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In Vitro Effects of Cyclosporine A and Tacrolimus on Regulatory T-Cell Proliferation and Function

Miroux, Céline1; Morales, Olivier1; Ghazal, Khaldoun2; Othman, Samia Ben2; de Launoit, Yvan1; Pancré, Véronique1; Conti, Filomena2,3,4,5; Delhem, Nadira1

doi: 10.1097/TP.0b013e3182590d8f
Basic and Experimental Research

Background Liver transplantation is the treatment of end-stage liver diseases, including hepatitis C. Immunosuppression prevents graft rejection but seems to accelerate the recurrence of hepatitis C. Regulatory T cells (Tregs) may be beneficial in tolerance but deleterious in recurrent hepatitis C. We evaluated the effects of cyclosporine or tacrolimus, the principal immunosuppressive drugs, on Treg proliferation and function.

Methods Human Tregs were isolated from healthy donors and cultured with cyclosporine, tacrolimus, or NIM811, a cyclosporine analog devoid of calcineurin-inhibiting activity. Treg proliferation and suppressive activity were assessed. The phenotype, cytokine production, and phosphorylation profile of nuclear factor of activated T cell of Tregs were also analyzed.

Results Cyclosporine and tacrolimus both decreased Treg proliferation, but only low doses of cyclosporine reduced Treg activity, by inducing the production of interleukin 2 proinflammatory cytokines in these cells. Moreover, NIM811 also inhibited Treg activity. The phosphorylation of nuclear factor of activated T cell in Tregs was not altered by cyclosporine, suggesting that the effects of this drug are independent of the calcineurin pathway.

Conclusion In summary, low doses of cyclosporine inhibit Treg activity, a finding that might explain the beneficial effect of this drug on hepatitis C recurrence. In contrast, by maintaining Treg activity, tacrolimus could be more helpful than cyclosporine in controlling rejection.

1CNRS UMR8161, Institut de Biologie de Lille, Universités Lille Nord de France, Lille, France.

2UPMC Univ Paris 06 & INSERM, UMR_S 938, CdR Saint-Antoine, F-75012, Paris, France.

3AP-HP, Hôpital Saint-Antoine, Pôle Digestif, Centre de Transplantation Hépatique, Paris, France.

4Faculté de Médecine René Descartes, Universitè Paris 5, Paris, France.

This work was supported by grants from the Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales to C.M. and N.D.; grants from Astellas France to K.G., S.B.O., and F.C.; and grants from the Comité du Nord de la Ligue contre le cancer to O.M. and V.P.

The authors declare no conflicts of interest.

5Address correspondence to: Filomena Conti, M.D., Ph.D., Centre de Transplantation Hépatique, Hôpital Saint-Antoine, Rue du Faubourg Saint-Antoine, 75012 Paris, France.


C.M., F.C., and N.D. participated in making the research design, writing the article, performing the research, contributing new reagents or analytic tools, and analyzing data. O.M. participated in performing the research and contributing new reagents or analytic tools. K.G. and S.B.O. participated in analyzing data. Y.D.L. participated in making the research design. V.P. participated in making the research design and analyzing data.

F.C. and N.D. contributed equally to this work.

Received 5 October 2011. Revision requested 31 October 2011.

Accepted 4 April 2012.

Liver transplantation (LT) is the treatment of end-stage liver diseases, and long-term survival rates are now excellent. The immunosuppressive therapies used at present after LT and intended to reduce the incidence of rejection, include calcineurin inhibitors such as cyclosporine A (CsA) and tacrolimus (Tac). Tac is more potent than CsA in reducing the incidence of acute and chronic rejection (1), and long-term follow-up has confirmed the benefits of Tac over CsA with respect to graft and patient survival (1, 2).

Graft rejection may be influenced by regulatory T cells (Tregs). Tregs account for 5% to 10% of peripheral CD4+ T cells; they constitutively express CD25 (3), can suppress host immune responses in the context of autoimmune diseases and transplantation (4, 5), and have been shown to be an essential component in immune tolerance (6). Treg suppressive mechanisms are mainly dependent on cell-to-cell contact, but the involvement of immunosuppressive cytokines such as interleukin (IL)-10 or transforming growth factor (TGF)–β (7) is not excluded. The transcription factor FoxP3, expressed by Tregs, seems to be a master gene controlling Treg development (8), most FoxP3+ Tregs being CD127/low (9, 10). Tregs are naturally anergic to in vitro antigenic stimulation, and this state is closely linked to their suppressive activity (11). The IL-2/IL-2 receptor pathway seems to be important in the activity and expansion of Tregs (12, 13).

In the post–organ transplantation setting, several experimental studies have demonstrated that Tregs may induce allograft tolerance (14, 15). It has also been shown that Treg levels fall significantly after LT, especially during allograft rejection (16), and that the reduction in circulating Tregs is counterbalanced by an intragraft increase (17). Tregs are influenced by immunosuppressive therapy; in particular, calcineurin inhibitors seem to reduce Treg function in vitro (18).

However, graft function and long-term survival are also largely affected by the recurrence of hepatitis C, the main indication of LT. Indeed, hepatitis C virus (HCV) infection of the graft is universal, and graft damage is accelerated, leading to cirrhosis in 20% to 30% of patients within 5 years (19) and reduced patient survival compared with other indications (20, 21). The mechanisms of accelerated HCV-induced liver damage after LT are poorly understood. The main factors determining the severity of recurrent hepatitis C include immunosuppressive therapy (22) and the proportion of Tregs (23). Concerning immunosuppressive therapy, a recent randomized pilot study evaluating CsA versus Tac in liver transplant recipients showed that a switch from Tac to CsA therapy could have a beneficial effect on hepatitis C recurrence by reducing HCV-RNA levels and favoring the response to antiviral therapy (24).

An increased frequency of Tregs was recently described in the blood of patients with persistent HCV infection when compared with those who had cleared HCV (25, 26). It has been proposed that Tregs contribute to HCV persistence by suppressing HCV-specific T-cell responses (27, 28). Some studies have shown a correlation between a reduced HCV-specific T-cell response and the secretion of IL-10 and TGF-β by liver-infiltrating Tregs (25) and that Tregs may inhibit HCV-specific T-cell activity in a cell-to-cell contact manner (25, 26). It has also been shown that Treg levels are significantly enhanced in recurrent hepatitis C and that Treg type 1 cell levels are specifically higher in severe recurrent hepatitis C (23). These findings suggest that Tregs may be implicated in the pathogenesis of HCV recurrence.

Tregs thus exert a dual effect after LT: a beneficial role in inducing tolerance but a deleterious effect in recurrent hepatitis C. During this study, we therefore evaluated the in vitro effects of CsA or Tac on Treg proliferation and function. We observed that low doses of CsA, not of Tac, impaired the function of Tregs, probably by inducing the secretion of cytokines such as IL-2 and interferon (IFN)-γ in these cells. These findings contribute to explaining the weaker progression of the viral disease and the enhanced antiviral response in liver transplant recipients under CsA during recurrent hepatitis C, while Tac could be superior to CsA in preventing rejection by maintaining Treg activity.

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Phenotypic Characterization of Freshly Isolated CD4+CD25+ Tregs

Flow cytometry and quantitative polymerase chain reaction (Q-PCR) analysis of Treg markers were used to discriminate between CD4+CD25+ Tregs and CD4+CD25 cells and to assess the purity of freshly isolated CD4+CD25+ Tregs. Treg-associated proteins CD25, CD127, and FoxP3 were assessed by flow cytometry. Based on the expression of CD4+ and CD25+, Treg purity was superior to 95% (Fig. 1A). Only 14% of CD25 cells were CD127, whereas 90% of Tregs were CD4+CD25+ and CD127. In addition, 80% of the isolated Tregs expressed FoxP3, whereas only 12% of the CD25 fraction was FoxP3+ (Fig. 1A).



The evaluation of Treg-specific gene expression by Q-PCR showed that most Treg-specific messenger RNA and particularly FoxP3, CD25, ICAM1, GITR, and OX-40 were overexpressed in the CD4+CD25+ Tregs isolated when compared with CD4+CD25 cells (Fig. 1B).

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Functional Characterization of Isolated Tregs

As previously described (11), we observed that isolated Tregs were anergic after activation with anti-CD3 and anti-CD28, whereas peripheral blood monocyte cells (PBMCs) were able to proliferate (Fig. 1C).

Isolated Tregs were able to significantly suppress PBMC proliferation (P<0.01) in a mixed leukocyte reaction (MLR) performed with activated PBMCs and Tregs at a 2:1 ratio. Tregs inhibited, in average, 80% of PBMC proliferation (Fig. 1C). This suppressive effect was associated with an inhibition of cytokine production in the MLR supernatant: the secretion of IL-2, IFN-γ, and IL-10 by PBMCs was inhibited by Tregs, but in contrast, TGF-β secretion was not inhibited (Fig. 1D).

The CD4+CD25 fraction did not suppress proliferation or cytokine secretion by activated PBMCs (data not shown).

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Calcineurin Inhibitors Reduced the Proliferation of PBMCs and Tregs

PBMCs and isolated Tregs were activated and cultured with different doses of CsA or Tac. CsA and Tac both inhibited the proliferative capacity of PBMCs in a dose-dependent manner (Fig. 2A,B). This inhibition was approximately 50% (SD, 12%) with 20- to 40-ng/mL CsA, 70% (11%) with 400-ng/mL CsA (Fig. 2A), 45% (6%) with 2- and 5-ng/mL Tac, and approximately 60% (4%) with 20- to 50-ng/mL Tac (Fig. 2B). Tregs proliferated in the presence of anti-CD3, anti-CD28, IL-2 (300 U/mL), and autologuous irradiated PBMCs (Fig. 2A), as previously shown (29, 30). The proliferation of isolated Tregs was inhibited by CsA in a dose-dependent manner. The inhibition of Treg proliferation was statistically significant approximately 35% (10%) with 40-ng/mL CsA (P<0.05) and 70% (6%) with 400-ng/mL CsA (P=0.008) (Fig. 2A). Treg proliferation was also significantly inhibited by Tac, approximately 70% at all concentrations (P<0.05) (Fig. 2B).



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Low Doses of CsA (20 and 40 ng/mL), Not Tac, Inhibit the Suppressive Activity of Tregs

In this study, Tregs inhibited PBMC proliferation by 33% (6.2%) in an MLR performed with anti-CD3/anti-CD28–activated PBMCs and autologous Tregs at a ratio of 2:1 (Fig. 2C). CsA inhibited Treg suppressive activity at a concentration of 20 ng/mL, but this inhibition became significant with 40 ng/mL (20% [6.2%] inhibition of PBMC proliferation with 20-ng/mL CsA vs. 33% [6.2%] in controls, P=0.06; 17% [8.4%] inhibition of PBMC proliferation with 40 ng/mL, P=0.034 vs. controls) (Fig. 2C). However, with 100- or 400-ng/mL CsA or with all doses of Tac used in the culture assay, no inhibition of Treg suppressive capacity was observed (Fig. 2C).

In mixed leukocyte allo-reaction (allo-MLR), we confirmed results obtained in anti-CD3/anti-CD28 activation conditions. Tregs inhibited PBMC proliferation by 37% (5.2%) in an MLR performed with PBMCs from donor 1, irradiated PBMCs from donor 2, and Tregs from donor 1, at a ratio of 2:1 (Fig. 2D). CsA inhibited Treg suppressive activity at a concentration of 40 ng/mL (22% [5.8%] inhibition of PBMC proliferation vs. 37% [5.2%] in controls) (Fig. 2D). However, with 400-ng/mL CsA, no inhibition of Treg suppressive capacity was observed (Fig. 2D).

In contrast, whatever the concentration of Tac used in the culture assay, no inhibition of Treg suppressive capacity was observed (Fig. 2E). Statistical analysis confirmed that, contrary to CsA, Tac addition (from 2 to 50 ng/mL) induced a highly significant decrease of PBMC proliferation in MLR condition (P<0.01). Note that such experiment did not allow concluding whether Tac potentiates the suppressive activity of Treg (Fig. 2E).

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Low Doses of Both CsA and Tac Do Not Change the Treg Phenotype

The expression of Treg-associated proteins, including CD25, CD127, and FoxP3, was assessed. Some insignificant changes were observed with low-dose CsA (Fig. 3A), such as a small increase in CD25 expression, particularly CD25high (19.6% vs. 12.8% in controls), and a small decrease in FoxP3 expression in the presence of 40-ng/mL CsA (26.1% vs. 27.4% in controls) (Fig. 3A). All Tac concentrations increased the CD4+CD25high fraction and did not induce significant modifications to CD127 or FoxP3 expression (Fig. 3B).



CsA (40 ng/mL) or Tac did not induce significant changes in the expression of the messenger RNA of Treg-associated markers (Fig. 3C,D).

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Low Doses of CsA, Not Tac, Switch the Cytokine Profile of Tregs to a Proinflammatory Profile

Tregs from three different donors were cultured in the presence or absence of CsA or Tac, and the supernatants were evaluated by enzyme-linked immunosorbent assay (ELISA) for cytokine secretion. We did not observe any significant changes in the secretion of IL-4, IL-10, or TGF-β between CsA-treated, Tac-treated, or untreated cells (Fig. 4A). By contrast, the levels of IL-2 and, particularly, IFN-γ proinflammatory cytokines were significantly higher in the supernatants of Treg cultures performed in the presence of 40-ng/mL CsA than it was in control cultures (Fig. 4B). The IL-2 level rose from 19.9 (5.14) in controls to 70.9 (23) pg/mL with CsA 40 ng/mL (P=0.029) in inactivated Tregs (Fig. 4B). IFN-γ levels rose from 90 (26.7) in controls to 302 (200) pg/mL in the presence of CsA 40 ng/mL (P=0.05) in activated Tregs (Fig. 4B). By contrast, 400-ng/mL CsA (Fig. 4B) and all doses of Tac (Fig. 4B) showed no effect on the production of these cytokines.



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Anti–IL-2 Restores the Suppressive Activity of Tregs in the Presence of Low Doses of CsA

MLR in the presence or absence of CsA (40 ng/mL) and in the presence or absence of anti–IL-2 or anti–IFN-γ was performed in two independent experiments. In these experiments (Fig. 4C), we confirmed that a low dose of CsA (40 ng/mL) inhibited Treg suppressive activity (unblocked condition). Very interestingly, in the same condition but in the presence of anti–IL-2, we clearly observed that blocking IL-2 can restore Treg suppressive activity. In contrast, in the presence of anti–IFN-γ, we did not observe any difference compared with CsA (40 ng/mL) basal condition, and we still observed an inhibition of Treg activity. Finally, using both the anti–IL-2 and anti–IFN-γ, we found a suppression level equivalent to the condition of classic MLR (PBMC in the presence of Treg).

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Low Doses of CsA Inhibit Treg Activity Independently of the Calcineurin Pathway

In T cells, CsA inhibits IL-2 transcription by blocking the calcineurin pathway through its bond to cyclophilin A. In order to determine whether the inhibitory effect of CsA on Treg activity also involves the calcineurin pathway, MLR was conducted in the presence of NIM811, a CsA analog that binds to and inhibits cyclophilins but is devoid of calcineurin-inhibiting activity (31). NIM811 inhibited Treg proliferation in a dose-dependent manner (Fig. 5A). Furthermore, similar to CsA, NIM811 was able to significantly inhibit Treg activity at the low dose of 40 ng/mL (21% [6%] PBMC inhibition with 40 ng/mL vs. 32% [2.5%] in controls; P=0.043) but not at higher doses (Fig. 5B).



In parallel, Western blotting analysis was performed to assess the phosphorylation profile of nuclear factor of activated T cells (NFAT) in activated Tregs, in the presence of CsA, Tac, and NIM811. The ratio of phosphorylated NFAT (NFAT P) to NFAT was evaluated using Multi Gauge analysis. No modifications to the NFAT protein phosphorylation profile were observed (NFAT P/NFAT=1) in the presence of NIM811 (Fig. 5C) or CsA (Fig. 5E). Only with 50-ng/mL Tac was a decrease seen in the NFAT P-to-NFAT ratio to 0.71, suggesting that Tac 50 ng/mL decreased the dephosphorylation of NFAT proteins (Fig. 5D).

Taken together, these data suggest that CsA impairs Treg activity independently of the calcineurin/NFAT pathway.

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This study was carried out to verify whether calcineurin inhibitors (Tac and CsA) reduced the number, proliferation, or activity of Tregs, which might impair the establishment of immune tolerance or even favor a recurrence of severe hepatitis C. Our data suggest that Tac and CsA inhibit Treg proliferation but that only CsA inhibits Treg activity as previously published (32–34). Our main finding is that CsA, used at low doses similar to those used long term after LT, probably affects Treg activity by inducing the production of proinflammatory cytokines in those cells, such as IL-2 and IFN-γ. These results are in line with those of a study in a rat cardiac allograft model, where differential effects of CsA were observed as a function of the concentrations (32). During our study, Tac never affected Treg activity.

In this study, Tregs have been activated with anti-CD3/anti-CD28 to calibrate the stimulation and to obtain a reproducible activation. However, results have been confirmed with human allo-MLR cultures to validate the physiologic relevance of the findings.

Neither CsA nor Tac treatment changed the Treg phenotype. In accordance with other studies (35, 36), isolated Tregs did not produce IL-10 but secreted basal levels of IFN-γ and IL-2, which were abrogated by anti-CD3 and anti-CD28 activation. No modification of suppressive cytokine (IL-10 and TGF-β) production was observed in the presence of CsA or Tac. However, with a low concentration of CsA, we observed a significant increase in the secretion of IL-2 and IFN-γ proinflammatory cytokines. Actually, IL-2 blocking experiments suggested that the inhibition of Treg activity by low-dose CsA may have resulted from an induction of IL-2 by these cells, which could also explain the increased proliferation of PBMCs during MLR.

The mechanism of the inhibition of Treg activity by CsA was then investigated. NIM811, a CsA analog that strongly binds to and inhibits cyclophilin but is devoid of calcineurin-inhibiting activity (31), also inhibited Treg activity. Moreover, the NFAT phosphorylation profile was not altered in the presence of CsA, suggesting that CsA reduces Treg function through a calcineurin-independent pathway.

These findings were unexpected insofar because the immunosuppressive effect of CsA is generally attributed to the prevention of IL-2 transcription (31). Analysis of the molecular and cellular mechanisms involved in the effects of low CsA doses on Tregs may provide a clearer understanding of how CsA abrogates Treg function. Bollyky et al. (37) showed that CD44 cross-linking promoted IL-2 secretion by Tregs in an NFAT-independent manner and that CD44 treatment bypassed CsA-mediated IL-2 inhibition. Moreover, FoxP3 is believed to inhibit IL-2 production through NFAT inhibition (38, 39), but in our hands, low doses of CsA did not induce any change in FoxP3 expression (Fig. 3). It now seems reasonable to determine whether CsA may promote IL-2 in an NFAT/calcineurin-independent manner. Especially because it has been shown in mice that the NFAT pathway is altered in Tregs with diminished calcineurin activity (40).

In summary, our data suggest that low doses of CsA, similar to those used in the context of long-term immunosuppressive therapy after LT, are able to inhibit Treg activity. Tregs play an important role in transplant tolerance, and the inhibition of Treg activity by CsA could explain why the latter does not accurately inhibit graft rejection when compared with Tac. By abrogating Treg function and favoring the development of effector T cells induced by the proinflammatory cytokines secreted by Tregs, CsA may favor the antiviral response against HCV and thus decrease the severity of HCV recurrence after LT, in accordance with the findings of recent studies where CsA was associated with less severe recurrences of hepatitis C and a better response to antiviral therapy (24, 41 42).

These findings could explain why Tac is superior to CsA in preventing rejection by maintaining Treg activity and why CsA may decrease the severity of recurrent hepatitis C and enhance the antiviral response.

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Isolation of Human CD4+CD25+ Cells

PBMCs from healthy donors were isolated using Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ). CD4+CD25+ T cells were isolated from the PBMCs using a magnetic-activated cell-sorting system (Miltenyi Biotec, Cologne, Germany). The study was approved by the Institut de Biologie de Lille (CNRS) and Etablissement Français du Sang institutional review boards, and informed consent was obtained in writing for each donor.

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Culture Conditions and Drug Treatment

All cultures were conducted in Roswell Park Memorial Institute 1640 medium supplemented with 2-mM L-glutamine 1%, 0.02-mM sodium pyruvate, 100-U/mL penicillin, 100-μg/mL streptomycin, and 10% heat-inactivated Human AB Serum. The cultures were incubated in a humidified incubator (37°C, 5% CO2). CsA (Sigma-Aldrich Saint-Quentin Fallavier, France) was used in cultures at 20, 40, 100, and 400 ng/mL, as previously published (43). Tac (Sigma-Aldrich) was used in cultures at 2, 5, 20, and 50 ng/mL. A CsA analog, NIM811 (Novartis Rueil-Malmaison, France) was used in the cultures at the same concentrations as CsA. The results are expressed as mean (SD) for four different experiments.

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Flow Cytometric Analysis

Cell phenotype was analyzed by flow cytometry (EPICS XL-MCL; Coulter, Villepinte, France). For membrane staining, the cells were washed and labeled with the following fluorochrome-conjugated monoclonal antibodies: CD4 PE-Cy5 (BD Pharmingen, San Diego, California), CD25-PE (Miltenyi, Bergisch Glabdach, Germany), and CD127–fluorescein isothiocyanate (Clinisciences, Naterre, France). For intracellular staining, cells were labeled with surface monoclonal antibodies and then fixed and permeabilized using FoxP3 Staining Buffer Kit with anti-FoxP3 fluorescein isothiocyanate Miltenyi.

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Mixed Leukocyte Reaction

The suppressive activity of Tregs was measured by their ability to inhibit the proliferative response of autologous PBMCs in an MLR of 105 responder PBMCs and 6.104 CD25+ cells (2:1 mixture). Assays were set up in a round-bottom 96-well plate cultured in triplicate for 48 hr. Cells were activated with plate-bound anti-CD3 antibodies (1 μg/mL), incubated at 37°C for 2 hr before the culture, and soluble anti-CD28 antibodies (100 ng/mL) (Clinisciences). Proliferation was measured after [methyl-3H]-thymidine (1 μCi per well; GE Healthcare, Aulnay sous Bois, France) incubation for the last 18 hr before harvesting. Radioactivity was determined using a β-counter (Wallac Trilux 1450; Turku, Finland). The results are expressed as a proliferative index and as a mean (SD) of four different experiments.

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Mixed Leukocyte Allo-Reaction

The suppressive activity of Tregs was measured by their ability to inhibit the proliferative response of autologous PBMCs in allo-MLR. Assays were set up with cultures of 105 responder PBMCs from donor 1 and 105 irradiated (50 Gy) PBMCs from donor 2 in a round-bottom 96-well plate cultured for 5 days. At day 5 of culture, we verified the formation of proliferation foci by microscopy, and then, 6.104 CD4+CD25+ Treg cells from donor 1 were added (2:1 mixture), with CsA (40 or 400 ng/mL) for another two days of culture. Each proliferation assay was carried out in triplicate. Proliferation was measured as previously described. The results are expressed as a proliferative index and as a mean (SD) of three different experiments.

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IL-2 and IFN-γ Blocking Assays

The suppressive activity of Tregs was measured by their ability to inhibit the proliferative response of autologous PBMCs in MLR assays in the presence or absence of CsA (40 ng/mL) and in the presence or absence of anti–IL-2 (50 ng/mL) or anti–IFN-γ (5 μg/mL), or combined anti–IL-2 (50 ng/mL) and anti–IFN-γ (5 μg/mL). The results are expressed as a proliferative index and as a mean (SD) of two independent experiments.

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Treg Proliferation Assay

CD4+CD25+ Tregs proliferated in the presence of anti-CD3 (150 ng/mL), anti-CD28 (100 ng/mL), IL-2 (300 U/mL; PreproTech Inc, Rocky Hill, NJ), and autologous irradiated (50 Gy) PBMCs (29, 30). Each proliferation assay was measured as described in the section on MLR.

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Enzyme-linked Immunosorbent Assay

IL-2, IL-4, IL-10, IFN-γ, and TGF-β production was measured by ELISA after 48 hr of culture. Methods are described in Carpentier et al. (23). The samples and standards were run in duplicate. The results are expressed as mean (SD) for the three different experiments.

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Real-time Quantitative PCR

CD25+ total RNA was isolated from fresh pellets using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France), and CD25 total RNA was isolated using TRIzol (Invitrogen, Cergy Pontoise, France), according to the manufacturer’s instructions. RNA concentrations were measured by spectrometry, and complementary DNA was synthesized from 2-μg/μL total RNA using the SuperScript II RNAse H Reverse Transcriptase kit (Invitrogen), according to the manufacturer’s procedure. We used a real-time Q-PCR technique to quantify the amount of CD25, CD127, FoxP3, GITR, OX-40, LAG-3, P-selectin, L-selectin, programmed death 1, IL-2, IL-4, IL-10, IFN-γ, TGF-β, and the housekeeping genes used were β-actin, ubiquitin, GAPDH, and HPRT. Q-PCR was applied using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Meylan, France). The target message was quantified by measuring the threshold cycle using the LightCycler system (Roche Diagnostics). For each gene, a standard curve was prepared, and the housekeeping genes were used in each sample to control sample-to-sample variations.

The results were analyzed using the “relative gene expression method” as described by Livak et al. (44) and briefly described elsewhere (23).

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Western Blotting

Protein lysis experiments on 200,000 activated Tregs were performed in 30 μL of lysis buffer by two freezing and thawing steps and 30 sec of sonication, after 48 hr of culture in the presence of the different drugs. Proteins were migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis 12% to 14% Bis-Tris acrylamide gel (Invitrogen) for 90 min at 120 V and then electrotransferred onto a nitrocellulose membrane. The membrane was blocked with 5% milk powder in 0.01% Tris-buffered saline/Tween 20 for 2 hr at room temperature. The membrane was then incubated overnight with the primary antibodies, rabbit anti-CD4 (SC-7219; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-NFAT (SC-7294; Santa Cruz Biotechnology), followed by incubation with the appropriate secondary antibody of either peroxidase-conjugated antimouse or antirabbit IgG (Amersham Biosciences). Immunoreactive proteins were visualized using Western Lightning Plus-ECL (PerkinElmer, Boston, MA) and using the LAS3000 Imaging system (LifeScience; Fujifilm, Dusseldorf, Germany). The results are the most representative of the three different experiments.

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Statistical Analysis

The relevance of proliferation assays MLR and ELISA results was validated using the statistical package SigmaStat for Windows 3.0.1 (SPSS, Ashburn, VA) and Sigma XL. All quoted P values are two-sided, with P<0.05 (*) and P<0.01 (**) being respectively considered statistically significant and highly significant.

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Regulatory T cells; Cyclosporine A; HCV; Transplantation

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