Mycophenolic acid-treated human dendritic cells have a mature migratory phenotype and inhibit allogeneic responses via direct and indirect pathways

Christine Lagaraine1, Cyrille Hoarau1, Valérie Chabot1,2, Florence Velge-Roussel1,3 and Yvon Lebranchu1

1 JE 2448, Cellules Dendritiques et Greffes, IFR 135, Imagerie et exploration fonctionnelles, Université François Rabelais, 2 bis Bd Tonnelle, 37032 Tours, France
2 Etablissement Français du Sang, Centre Atlantique, Tours, France
3 UFR des Sciences Pharmaceutiques, 31 Avenue Monge, 32200 Tours, France

Correspondence to: Y. Lebranchu; E-mail: lebranchu{at}med.univ-tours.fr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immature dendritic cells (DCs) can induce T-cell hyporesponsiveness, thus interfering with the process of DC maturation in a pro-inflammatory context, may therefore provide a novel approach to inducing allograft tolerance. We have studied the effects of mycophenolic acid (MPA), an immunosuppressive agent currently used in transplantation, using an in vitro model of a mixed human DC/alloreactive CD4+ T lymphocyte culture. DCs differentiated from monocytes were exposed to MPA during maturation. MPA treatment affected the maturation of DCs, and this was reflected both in the impairment of the up-regulation of co-stimulatory molecule expression and the maintained endocytic capacity. However, MPA-DCs exhibited a distinctive microscopic morphology and secreted IL-10 and so could no longer be regarded as immature DC. Moreover, MPA-DCs had a mature phenotype for chemokine receptor expression, exhibiting down-regulation of CCR5 and up-regulation of CCR7. Interestingly, the abilities of the MPA-DCs to induce CD4+ T-cell proliferation in response to alloantigens was impaired not only via direct but also via indirect pathways. The maintenance of endocytosis and the inhibition of syngeneic T-cell activation suggest that these cells could have a potential role to avoid chronic rejection. All these characteristics suggest that MPA-DCs may be used in cell therapy to induce allograft tolerance.

Keywords: Cell therapy, Immunosuppressive agent, Tolerance, Transplantation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) and play a central role in initiating the primary immune response due to their unique ability to stimulate naive T cells (1). The functional properties of myeloid DCs derived from bone marrow progenitors depend on their stage of maturation. Immature DCs act as sentinels in non-lymphoid organs (2) and have a high capacity to capture and process antigens before transporting them to secondary lymphoid organs where they present antigens to naive T cells (3). In the presence of danger signals, DCs mature during migration, progressively losing their ability to capture and process antigens, but increasing the expression of surface class II MHC and co-stimulatory molecules, and up-regulating their production of cytokines such as IL-12 to become fully potent APCs (4).

Nevertheless, DCs not only induce immunogenic reactions but can also exhibit tolerogenic properties (5), and the balance between immunogenicity and tolerance seems to depend directly on their functional state of maturation and degree of activation. It has been shown that depending on the nature and intensity of the expression of co-stimulatory molecules, immature DCs may induce T-cell hyporesponsiveness (611) or regulatory T cells (12, 13). This means that interfering with the process of DC maturation can profoundly modify the outcome of the immune response, and manipulating DCs may provide a novel approach to preventing acute and chronic allograft rejection. In order to keep DCs at an immature stage after in vivo administration in a pro-inflammatory context that would normally lead to DC maturation and activation, it is essential to investigate drugs able to inhibit DC maturation which can be used in vivo in transplanted patients. Recent in vitro studies have reported the ability of agents such as deoxyspergualin, glucocorticosteroids, vitamin D3 analogs, rapamycin and N-acetyl-cysteine to inhibit DC maturation and to impair the ability of DCs to induce allogeneic T-cell proliferation [reviewed in Lagaraine and Lebranchu (14)]. In particular, mycophenolate mofetil (MMF), an immunosuppressive agent currently used to prevent acute cellular rejection after transplantation and in the treatment of autoimmune diseases (15, 16), impairs the maturation of DCs. Mycophenolic acid (MPA), the active metabolite of MMF, inhibits lymphocyte inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of the first committed step of de novo synthesis of the guanosine nucleotides required for DNA synthesis and T and B cell proliferation (17). However, the effects of MPA on DCs and the consequences of MPA-DC on T-cell activation remain to be determined. In mice, MMF inhibits the up-regulation of DC co-stimulatory and adhesion molecules in response to maturing agents, reduces the synthesis of cytokines such as IL-12 and inhibits the ability of DC to stimulate allogeneic T-cell proliferation (18). Similarly, Colic et al. recently reported that in humans MMF inhibited the maturation of DCs, which exhibited impairment of the expression of co-stimulatory molecules, of cytokine secretion and of the ability to induce allogeneic T-cell proliferation (19). It is therefore necessary to characterize more precisely the maturation and endocytic capacities, activation and functions of human MPA-treated DCs with a view to their potential use in cell therapy.

We analyzed the effects of MPA on DC maturation with regard to phenotype, migration profile and ability to capture antigens and induce the proliferation of allogeneic T cells via the direct and indirect pathways. We report here an inhibitory effect of MPA on monocyte-derived DCs in terms of maturation markers and co-stimulatory abilities. These MPA-treated DCs had the ability to synthesize considerable quantities of IL-10. Interestingly, the morphological characteristics of these MPA-DCs were widely different from those of both immature and mature DC. Moreover, we have shown here for the first time that MPA-DC inhibit the proliferation of CD4+ T cells not only via the direct but also via the indirect pathway. Surprisingly, investigation of MPA-DCs chemokine receptor expression to determine their migratory ability revealed that MPA-DCs have fairly a mature phenotype, probably allowing their migration to the lymphoid organs.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Media, cytokines and reagents
The culture medium used consisted of RPMI 1640 (GIBCO, Cergy Pontoise, France) supplemented with 50 IU ml–1 penicillin (ICN, Orsay, France), 50 IU ml–1 streptomycin (ICN), 2 mM glutamine (Bio Whittaker, Vervier, Belgique), 1 mM sodium pyruvate (GIBCO) and 10% heat-inactivated FCS (GIBCO). X-VIVO 15 culture medium (Bio Whittaker) was used for indirect pathway experiments, supplemented with 50 IU ml–1 penicillin, 50 IU ml–1 streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 2% human serum albumin (HSA) (kind gift of LFB, Courtaboeuf, France). Recombinant human IL-4 and recombinant human tumor necrosis factor-{alpha} (TNF-{alpha}) were obtained from R&D Systems (Abingdon, UK). Granulocyte macrophage colony-stimulating factor (GM-CSF) was obtained from Novartis (Reuil Malmaison, France). MPA purchased from Sigma–Aldrich (St Quentin Fallavier, France) was re-constituted in methanol. Zymosan and poly I : C were obtained from Invivogen (Toulouse, France), and LPS was purchased from Sigma–Aldrich. N-2,2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate sodium salt (dbGMPc) and Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-AMPS) were obtained from Sigma.

Generation of DCs
Blood from healthy donors was obtained by cytapheresis (informed consent was obtained from volunteers). Human PBMC were then isolated by centrifuging over Ficoll hypaque (d = 1.077, Lymphoprep; Nycomed, Oslo, Norway). The PBMC were washed twice and re-suspended in culture medium with 10% heat-inactivated FCS and then plated in 75-cm2 flasks (Falcon, Becton Dickinson, Mountain View, CA, USA) to ensure monocyte adherence. After 45 min at 37°C, any non-adherent cells were removed, and the adherent cells were cultured in the presence of 25 ng ml–1 human IL-4 and 1000 IU ml–1 GM-CSF at 37°C in a humidified 5% CO2 atmosphere as previously described (20). On day 3, fresh medium and cytokines were added (at the same concentrations as previously). On day 5, immature DCs were harvested, washed and re-suspended in culture medium with IL-4 and GM-CSF. To induce maturation, TNF-{alpha} was added at 20 ng ml–1 for 2 days, in the presence or absence of increasing doses of MPA. When specified, DCs were matured with zymosan at 10 µg ml–1, LPS at 50 ng ml–1 or poly I : C at 10 µg ml–1 for 2 days. When specified, dbGMPc (10 mM) or Rp-AMPS (10 µM) was added to the culture medium at the same time as the TNF-{alpha} and MPA. On day 7, the DCs were harvested, washed and used for subsequent experiments after evaluating their viability by trypan blue exclusion.

Analysis of the expression of cell-surface molecules by flow cytometry
DCs were harvested, and 2 x 105 cells per sample were re-suspended in PBS. Cells were then incubated with saturating concentrations of the various fluorochrome-conjugated mAbs for 30 min at 4°C. The stained cells were washed twice in PBS and fixed in 0.5% PFA PBS solution until analyzed by flow cytometry. Cell-surface expression was then analyzed using a 488-nm laser flow cytometer (FACSCalibur®, Becton Dickinson) using CELLquest® software (Becton Dickinson). Data were analyzed for geometric mean fluorescence intensity (MFI) (MFI of antibody of interest/MFI of isotype control) and for the percentage of marker-positive cells (at least 5000 cells per sample were analyzed). The following mouse anti-human mAbs were used: PE-anti-CD40 (IgG1, mAb86), PE-anti-CD54 (IgG1, 84H10), PE-anti-CD80 (IgG1, mAb104), PE-anti-CD58 (IgG2a, AICD58), PE-anti-CD83 (IgG2b, HB15A), PE-anti-CD86 (IgG2b, HA5), PE-anti-HLA-DR (IgG2b, B8.12.2) purchased from Immunotech (Marseille, France) and PE-anti-CCR5 (IgG2a, 150503) purchased from Becton Dickinson (Grenoble, France), and PE-anti-CCR7 purchased from R&D Systems (Lille, France). Control cells were stained with corresponding isotype-matched control mAbs (Immunotech, Becton Dickinson and R&D Systems).

DC endocytosis
Mannose receptor-mediated endocytosis was measured as the cellular uptake of dextran and was quantified by flow cytometry: 2 x 105 DCs per sample were incubated with 1 mg ml–1 of FITC-dextran (Sigma–Aldrich) for 60 min at 37°C. Cells were washed twice with PBS to remove excess dextran, and at least 5000 cells per sample were analyzed.

Cytokine measurements
Immature DCs (2 x 106 cells) were matured with TNF-{alpha} in the presence or absence of MPA for 48 h. The supernatants were then collected and stored at –20°C until analysis. Measurements of IL-12 p70 and IL-10 levels were performed by appropriate ELISA using commercially available antibodies and standards according to the manufacturer's protocols (eBiosciences, Montrouge, France).

Chemokine receptor mRNA analysis by RNase protection assay
Immature, mature and MPA-DCs were harvested, and RNA samples were purified by a Trizol technique according to the manufacturer's instructions (Invitrogen, Cergy Pontoise, France). Concentrations of RNA were determined (260/280 nm ratio) by spectrophotometry. Purified RNA (5 µg) was then used for RNase protection assay (RPA) analysis using hCR-5 (CCR1, CCR2, CCR5) and hCR-6 (CXCR1, CXCR4, CCR7) Riboprobe Template Sets, and the RiboQuant multiprobe RPA System Kit (Pharmingen, Le pont de Clair, France) according to the manufacturer's protocol. Briefly, purified RNA samples were hybridized overnight with 32P-labeled mRNA products from the template set, and afterwards digested with RNase A for 45 min at 30°C. Protected RNA samples plus 32P-labeled probes were run on 5% polyacrylamide electrophoresis gel. The gels were dried at 80°C for 1 h then autoradiographed (at different times to avoid the saturation band density), and the mRNA band densities were quantified with NIH Image 1.61 per ppc software. Results were normalized with the housekeeping gene glyseraldehyde-3-phosphate dehydrogenase (GAPDH) L32 band densities included in the probe template set, and expressed as arbitrary densitometry units.

Scanning electron microscopy
The morphology of monocyte-derived DCs was assessed by scanning electron microscopy. DCs were suspended in PBS and added to polylysine-coated cover slips before being fixed in 1% glutaraldehyde, 4% PFA in 0.1 M phosphate buffer, pH 7.4, and post-fixed in 2% osmium tetroxide. Cells were then dehydrated in graded acetone, critical point dried using carbon dioxide and coated by gold sputtering. Immature, mature and MPA-treated DCs were examined using a GEMINI 982 LEO Scanning electron microscope.

Isolation of CD4+ T lymphocytes
PBMC from heparinized blood of healthy human volunteers were isolated using Ficoll hypaque, as described above. PBMC were first depleted of adherent cells by two 45-min adhesion cycles on plastic. CD4+ T lymphocytes were isolated using anti-CD4-coated Dynabeads® followed by Detachabeads® (Dynal, Compiegne, France) according to the manufacturer's instructions. Briefly, peripheral blood lymphocytes were re-suspended in 1 ml PBS–1% FCS and Dynabeads® were added, depending on the estimated number of target CD4+ T cells in the sample (using a bead to target cell ratio of 3 : 1) for 45 min at 4°C. The cells were detached by incubating with Detachabeads® for 45 min at room temperature.

Allogeneic mixed leukocyte reaction
Monocyte-derived DCs were harvested at day 7, washed and distributed in 96-well flat-bottomed culture plates (Falcon, Becton Dickinson) at 3 x 104 cells in 100 µl per well (unless otherwise indicated) before being irradiated at 20 Gy. CD4+ T cells (105 cells per 100 µl) were added to each well and co-cultured with control or MPA-treated DC in RPMI–10% FCS at 37°C in a humidified 5% CO2 atmosphere. Cell proliferation was assessed by the incorporation of 1 µCi [3H]thymidine (Amersham Pharmacia Biotech, Little Chalfont, UK) during the last 18 h of a 5-day culture and measured by liquid scintillation counting (Tri-Carb 2550 TR/LL, Packard, Rungis, France). Results are expressed as mean counts per minute ± SD obtained from triplicate wells. When indicated, 20 µg ml–1 neutralizing anti-IL-10 antibodies (clone 23738, R&D Systems) were added to the co-culture.

Detection of DC apoptosis
Cells (5 x 105) were washed twice in PBS, and then re-suspended with 500 µl of Ca2+-binding buffer. One microgram of 7-amino actinomycin D (Sigma–Aldrich) and 1 µl of FITC-labeled Annexin V (Immunotech) were then added, before incubating for 10 min in the dark at 4°C. Flow cytometry analysis (FL3-H and FL1-H) was performed immediately after incubation.

Pulsing of DCs with a lysate of allogeneic PBMC
Immature DCs were generated as described above except that we used a X-VIVO 15 serum-free medium supplemented with 2% HSA.

Lysate from allogeneic PBMC was prepared by three freeze (–80°C) and thaw (37°C) cycles. The lysate was centrifuged to remove the debris, and the supernatant was collected and passed through a 0.2-µm filter (Pall Corporation, St Germain-en-Laye, France). Immature DCs were pulsed with lysate at a ratio of 3 : 1 PBMC equivalents and matured with TNF-{alpha} before use (3 x 104 cells irradiated at 20 Gy per well) in a 7-day mixed leukocyte reaction (MLR) with 105 syngeneic CD4+ T cells. CD4+ T-cell proliferation was assessed as in the allogeneic MLR.

Statistical analysis
The microscopic analysis data are presented as percentages and were compared by {chi}2 test. Data of proliferation analyses are presented as the mean ± SD and were compared by Student's t-test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MPA inhibits the maturation of DCs
First, to test the effects of MPA on DC maturation, monocyte-derived immature DCs were stimulated by TNF-{alpha} in the presence or absence of MPA (MPA-DC). As shown in Fig. 1, MPA significantly reduced the up-regulation of co-stimulatory and maturation molecules in response to TNF-{alpha}, the MFI being reduced 2-fold for CD40, CD54 and CD58, and 3-fold for CD86 and HLA-DR expression. Moreover, only 32% of cells were positive for CD83, compared with 62% for control DCs. Likewise, 65% of the cells treated with MPA expressed HLA-DR, compared with 92% of the control DCs.



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Fig. 1. Pre-treatment of DCs with MPA affects the up-regulation of cell-surface expression of co-stimulatory and adhesion molecules induced by TNF-{alpha}. Immature DCs and DCs matured in the presence (MPA-DC) or absence (control DC) of 100 µM MPA were harvested and the surface expression of molecules was analyzed by FACS. Empty histograms correspond to the control isotypes, and solid histograms represent specific staining of the cell-surface markers indicated. MFIs of one representative experiment out of 10 are indicated.

 
We have also verified that the doses used in our study were consistent with those used for transplanted patients. The lowest dose (10 µM, i.e. 3.2 µg ml–1) was comparable to the blood trough levels observed in patients treated with MMF (mean concentrations between 2.5 and 5 µg ml–1, area under the curve: 30–60 µg h ml–1) and some peak values corresponded to our highest dose (100 µM, i.e. 32 µg ml–1), explaining our concentration range (21, 22).

As a major functional characteristic of immature DCs is their endocytosis capacity, we analyzed the capacity of monocyte-derived immature DCs stimulated by TNF-{alpha} to internalize FITC-dextran in the presence or absence of MPA. As shown in Fig. 2, the percentage of DCs able to internalize FITC-dextran, and the MFI was greater for MPA-DCs than mature DCs, indicating that the uptake of antigen through the mannose receptor was maintained by this treatment. Therefore, MPA-DCs have some characteristics of immature DCs.



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Fig. 2. MPA-treated DCs display maintained endocytic ability. FITC-dextran internalization at 37°C (black lines) and 4°C (negative control, gray lines) for 30 min was analyzed on immature DC, control mature DC and MPA-DC (100 µM) by flow cytometry.

 
MPA-DCs have a distinctive morphology
Using scanning electron microscopy, we also compared the morphology of MPA-DCs with that of immature and mature DCs. Three distinct morphologies were observed and described as knob-like projections, large prominent ruffles and needle-like dendrites (Fig. 3a). In the immature population, 75% of the DCs were rounded with dense centers and small knob-like projections (Fig. 3b), corresponding to the morphology of immature DCs (23). In contrast, 47% of TNF-{alpha}-matured DCs had a more irregular shape with numerous long needle-like projections, as reported by Steinman (1). The MPA-DC population presented a specific profile, most of these cells having large prominent ruffles and a shrunken folded membrane structure (56%). A few (13%) had needle-like projections, and the others (31%) retained the immature-type morphology. Comparisons between the percentages of the three DC populations demonstrated that MPA-DCs were significantly different from immature DCs (Fig. 3a, I) in the ‘knob-like projection’ form ({chi}2 test, P < 0.0001), and from mature DCs (Fig. 3a, III) in the ‘needle-like dendrites’ form ({chi}2 test, P < 0.001). Similarly, a significantly higher proportion of the large prominent ruffle form was present in the MPA-DC population than in the other populations ({chi}2 test, P < 0.001). The above analysis highlights the fact that MPA-DCs displayed a distinctive profile which differed from those of both immature and mature DCs.



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Fig. 3. (a) Analysis of DC morphology. Scanning electron microscopy analysis of monocyte-derived DCs. (I) DC with knob-like projections, (II) DC with large prominent ruffles and (III) DC with needle-like dendrites. Original magnification: I and II x2000, III x5000. (b) Analysis of DC morphology. Almost 300 cells were observed and assigned to three categories according to their morphologic features.

 
MPA-DCs display impaired IL-12 synthesis and increased production of IL-10
To find out whether MPA-DCs had a tolerogenic phenotype in terms of cytokine production, we assessed the synthesis of cytokines by DCs that had been exposed to this immunosuppressive drug. As shown in Fig. 4, MPA-DCs produced less IL-12 compared with mature DCs. Conversely, MPA-DCs had increased secretion of IL-10 compared with mature DC. This secretion of cytokines by the MPA-DCs differed from the cytokine secretion profiles of mature DCs and immature DCs, which secreted very little IL-12 and IL-10.



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Fig. 4. Cytokine synthesis of MPA-DCs. Levels of cytokines produced by immature DCs and DCs matured by TNF-{alpha} in the absence or presence of MPA (100 µM) for 48 h were determined by ELISA. The results are expressed in picograms per milliliter as the mean ± SD of a triplicate determination. Data are representative of one out of six experiments. *P < 0.005, **P < 0.0001 compared with mature DCs.

 
MPA-DCs have a chemokine receptor phenotype similar to that of mature DCs
To gain insight into the effects of MPA on DC maturation, we investigated the expression of chemokine receptors. Using an RPA, we analyzed the expression of CCR1, CCR2, CCR5, CCR7, CXCR1 and CXCR4 mRNA of immature, mature and MPA-DCs. As shown in Fig. 5, immature DCs expressed high levels of CCR1, CCR2, CCR5 and CXCR1 mRNA, whereas these were all strongly down-regulated in mature DCs. In contrast, CXCR4 and CCR7 mRNA were present at low levels in immature DCs and up-regulated in mature DCs. MPA-DCs still expressed high levels of CXCR1 mRNA, which corresponded to an immature profile. Nevertheless, the chemokine receptor expression of MPA-DCs closely resembled that of mature DCs, with down-regulation of CCR1, CCR2 and CCR5 mRNA expression, and up-regulation of CXCR4 and CCR7 mRNA expression, although for some of the donors tested, levels of CXCR4 and CCR7 mRNA remained lower than in mature DCs.



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Fig. 5. MPA-DCs have a mature profile for chemokine receptors mRNA synthesis. Chemokine receptor mRNA levels (A and B) were measured by RPA on immature (i), mature (m) and MPA-DCs (100 µM)(MPA). Chemokine band densities were quantified and normalized with those of GAPDH and L32. Results are expressed as arbitrary densitometry units (C) and are representative of five out of seven donors tested.

 
We also studied the surface expression of chemokine receptors CCR5 and CCR7. As expected from the RPA analysis, and as in mature DCs, the MPA-DCs expressed CCR7 and not CCR5 (Fig. 6).



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Fig. 6. Pre-treatment of DCs with MPA does not affect the regulation of chemokine receptor expression induced by TNF-{alpha} on immature DCs. Monocyte-derived DCs matured in the presence (MPA-DC) or absence (mature DC) of 100 µM MPA and immature DCs were harvested, CCR5 (A) and CCR7 (B) expression was analyzed by FACS. Empty histograms correspond to the control isotypes, and solid histograms represent specific staining of the cell-surface markers indicated. Data are representative of >10 experiments.

 
MPA inhibits the ability of human DCs to induce proliferation of allogeneic CD4+ T cells via the direct pathway
As MPA was shown to inhibit DC maturation, we next tried to find out whether this inhibition impaired co-stimulatory ability. Compared with immature DCs, mature DCs are potent stimulators of allogeneic T cells. The allostimulatory ability of MPA-DCs was determined by ascertaining their ability to induce proliferation of purified allogeneic CD4+ T cells in 5-day MLR cultures. As shown in Fig. 7A, MPA-DCs exhibited a considerably reduced ability to stimulate T-cell proliferation; this inhibition of T-cell proliferation was dose-dependent. Moreover, pre-exposure to MPA reduced the ability of DC to stimulate a primary alloreactive T-cell response at all the DC : T-cell ratios tested (Fig. 7B).



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Fig. 7. (A) Pre-treatment of DCs with MPA inhibits their ability to stimulate allogeneic CD4+ T cells. Monocyte-derived DCs matured with TNF-{alpha} in the absence (mature DC) or presence of increasing doses of MPA (10–100 µM) were collected on day 7, washed, irradiated (20 Gy) and then co-cultured with allogeneic CD4+ T lymphocytes at a cell ratio of 1 : 3 for a total of 5 days. T-cell proliferation was assessed by incorporation of [3H]thymidine ([3H]TdR) during the last 18 h of the assay (mean ± SD of a triplicate). Date are representative of one out of >10 experiments. *P < 0.01, **P < 0.001 compared with control. (B) Inhibition of CD4+ T-cell proliferation by MPA-DCs at various DC/T-cell ratios. Allogeneic CD4+ T cells (105) were co-cultured with increasing numbers of MPA-treated (square) or mature (closed circles) DCs (2.5 x 103–30 x 103) for 5 days. T-cell proliferation was assessed by incorporation of [3H]TdR during the last 18 h of the assay (mean ± SD of triplicate). Data are representative of one out of three experiments. (C) MPA inhibits DC maturation whatever the maturing agent. Allogeneic CD4+ T cells (105) were co-cultured with DCs matured with TNF-{alpha}, LPS, Zymosan or poly I : C in the presence or absence of MPA (100 µM). Proliferation was assessed by incorporation of [3H]TdR during the last 18h of the assay. Values are the means ± SD of a triplicate. Data are representative of one out of three experiments. (D) and (E) Effect of MPA on DCs is not due to an imbalance in purine synthesis. Monocyte-derived DCs matured with TNF-{alpha} treated with dbGMPc (C) or Rp-AMPS (D) or left untreated in the absence (mature DC) or presence of MPA (100 µM) were collected on day 7, washed, irradiated (20 Gy) and then co-cultured with allogeneic CD4+ T lymphocytes at a cell ratio of 1 : 3 for a total of 5 days. T-cell proliferation was assessed by incorporation of [3H]TdR during the last 18 h of the assay (mean ± SD of a triplicate). Data are representative of one of three experiments. *P < 0.001 compared with control.

 
Knowing that MPA has a direct action on lymphocytes, we checked to make sure that treated DCs had not released MPA into the supernatant during co-culture with CD4+ T cells. DCs treated with 100 µM MPA during maturation were washed and re-suspended in RPMI–10% FCS. The supernatant collected after 24 h and used in MLR with control DC did not induce any inhibition of CD4+ T-cell proliferation (73 500 c.p.m. in control supernatant versus 78 200 c.p.m. in MPA-DC supernatant; data not shown).

Having demonstrated the inhibitory effect of MPA on the TNF-{alpha}-induced maturation of DCs, we then investigated whether this inhibition occurred when other maturing agents, such as LPS, zymosan and poly I : C were used. Similarly, MPA-DCs treated during maturation with LPS, zymosan or poly I : C exhibited an impaired ability to stimulate allogeneic CD4+ T cells (Fig. 7C).

In order to assess the role of IL-10 synthesis by DCs in their reduced ability to stimulate CD4+ T-cell proliferation, we performed assays in the presence of neutralizing anti-IL-10 antibodies, and we did not observe any significant difference between T-cell proliferation induced by MPA-DC in the presence or absence of anti-IL-10 antibodies (data not shown).

MPA is an inhibitor of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. This inhibition leads to the intracellular depletion of GMP, GTP and dGTP. We therefore investigated whether supplementation with dbGMPc could prevent the effects of MPA on DCs. We also used Rp-AMPS, an inhibitor of cyclic adenosinch monophosphate (cAMP), to counter any potential excess of adenosine nucleotides. The action of MPA was not overcome by adding guanosine nucleotides or by blocking the adenosine nucleotides. DCs exposed to dbGMPc or Rp-AMPS in addition to MPA induced very weak proliferation of CD4+ T lymphocytes (Fig. 7D and E). The action of MPA on DC therefore seems to be independent of IMPDH inhibition.

MPA does not induce increased apoptosis of DC
In order to understand the mechanisms of this reduced co-stimulatory ability of MPA-DCs, we checked whether this impairment of DC function was due to increased apoptosis of treated DCs. The apoptotic status of the DCs was determined by quantification of phosphatidylserine externalization using Annexin V. Figure 8 shows that the percentage of apoptotic cells was low and similar in MPA-DCs and control DCs (3.3 and 3.0%, respectively). These findings indicate that MPA does not induce apoptosis of DC.



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Fig. 8. MPA does not induce apoptosis in monocyte-derived DCs. Monocyte-derived DCs stimulated with TNF-{alpha} for 48 h in the presence (MPA-DC) or absence (mature DC) of 100 µM MPA were harvested and analyzed by flow cytometry. The percentages of necrotic [Annexin V+/7-amino actinomycin D (7-AAD+)] and apoptotic (Annexin V +/7-AAD–) cells were determined using Annexin V-FITC/7-AAD staining, as described in Methods. Data are representative of one out of three experiments.

 
MPA inhibits the ability of human DCs to induce proliferation of allogeneic CD4+ T cells via the indirect pathway
As MPA-DCs have reduced allostimulatory ability via the direct pathway and also display high endocytosis capacities, we evaluated the ability of MPA-DCs to induce T-cell proliferation via an indirect pathway model in order to obtain further insight into the influence of MPA on the functional immunostimulatory capacities of DCs. DCs were pulsed with lysate from allogeneic PBMC and matured in the presence or absence of MPA under serum-free conditions. We next assessed the T-cell stimulating ability of lysate-pulsed DCs in autologous co-culture. As shown in Fig. 9, DCs pulsed and matured with TNF-{alpha} in the presence of MPA exhibited impaired ability to induce T-cell proliferation in response to allogeneic peptides. These findings show that MPA reduces the ability of DC to induce allogeneic proliferation, via both the direct and indirect pathways.



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Fig. 9. Pre-treatment of DCs with MPA inhibits their ability to stimulate syngeneic T cells. Monocyte-derived DCs were pulsed or not with lysate from allogeneic PBMC in X-VIVO–2% HSA and matured with TNF-{alpha} in the presence (MPA-DC) or absence (mature DC) of 100 µM MPA. Then on day 7 DCs were collected, washed, irradiated (20 Gy) and cocultured with syngeneic CD4+ T lymphocytes at a cell ratio of 1 : 3 for a total of 5 days. T-cell proliferation was assessed by incorporation of [3H]thymidine during the last 18 h of the assay (mean ± SD of a triplicate). *P ≤ 0.001 compared with control. Data are representative of one out of four experiments.

 
MPA-DCs alter expression of activation molecules on allogeneic CD4+ T cells
As CD4+ T cells co-cultured with MPA-DCs exhibited impaired proliferation capacities, we next assessed the expression of activation molecules on the surface of T cells at the end of the primary MLR. As shown in Fig. 10, CD4+ T cells activated by mature DCs exhibited a high level of expression of CD25. In contrast, the expression of CD25 was weaker on CD4+ T cells stimulated by MPA-DCs (29 versus 57% of positive cells). We then analyzed the expression of CD69, a very early activation molecule, and observed that CD69 expression was similar in the two groups of CD4+ T cells; they were strongly CD69-positive. These findings show alterations of the activation-signaling events in CD4+ T cells co-cultured with MPA-DCs.



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Fig. 10. Impaired activation of CD4+ T cells stimulated by MPA-DCs. At the end of a 5-day co-culture with MPA-DCs versus mature DCs, CD4+ T cells were harvested and the expression of activation marker was performed by FACS. CD4+ T cells were characterized by anti-CD3 antibody and analyzed for CD25 or CD69 expression. Data are representative of one out of six experiments.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Depending on the maturation process, and the nature and intensity of the expression of co-stimulation molecules, myeloid DCs may induce either alloagression or tolerance. The blocking of DC co-stimulation properties may therefore offer a means of inducing graft tolerance.

In this study we analyzed the effects of MPA, an immunosuppressive drug, on DC maturation and showed that MPA affects the maturation and activation of human DCs ex vivo, conferring special properties on MPA-DCs. We demonstrated that the profile of DCs exposed to MPA corresponded to neither the immature nor the mature profiles. They presented a distinctive morphological appearance, maintained their endocytosis capacity, displayed impaired expression of co-stimulatory molecules and secreted increased amounts of IL-10. Moreover, their stimulatory abilities were also impaired, and we observed that treated DCs had an inhibitory effect on T-cell proliferation induced via both the direct and indirect pathways.

As mature and immature DCs have distinct morphological appearances, we also investigated the morphological characteristics of MPA-treated DCs. The fact that the DCs were not completely mature was confirmed by morphological analysis using scanning electron microscopy. The MPA-DCs had a distinctive structure corresponding to neither the mature nor immature DC structure but to a specific intermediate stage. These partially matured cells could correspond to the semi-mature DCs described by Lutz and Schuler (24).

We then studied maturation markers and observed that the up-regulation of co-stimulatory molecules (CD86, CD40, HLA-DR) and adhesion molecules (CD54, CD58) in the course of DC maturation was impaired when DCs were treated with MPA during this period. A decrease in both the MFIs and in the percentage of cells responding to maturing agents was observed in DCs treated with this immunosuppressive drug. Our findings, showing that the maturation of human DCs is inhibited by MPA, are in accordance with previous studies demonstrating a similar inhibitory effect of MMF (the pro-drug of MPA) on CD40, CD80, CD86 and CD54 expression in mouse (18) and human (19) models. However, in contrast with the latter study, MPA-treated cells maintained their endocytic capacity. These differences between our results and those of the authors mentioned above could be explained by the dose range of MPA and the fact that we treated the DCs only during the maturation phase and not throughout the differentiation and maturation processes. Moreover, our dose range matched the physiological doses reported for patients treated by MMF (10–100 µM) (21, 22), and the doses of MPA we used induced no DC apoptosis, in contrast to Colic's study (53% survival) (19). Our culture conditions may have induced a particular maturation process, resulting in DCs with a specific profile.

We also analyzed the levels of cytokines produced by MPA-DCs. In accordance with their non-mature phenotype, MPA-DCs secreted only small amounts of IL-12 but increased amounts of IL-10. This shift of the IL-12/IL-10 balance toward IL-10 was also observed with vitamin D3/dexamethasone-treated DCs (25), and this is interesting as the secretion of large quantities of IL-10 might correspond to a tolerogenic profile of DC. IL-10 has an autocrine effect on DC by reducing up-regulation of the expression of co-stimulatory molecules and by inhibiting inflammatory cytokine production (26, 27). IL-10 also has a direct effect on T cells, leading to the suppression of proliferative, effector and cytokine T-cell responses, and the induction of anergy (28). Nevertheless, the impairment of co-stimulatory abilities of MPA-DCs cannot be explained solely by the secretion of IL-10 because their co-stimulatory abilities were not significantly affected by neutralizing anti-IL-10 antibodies. This probably results from the combined characteristics of MPA-DCs, i.e. their inhibition of co-stimulatory molecule expression, the maintenance of endocytic capacities, their particular morphology which might affect interactions between DC and T cells and the inhibition of IL-12 secretion.

The functional relevance of the effects of MPA on DC was established during MLR experiments in which we observed that this inhibition of DC maturation led to the impairment of the ability of DC to induce proliferation of human allogeneic CD4+ T cells. The functional properties of MPA-treated DCs were similar to those observed for immature DCs (10, 11, 24, 26, 29, 30) and with DCs treated by agents such as glucocorticosteroids, vitamin D3, N-acetyl-cysteine, rapamycin, deoxyspergualin and IL-10 (3137).

However, our results demonstrated for the first time that the indirect pathway could be inhibited by ex vivo pre-treated DC. MPA-treated DCs inhibited the proliferation not only of allogeneic T cells but also of syngeneic T cells when DCs were pulsed with a lysate of allogeneic PBMC. The fact that MPA-DCs were still able to endocyte alloantigens and induced a blockade of the indirect pathway are particularly relevant to their potential role to induce allograft tolerance. Indeed, the indirect pathway, by which recipient DCs present donor antigens, plays an essential role in the chronic phase of graft rejection (38, 39). The inhibition of DC maturation by MPA in vivo might therefore induce inhibition of the proliferation of T cells specific for allogeneic peptides and thus provide more effective control of chronic rejection. Such an effect of MMF on long-term improvement of graft survival has already been reported (40). Finally, we have demonstrated that MPA-DCs activate allogeneic CD4+ T cells in an incomplete manner, with reduced CD25 expression. This decrease in CD25 expression could explain the impaired proliferative responses in T cells because its expression is required for the progression to the S phase. The surface expression of CD25 requires gene transcription and new protein synthesis (41). In contrast, CD69 expression, which is expressed within a few hours of activation and does not require RNA or protein synthesis, is maintained (42, 43). The differential regulation of the expression of CD69 and CD25 indicates that MPA-DCs do not induce the correct intracellular signal transduction pathways and alter the transcription of factors necessary to the full activation of CD4+ T cells. The precise consequences of CD4+ T-cell activation by MPA-DCs are currently under investigation and preliminary findings have shown a donor-specific hyporesponsiveness of the allogeneic CD4+ T cells (data not shown; C. Lagaraine, manuscript in preparation).

To give a ‘tolerogenic’ signal to CD4+ T cells, the DCs have to reach secondary lymphoid organs. RPA analysis of chemokine receptors has allowed to characterize the migratory profile of MPA-DCs, which was similar to that of mature DCs, with marked synthesis of CCR7 and CXCR4 mRNA and little synthesis of CCR1, CCR2 and CCR5 mRNA (44, 45). The same profile was found by flow cytometry analyses, MPA-DCs exhibiting down-regulation of CCR5 and up-regulation of CCR7 compared with immature DCs. We are currently analyzing the migratory capacities of MPA-DCs using chemotaxis assays, and preliminary findings have shown that MPA-DCs migrate in response to CCL19, a ligand of CCR7, to the same extent as mature DC (data not shown). These characteristics may suggest an effective migratory potential that is promising for cell therapy based on the infusion of MPA-DCs.

The precise molecular effects of MPA on DCs remains unknown. In DCs, in contrast to lymphocytes, the inhibitory effect of the drug does not seem to be due to inhibition of inosine monophosphate dehydrogenase. Indeed, the action of MPA was not overcome by adding guanosine nucleotides or by blocking adenosine nucleotides. MPA might have no impact on the phosphatidyl inositol-3-kinase pathway, as we observed no inhibitory effect on the migration profile, the key role of this enzyme hitherto reported (46). We finally demonstrated that MPA inhibits full maturation of DCs whatever the maturing agent used, Toll-like receptor (TLR) activators or TNF-{alpha}, although these agents are known to induce maturation via two different signaling pathways (4749). This suggests that the impact point of MPA on DCs is probably located at a common stage of the TNF- and TLR-signaling pathways.

Overall, these results reveal another mechanism of action of MPA which, in addition to its role in the inhibition of T-cell proliferation, has a direct inhibitory effect on human DCs. The fact that MPA inhibits the maturation of DCs, without affecting their mature migratory phenotype, could prove valuable for cell therapy. These effects observed in vitro are promising, as MMF is widely used in transplantation and a recipient's treatment might maintain the ‘tolerogenic’ nature of infused MPA-DCs despite the pro-inflammatory context of the graft.


    Acknowledgements
 
We would like to thank the staff of the Etablissement Français du Sang (EFS) for the removal of donors' blood from cytapheresis. We thank the Department of Microscopie électronique of Tours University, and particularly Brigitte Arbeille and Claude Lebos for performing the scanning electron microscopy studies and Mathieu Benoit Voisin for RPA analyses, Angélique Rico (EFS) for technical assistance, Hervé Groux for stimulating discussion and Doreen Raine for editing the English language.


    Abbreviations
 
APC   antigen-presenting cells
dbGMPc   N-2,2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate sodium salt
DC   dendritic cell
EFS   Etablissement Français du Sang
GAPDH   glyseraldehyde-3 phosphate dehydrogenase
GM-CSF   granulocyte macrophage colony-stimulating factor
HSA   human serum albumin
IMPDH   inosine monophosphate dehydrogenase
MFI   mean fluorescence intensity
MLR   mixed leukocyte reaction
MMF   mycophenolate mofetil
MPA   mycophenolic acid
poly I:C   polyinosine-polycytidylic acid
RPA   RNase protection assay
Rp-AMPS   Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt
TLR   Toll-like receptor
TNF-{alpha}   tumor necrosis factor-{alpha}

    Notes
 
Transmitting editor: E. Vivier

Received 12 July 2004, accepted 10 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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