent advances in T-cell differentiation in alloimmunity.23,24,36-38 The culture system we chose was already activated alloreactive CD4+CD45RA/CD45RO+ T cells being restimulated nonspecifically by subthreshold levels of anti-CD3 plus autologous APCs or anti-CD28 plus high concentration of IL-223,24 in the presence of TAC and SRL. However, because we depleted the CD45RA+ cells to purify the responders, and most of the cells were found to be CD45RO+ by flow cytometric analysis (data not shown), the presence of minor subsets of CD45RB and CD45RC could not be ruled out. Unlike in our previous studies assessing the generation of Tregs in primary MLR,22 the current assay system evaluating the differentiation and expansion of Treg subsets required the addition of high concentrations of IL-2. This provided us with an ideal model to study the mechanism of action by the immunosuppressive drugs on these alloimmune T-cell subsets.

Tacrolimus and SRL at therapeutic or even lower concentrations strongly inhibited the proliferative capacity of human alloantigen-activated CD4 Th cells in vitro (Figure 2). On the average, over 90% of both FN-γ+–, IL-17–producing cells were blocked by TAC and a relatively lower percentages by SRL (Figure 3A and B). On the other hand, SRL markedly increased the proportion of FOXP3+Tregs compared to a dose-dependent decline mediated by TAC (Figure 4A), akin to the observations made in vivo in transplant patients converted from TAC to SRL treatment.39–42 Additionally, SRL was able to abrogate the inhibition of Treg expansion mediated by TAC (Figure 4). However, both agents significantly inhibited the expansion of the absolute number of cells in the cultures (Figure 4B).

Allotransplantation generates an intrinsic inflammatory environment, and the outcome is determined by how the immunosuppressive drugs modulate the T-cell subsets. Our observations that even under intense inflammatory conditions, TAC was able to universally block T cell proliferation (Figure 5)22,43 have profound implications in selecting this class of immunosuppression in transplant patients especially in the early posttransplant period when the inflammation is high. In contrast, SRL demonstrated an inhibitory effect on Th1 and TH17 while it amplified FOXP3+ Tregs. Concomitant use of TAC and SRL was able to suppress the proliferation of alloreactive Th1 and Th17 while maintaining Tregs. These in vitro data have important implications when selecting immunosuppressive drugs during the posttransplant period.

Although Th cell subsets preferentially express particular transcription factors and produce distinct cytokines, recent studies suggest considerable levels of plasticity between different T-cell lineages and in vivo reprogramming.2,3,44 For instance, peripheral mature Tregs can be converted in vitro into IFN-γ–secreting (IFN-γ+FOXP3+), IL-17–secreting (IL-17+FOXP3+) or even into highly differentiated Th1 or Th17 cells in the presence of polarizing cytokines IL-12, Th17, IL-1β, or IL-6.30,31 Similarly, in vivo proinflammatory microenvironments also promote reprogramming of Tregs.32 Functionally, IL-17+Foxp3+ or IFN-γ+Foxp3+ T cells retained their suppressive capacity, but they were not as strong as IL-17Foxp3+ or IFN-γFoxp3+ Tregs. They express moderate levels of effector cytokines and may even home to various tissues and play conventional T-cell roles in the local inflammatory microenvironment.3 In alloimmunity a recent study by Blazar group showed that high concentration of SRL (>100 ng/mL) not only completely inhibited the differentiation of IFN-γ–secreting T cells but also inhibited Foxp3+ Tregs to produce inflammatory cytokines (IFN-γ or IL-17).17 However, this work was done with concentrations that were not applicable in clinical practice, and the authors were unable to study the effect of SRL on the IL-17+Tregs because of the scarcity of this subset in their system. In our study, even in an “inflammatory environment,” clinically therapeutic doses of SRL (e.g., 5 ng/mL) effectively expanded functionally alloantigen-specific Tregs without amplifying IFN-γ+FOXP3+ or IL-17+FOXP3+ Treg subsets, and in combination with low dose of TAC, prevented the augmentation of these proinflammatory Treg subsets (Figures 4 and 5). Additionally, the Tregs amplified in presence of SRL strongly inhibited the proliferation of naïve CD4 cells in a quasi-antigen specific manner as well as prevented the generation and functions of Th1 and Th17 cell subsets (Figure 6D and E). Thus, our study confirmed the view that the Treg phenotype in alloimmunity was not terminally differentiated29,45 and extended it to show that therapeutic concentrations of SRL would stabilize true regulatory cells.

As the importance of Foxp3 stabilization becomes apparent, any protocol aiming to generate in vitro FOXP3+ Tregs for future clinical transplant usage should take into account their lineage stability.46 Unlike the in vivo converted induced Tregs, which possessed complete demethylation and stable FOXP3 expression,46-48 in vitro generated induced Tregs had only transient and unstable FOXP3 expression lacking in demethylation of the Foxp3 locus.46–48 The present study demonstrated that SRL affects FOXP3 locus methylation as assessed by DNMT inhibition (Figure 7). Therefore, it is reasonable to speculate that SRL itself could function as the induction and stabilization agent for de novo FOXP3 expression in in vitro Treg cultures.

In summary, SRL and TAC differentially affect differentiation/expansion of alloreactive Th1 and Th17 cells and graft-protective Tregs. Tacrolimus, even at lower than clinically therapeutic doses, is more effective than therapeutic concentrations of SRL for inhibiting alloreactive Th1 and Th17 cells, even in inflammatory microenvironment. However, SRL promotes the differentiation and expansion of donor-specific Tregs without supporting Treg reprogramming to IFN-γ+FOXP3+ and IL-17+FOXP3+Treg subsets. Mechanistically, SRL binds to mTOR and stabilizes Tregs by inhibiting DNA methyltransferase and promoting Foxo3/FOXP3 interactions. These results, although performed in an artificial in vitro model, add important information of how CNI and mTOR inhibitors, alone or in combination affect T cell subpopulations. Future clinical trials should be designed based on the present data to confirm our findings to help in guiding the clinical use of these immunosuppressants.

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ACKNOWLEDGMENT

The authors thank Dr. Li Zhang of the Department of Medicine, Division of Rheumatology for helpful discussions.

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REFERENCES

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