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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Leflunomide, a potent disease-modifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen-presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen-specific formation of stable T cell/APC conjugates.

Methods

Effects of pharmacologic concentrations of teriflunomide on CD3/CD28- and lymphocyte function–associated antigen 1–induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen- and antigen-pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation.

Results

Teriflunomide inhibited T cell receptor (TCR)/CD3–mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF-κB, remained unaltered. In contrast, inhibition of TCR/CD3-triggered β1,2 integrin avidity and integrin-mediated costimulation (outside-in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates.

Conclusion

These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune-mediated disorders such as rheumatoid arthritis.

Leflunomide is a synthetic, low molecular weight isoxazole derivative that is clinically applied for the treatment of rheumatoid arthritis (RA) due to its potent antiinflammatory and immunosuppressive properties (1–3). Furthermore, it is currently being evaluated for the treatment of inflammatory bowel disease (4) and as a novel immunosuppressive agent for the amelioration of chronic allograft rejection (5). Leflunomide is a prodrug that is rapidly converted into its active metabolite A77 1726, called “teriflunomide,” which constitutes >95% of the drug in the circulation. Two major mechanisms have been reported to underlie the inhibitory effects of leflunomide on T cell activation. First, teriflunomide blocks de novo pyrimidine synthesis by inhibiting dihydroorotate dehydrogenase (DHODH), which leads to diminished DNA synthesis and thus impaired proliferative capacity (6). Second, teriflunomide is an inhibitor of the Src family tyrosine kinases LCK and Fyn and, as such, brought about a diminished CD3-induced calcium response in Jurkat cells (7) and diminished interleukin-mediated signaling by inhibition of JAK-3 phosphorylation, as demonstrated in a murine cell line (8). However, LCK was shown to be dispensable for antigen-presenting cell (APC)–induced T cell activation and even to be a negative regulator of T cell receptor (TCR)–induced signals (9). Moreover, it is unknown whether teriflunomide exerts any effects on untransformed T cells, independent of de novo pyrimidine synthesis.

Successful T cell activation depends on prolonged stimulation by professional APCs, which is enabled by the formation of a so-called immunologic synapse (IS) (10). The IS is a supramolecular structure initiated by congregating of specific adhesion and signaling molecules at the contact site between the T cell and the APC, which facilitates activation and further differentiation of T cells (11). Stabilized T cell/APC conjugates are characterized by clustering of CD3 within the IS (12). Relocalization of T cell molecules to the T cell/APC interface is an active and highly regulated process, which is induced by the integration of signals derived from the TCR/CD3 complex and costimulatory receptors and requires cytoskeletal rearrangements (10, 13, 14). The β2 integrin lymphocyte function–associated antigen 1 (LFA-1) plays a dual and crucial role in the formation of T cell/APC conjugates. First, due to TCR-mediated signals, LFA-1 alters its adhesive state mainly by enhanced avidity, which is controlled by the actin cytoskeleton (15–17). This process of “inside-out signaling” leads to enhanced binding of LFA-1 to its ligand intercellular adhesion molecule 1 (ICAM-1) and thus adhesion to APCs (18,19). Second, ligand binding of LFA-1 itself transduces critical signals (“outside-in signaling”), which augment TCR/CD3-induced T cell adhesion, surface molecule expression, and interleukin production, and then lead to Th1 differentiation (20–22).

In the present study, we demonstrated a novel mechanism of action of the disease-modifying antirheumatic drug (DMARD) leflunomide, which is independent of its activity as an antimetabolite. Despite only moderate inhibition of proximal T cell signaling, the active leflunomide metabolite teriflunomide suppressed integrin avidity after TCR/CD3 engagement. Furthermore, strong abrogation of ICAM-1–mediated costimulation of T cells by teriflunomide was observed. Consistent with these alterations, the formation of the mature IS was profoundly disrupted by teriflunomide, culminating in the abrogation of antigen-specific conjugate formation occurring between APCs and T cells. Since undisturbed interaction of T cells with APCs is a prerequisite for eliciting an effective immune response, these results could explain the potency of leflunomide in the treatment of a variety of disorders involving activated T cells, independent of its antimetabolic activity.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Cell isolation and culture.

To obtain peripheral blood T lymphocytes (PBTLs), mononuclear cells were purified from buffy coats of peripheral blood provided by healthy volunteers, using Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation. Subsequently, PBTLs were isolated by rosetteing with neuraminidase-treated sheep erythrocytes (Dade Behring, Marburg, Germany). Cells were cultivated in RPMI 1640 medium (Invitrogen, Groningen, The Netherlands) supplemented with 2 mML-glutamine, 50 μg/ml streptomycin, 50 units/ml penicillin, and 10% fetal calf serum (Hyclone, Logan, UT). Plasma concentrations (mean ± SEM) of the active leflunomide metabolite A77 1726 (in RA patients who received a leflunomide maintenance dosage of 20 mg/day) were 46 ± 31 μg/ml (∼150 ± 100 μM) (23). Therefore, we chose concentrations ranging from 12.5 to 200 μM of A77 1726 or its free acid amide HMR 1726 (Aventis Pharmaceuticals, Strasbourg, France) for overnight incubation of cells. Both compounds exerted the same effects and are referred to as “teriflunomide” throughout this report. In some experiments, uridine was added to test the reversibility of the observed effects of teriflunomide. The generally used uridine concentration (50 μM) was shown to be sufficient and equally effective as higher uridine concentrations (up to 200 μM) to bypass DHODH inhibition, according to the literature (24) and as assessed by partial reversal of inhibited CD3/CD28-induced T cell proliferation at low teriflunomide concentrations. Like the 50 μM concentration, 200 μM of uridine could not reverse the effects of teriflunomide, as measured in various assays (surface marker expression, homotypic cell aggregation, IS formation) (data not shown).

Calcium response.

Cells were labeled with the fluorescence Ca++ indicator Indo 1-AM (Molecular Probes, Eugene, OR) by incubating cells (2 × 106/ml) at 37°C for 30 minutes in culture medium containing 2 μg/ml Indo 1-AM. After washing, samples (106 cells in 200 μl culture medium) were equilibrated to 37°C for 7 minutes. During flow cytometric measurement, cells were stimulated using CD3 by adding 10 μg/ml anti-CD3 monoclonal antibody (mAb) (clone OKT3; Ortho Diagnostics, Raritan, NJ) followed after 1 minute by 20 μg/ml F(ab′)2 fragments of goat anti-mouse IgG (Sigma, St. Louis, MO) for crosslinking. Measurement of cytoplasmic Ca2+ concentration by flow cytometry was performed at a constant temperature of 37°C with a FACStar Plus (Becton Dickinson, Mountain View, CA) equipped with an argon laser (excitation 333–363 nm, detection 530 nm for calcium-free Indo 1-AM, 395 nm for the calcium-bound form). The ratio of fluorescence intensity channel 1 to fluorescence intensity channel 2 at both wavelengths was computed as a direct estimate of the cytoplasmic calcium concentration and was measured for 5 minutes following stimulation.

Biochemical analyses of early signaling.

Early signaling events were analyzed essentially as previously described (25). Briefly, PBTLs (5 × 107/ml in original culture medium) were stimulated by incubation with anti-CD28 mAb (clone Leu28) (10 μg/ml; BD Biosciences, San Jose, CA) and anti-CD3 mAb (10 μg/ml) and subsequent crosslinking for 15 minutes. Stimulation was stopped by the addition of ice-cold washing buffer. Cells were pelleted by a short centrifugation (12,000g for 20 seconds) and lysed on ice for 30 minutes in Tris buffered saline (pH 7.4) containing 1% Nonidet P40 (Pierce, Rockford, IL), phosphatase, and protease inhibitors. Nuclei were removed by short centrifugation. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK). Antibodies directed against phosphorylated forms were used to assess the phosphorylation of ERK (Santa Cruz Biotechnology, Santa Cruz, CA) and p38 (Cell Signaling Technology, Beverly, MA). Antigens were detected using horseradish peroxidase–labeled secondary antibodies (Sigma). Chemiluminescence was generated with BM chemiluminescence substrate (Roche Diagnostics, Mannheim, Germany) and quantified on a Lumi-Imager (Roche Diagnostics). Membranes were stripped and reprobed for nonphosphorylated ERK-2 and p38 as a quantity control (mAb from Santa Cruz Biotechnology). IκBα was detected using rabbit polyclonal antibody from Merck Biosciences (Darmstadt, Germany).

Adhesion to ICAM-1 and fibronectin.

Culture plates (96-well) were coated by overnight incubation at 4°C with 5 μg/ml ICAM-1/Fc chimera (R&D Systems, Minneapolis, MN) or 20 μg/ml fibronectin (Sigma) in phosphate buffered saline (PBS). Cells (3 × 105/well) were plated on ice for 45 minutes in Iscove's modified Dulbecco's medium supplemented with 0.4% bovine serum albumin in PBS and stimulated for 15 minutes at 37°C with anti-CD3 mAb plus a crosslinker (10 and 20 μg/ml), phorbol myristate acetate (PMA) (100 ng/ml; Alexis, San Diego, CA) plus ionomycin (1.5 μM), or Mg2+ (5 mM) plus EGTA (2 mM). After 3 washes with PBS, cells were detached with trypsin/EDTA (Invitrogen) and counted using a flow cytometer (FACSCalibur; BD Biosciences). The cell numbers obtained were related to the total cell numbers determined from respective unwashed wells.

Homotypic cell aggregation.

PBTLs (1 × 106/ml) were incubated in 24-well culture plates and stimulated with PMA (100 ng/ml) and ionomycin (1.5 μM) or with immobilized anti-CD3 mAb (overnight incubation of 24-well plates in 0.1 μg/ml OKT3 in PBS) and anti-CD28 mAb (0.5 μg/ml) for 24 hours. Cell clustering was determined by light microscopy.

Assessment of T cell activation markers and intracellular interleukin-2 (IL-2).

To assess IL-2 production, cells were stimulated with immobilized anti-CD3 mAb (overnight incubation of 24-well plates with 0.1 μg/ml OKT3 in PBS) and anti-CD28 mAb (0.5 μg/ml) with or without the ICAM-1/Fc chimera (5 μg/ml), or with PMA (100 ng/ml) plus ionomycin (1.5 μM; Sigma) at 37°C for 8 hours in the presence of brefeldin A (2 μg/ml; Sigma). After washing with PBS, the cells were fixed with 2% formaldehyde solution for 20 minutes, washed, and incubated with phycoerythrin (PE)–labeled rat anti-human IL-2 (Becton Dickinson) for 25 minutes at room temperature in the presence of 0.1% saponin (Sigma). To induce CD25 and CD69 surface expression, cells were stimulated in the same manner but for 24 hours. Fluorescein isothiocyanate (FITC)– and PE-labeled antibodies (Becton Dickinson) were used for flow cytometry. To determine integrin expression after teriflunomide incubation, cells were stained with FITC- or PE-labeled mAb to the LFA-1 α-chain (CD11a) and β-chain (CD18; all from BD Biosciences). Samples were analyzed with a FACSCalibur.

Formation of immunologic synapses and antigen-induced T cell/APC conjugate formation.

For superantigen stimulation, 5 × 106/ml Raji cells were labeled with 0.5 μM of CellTracker Orange CMTMR (Invitrogen) and pulsed with 5 μg/ml staphylococcal enterotoxin E (Toxin Technology, Sarasota, FL) in Hanks' balanced salt solution (HBSS) at 37°C for 20 minutes. After washing, Raji cells were incubated with teriflunomide-treated Jurkat cells (JE6-1; American Type Culture Collection, Manassas, VA) at 37°C at a ratio of 1:1 for 1 or 15 minutes. The reaction was stopped by adding ice-cold HBSS. Cells were plated on poly-L-lysine–coated slides (Marienfeld, Lauda-Koenigshofen, Germany) and allowed to settle for 15 minutes on ice. To stain filamentous actin (F-actin; Invitrogen) and CD3ε, cells were fixed with 4% formaldehyde in PBS for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature (except for CD3 staining), and stained with anti-CD3ε (clone UCH-T1; Santa Cruz Biotechnology) or Alexa Fluor 488–labeled phalloidin (to stain F-actin) for 30 minutes at room temperature. To stain LFA-1α, cells were air-dried for 1 hour, fixed with ice-cold (−20°C) methanol, and treated with anti–LFA-1α (clone 27; BD Transduction Laboratories) for 30 minutes at room temperature. Secondary antibodies were labeled with Alexa Fluor 488 (Invitrogen). The percentage of conjugates that showed relocalization of the respective molecules was determined by 2 individuals (MZ, RG), who counted at least 100 conjugates per sample in a blinded manner.

For antigen-specific stimulation, 5 × 106/ml HOM-2 cells were labeled at 37°C for 3 hours with 0.5 μM of CellTracker Orange CMTMR and 200 μg/ml hemagglutinin (HA) 307-319 peptide, inactive peptide (HA K316E), 20 μg/ml staphylococcal enterotoxin B (Sigma), or were left unpulsed. After washing, HOM-2 cells were incubated with CH7C17 T cells (transfected with HA 307-319–specific TCR [26,27], generously provided by L. Wedderburn, Institute of Child Health and Great Ormond Street Hospital for Children NHS Trust, London, UK) at a ratio of 1:1 at 37°C for 1 or 15 minutes. Cells were plated on poly-L-lysine–coated slides and stained for CD3, as described above. The percentage of T cells that formed conjugates with APCs was determined by 2 individuals (MZ, RG), who counted at least 300 T cells per sample in a blinded manner.

Statistical analysis.

Data are expressed as the mean ± SEM. Comparisons were performed by Student's unpaired 2-tailed t-test. P values less than 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Effect of teriflunomide on early T cell signaling events.

To determine whether the effects of leflunomide on primary human T cells involve the inhibition of early signaling events, we investigated the effects of the active leflunomide metabolite teriflunomide on essential TCR/CD3-mediated signaling in PBTLs. Teriflunomide impaired the CD3-induced rise of cytoplasmic calcium in PBTLs by 56% compared with untreated controls (Figure 1a). To test whether inhibition of this very early signaling event is reflected by altered downstream signaling, we analyzed MAPK (p38, ERK-1/2) phosphorylation and the NF-κB activating pathway. Interestingly, activation-induced phosphorylation of p38 and ERK-1/2, as well as the degradation of IκBα that is essential for NF-κB activation, remained unaltered in teriflunomide-treated PBTLs (Figure 1b). Thus, teriflunomide affected only distinct proximal signaling components in primary human T cells, but whether teriflunomide had any impact on subsequent manifestations of T cell functions remained unclear.

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Figure 1. Effects of leflunomide on early signaling in primary T cells. a, Human peripheral blood T lymphocytes (PBTLs) were pretreated with 200 μM teriflunomide (Tef) and stimulated by crosslinking (XL) of CD3 (OKT3). Dot blots show the typical time course of cytoplasmic calcium concentration, and bar graphs show the maximum induced rise of the calcium concentration as a percentage of untreated control (Ctr) (mean and SEM from 8 independent experiments). ∗∗∗ = P < 0.001. b, PBTLs were treated with 200 μM teriflunomide, left unstimulated (unst), or stimulated by crosslinking of CD3 plus CD28 for 15 minutes. The indicated proteins and phosphorylated (p) proteins were detected by immunoblotting. Blots shown are representative of 4 independent experiments.

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Inhibition of T cell adhesion by teriflunomide.

Efficient T cell activation critically depends on integrin-mediated adhesion. Whereas adhesion to the cell surface protein ICAM-1 is crucial for T cell/APC interactions (18,19), β1 integrin–mediated adhesion to extracellular matrix proteins such as fibronectin determines the capacity of T cells to migrate and infiltrate inflamed tissues (28, 29). As shown in Figure 2a, teriflunomide caused a significant and concentration-dependent reduction of CD3-induced T cell adhesion to ICAM-1. When LFA-1 was directly stimulated by Mg2+ and the removal of Ca2+ with EGTA (30), T cell adhesion was also significantly reduced by teriflunomide treatment, although to a lesser extent (Figures 2a and b). Surface expression of LFA-1 α- and β-chains (CD11a and CD18) was not decreased (Figure 2c), which showed that diminished integrin expression was not responsible for the effect. Teriflunomide also inhibited adhesion to ICAM-1 when most proximal signaling steps were bypassed with PMA plus ionomycin (Figure 2b), which indicated effects on downstream signaling. To test whether inhibition of stimulated adhesion was due to diminished pyrimidine synthesis, we added uridine to the cultures and found no abrogation of the effects of teriflunomide on adhesion to ICAM-1 (Figure 2b). Importantly, adhesion to fibronectin was affected by teriflunomide in a similar manner (Figure 2d).

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Figure 2. Inhibition of T cell adhesion by teriflunomide. a, Human peripheral blood T lymphcytes (PBTLs) were pretreated with the indicated concentrations of teriflunomide (Tef), and the adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) induced by crosslinking of CD3 or by treatment with Mg2+ plus EGTA for 15 minutes was analyzed. The mean ± SEM percentage of adherent cells in 2 independent experiments is shown. b, PBTLs were pretreated with 200 μM teriflunomide with or without 50 μM uridine (U) or were left untreated (control [Ctr]), and adhesion to ICAM-1 was induced without further stimulation (unst) or by the indicated stimuli. The percentage of total cells that adhered to immobilized ICAM-1 in 8 independent experiments is shown. PMA + iono = phorbol myristate acetate + ionomycin. c, Surface expression of lymphocyte function–associated antigen 1 chains CD11a and CD18 after 200 μM teriflunomide treatment in 6 independent experiments is shown as the mean immunofluorescence versus untreated control cells. d, PBTLs were pretreated with 200 μM teriflunomide with or without 50 μM uridine, and adhesion to fibronectin was induced by the indicated stimuli. The percentage of total cells that adhered to immobilized fibronectin in 6 independent experiments is shown. Values in b–d are the mean and SEM. ∗ = P ≤ 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. ns = not significant. e, PBTLs were pretreated with 100 μM or 200 μM teriflunomide with or without (w/o) 200 μM uridine and stimulated for 24 hours with PMA plus ionomycin. Cell clustering was determined by light microscopy. Representative photographs from 1 of 6 independent experiments are shown.

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To study whether these short-term effects (15 minutes' stimulation) are also evident after a longer period of stimulation, we investigated the effects of teriflunomide on a hallmark LFA-1/ICAM-1–dependent T cell activation event, i.e., homotypic cell–cell aggregation after 24 hours of stimulation (Figure 2e). When cells were stimulated with PMA/ionomycin, large dense clusters were formed with only a few solitary cells remaining. In the presence of 100 μM teriflunomide, clusters were markedly smaller and less packed, with many solitary cells remaining. In the presence of 200 μM teriflunomide, essentially no clusters were observed (Figure 2e). Cell–cell aggregation following CD3/CD28 stimulation was inhibited by teriflunomide to a similar extent (results not shown). The addition of uridine also did not reverse the inhibition of homotypic aggregation (Figure 2e). Thus, teriflunomide inhibited essential T cell functions, such as adhesion to ICAM-1 and fibronectin induced by various stimuli, and this inhibition was not reversed by exogenous uridine.

ICAM-1–mediated costimulation of T cells is diminished by teriflunomide.

LFA-1 tranduces signals and acts as an important costimulatory molecule (20–22). Inhibition of T cell adhesion to ICAM-1 after selective activation of LFA-1 with Mg2+ plus EGTA by teriflunomide indicated a block of LFA-1 outside-in signaling. Hence, we further investigated the effect of teriflunomide on LFA-1–mediated activation events in PBTLs.

As shown in Figure 3, additional costimulation via ICAM-1/LFA-1 interaction augmented suboptimal CD3 plus CD28–mediated T cell stimulation. Surface expression of the early T cell activation markers CD69 and CD25 was drastically blocked by teriflunomide in CD3 plus CD28–activated T cells, particularly when T cell activation was strongly boosted by ICAM-1–mediated costimulation (Figure 3a). Importantly, teriflunomide also inhibited activation marker expression following stimulation with PMA plus ionomycin (Figure 3a). Teriflunomide treatment tended to inhibit CD3 plus CD28–induced IL-2 production, although not to a degree that was statistically significant (P = 0.38 for 100 μM and P = 0.26 for 200 μM teriflunomide) (Figure 3b), but strongly blocked CD3 plus CD28 plus ICAM-1–induced IL-2 expression (Figure 3b). None of these inhibitory effects of teriflunomide could be reversed by the addition of uridine (Figures 3a and b), which showed that altered pyrimidine synthesis does not account for these effects. Thus, teriflunomide affected distinct CD3/CD28-induced downstream T cell activation events and effectively blocked integrin function at various stages of the T cell activation process.

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Figure 3. Effect of teriflunomide on CD3 plus CD28 plus ICAM-1/lymphocyte function–associated antigen 1 costimulation–induced T cell activation. a, PBTLs were pretreated with 200 μM teriflunomide with or without 50 μM uridine or left untreated and stimulated for 24 hours with the indicated stimuli. Surface expression of CD25 and CD69 was detected by flow cytometry. Mean immunofluorescence (IF) intensities in 6 independent experiments are shown. b, PBTLs were pretreated with 100 μM or 200 μM teriflunomide with or without 50 μM uridine, as indicated, and stimulated for 8 hours with the indicated stimuli in the presence of brefeldin A. Production of phycoerythrin-labeled interleukin-2 (IL-2–PE) was determined by flow cytometry. Representative dot blots and a diagram summarizing 6 independent experiments are shown. Values are the mean and SEM. ∗ = P ≤ 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. See Figure 2 for other definitions.

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Teriflunomide-induced inhibition of immunologic synapse formation.

T cell activation induced by antibodies against distinct receptors or by immobilized ligands only fairly mimics physiologic stimulation, since it completely disregards the specific interaction between T cells and APCs. Prolonged T cell stimulation, which is essential for full T cell activation and further immune responses, critically depends on the formation of a mature IS characterized by relocalization of T cell adhesion and signaling molecules to the T cell/APC interface (10, 31). To determine whether teriflunomide affected IS formation, we first used a model system with Jurkat cells and Raji cells as APCs. These cells spontaneously form conjugates and build IS when the Raji cells are loaded with superantigen. Teriflunomide treatment of T cells profoundly diminished the superantigen-induced relocalization of F-actin, LFA-1α, and, notably, CD3 to the T cell/APC interface (Figure 4). These results demonstrated that teriflunomide exerts a strong inhibitory effect on the formation of the mature IS.

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Figure 4. Inhibition of superantigen-induced immunologic synapse formation. Jurkat cells were incubated with the indicated concentrations of teriflunomide (Tef) and stimulated for 15 minutes with superantigen-pulsed antigen-presenting cells (APCs) (staphylococcal enterotoxin E [SEE]) or unpulsed APCs, as indicated. Proteins (CD3, filamentous actin [F-actin], and lymphocyte function–associated antigen 1α [LFA-1α]) were visualized by indirect immunofluorescence. a, Typical examples of conjugates with teriflunomide-treated (100 μM) or untreated T cells stimulated with superantigen-pulsed APCs (red-stained cells). Arrowheads show conjugates positive for protein relocalization (original magnification × 40). b, A summary of 4 independent relocalization experiments. Values are the mean and SEM. ∗ = P ≤ 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus solvent (solv) control. med = medium.

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In vivo, the evolution of an IS between T cells and APCs resulted in the formation of stable conjugates that are needed for prolonged stimulation and full activation of T cells (32). To investigate whether the inhibitory effects of teriflunomide on integrin activation and IS formation could result in impaired physiologic T cell activation by APCs, we investigated antigen-induced conjugate formation of a peptide-specific T cell line (CH7C17) with antigen-presenting B cells. In this antigen-specific system, conjugate formation occurs only after priming of APCs with the specific antigen, but not with the mutated peptide (Figure 5). Teriflunomide treatment significantly blocked antigen-induced formation of T cell conjugates with APCs (Figure 5). Taken together, these findings indicate that teriflunomide disrupts T cell/APC interactions on the level of the IS and subsequent antigen-specific conjugate formation, which are early key events in promoting T cell–driven immune responses.

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Figure 5. Inhibition of formation of antigen-induced T cell/antigen-presenting cell (APC) conjugates by teriflunomide (Tef). T cells (CH7C17) were treated with 100 μM teriflunomide and stimulated for 15 minutes by incubation with APCs (HOM-2) pulsed with hemagglutinin peptide (HA 307-319), or left unstimulated (incubation with inactive peptide [HA K316E]–pulsed APCs). a, Teriflunomide-treated and untreated T cells (green) stimulated with antigen peptide–pulsed APCs (red). Arrowheads indicate conjugates (original magnification × 40). b, Proportion of T cells forming conjugates with APCs in 4 independent experiments. Values are the mean and SEM. ∗∗∗ = P < 0.001 versus solvent control.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In this study, a novel aspect of the immunosuppressive action of leflunomide distinct from its proposed action as an antimetabolite was revealed, namely, the profound interference of the active leflunomide metabolite teriflunomide with T cell/APC interactions. By mimicking in vivo stimulation of T cells by APC, we showed that teriflunomide disrupts not only maturation of the IS by relocalization of molecules to the contact site, but also antigen-specific T cell/APC conjugate formation. In human T cells, teriflunomide moderately inhibited antibody-mediated T cell activation via CD3 and CD3/CD28, but strongly interfered with physiologically relevant T cell activation via APCs. As a molecular basis of this phenomenon, the activation-driven increase in integrin avidity and costimulation via integrins were strongly impaired by teriflunomide.

These findings may have a substantial impact on our understanding of the influence of teriflunomide as a DMARD, since the completion of an immune response critically depends on the successful interaction of T cells with APCs (32). Furthermore, infiltration of the synovial membrane with leukocytes is a pivotal event in RA, and recent data have demonstrated decreased synovial cellularity in patients treated with leflunomide (33). Also, inhibited activation of monocytic cells by A77 1726–treated T cells (34) may be related to altered integrin avidity. Our data showing a complex influence of teriflunomide on integrin function are consistent with recent data that show that this drug also interferes with leukocyte/endothelial interactions (35). This peculiar property, which is not shared by most other currently used immunosuppressive drugs, such as calcineurin inhibitors, might also be involved in the beneficial effects of malononitrilamides on chronic allograft rejection (5, 36).

While murine lymphocytes are highly sensitive to DHODH inhibition by teriflunomide (1, 8), its 50% inhibition concentration (IC50) for DHODH is ∼150 μM in human cells, similar to the IC50 for LCK and Fyn in Jurkat cells (7). Hence, the concentrations used in this study are the optimal inhibition concentration for tyrosine kinase inhibition and within the range of plasma concentrations in leflunomide-treated patients (23). As shown for several other leflunomide-mediated effects, such as repression of viral replication, response to IL-2, and effector cell functions in an autoimmune encephalomyelitis model (8, 37, 38), the inhibitory effects of teriflunomide in our study are not reversible by the addition of exogenous uridine. Thus, the effects of a diminished pyrimidine pool due to DHODH inhibition, such as reduced production of glycoproteins that are involved in cell/cell contact and adhesion (1) as well as inhibited tumor necrosis factor–induced cellular responses and promotion of Th2 cell differentiation (39, 40), probably do not underlie the effects observed in this study.

Src family kinases play a pivotal role in early T cell signal transduction, as well as in cytoskeletal rearrangements, integrin activation, adhesion, and T cell/APC conjugate formation (41, 42). Hence, the lack of inhibition by the Src family kinase inhibitor teriflunomide on CD3/CD28-mediated signaling via MAPK activation and IκB degradation is astonishing at first glance. However, the roles of Src family kinases in TCR signaling are complex, and LCK even acts as a negative regulator of T cell activation (9, 43, 44). Moreover, inhibition of PMA plus ionomycin–induced activation events by teriflunomide indicates that interference with crucial activation events is not limited to most proximal CD3-induced signaling events, but involves steps downstream of protein kinase C/Ras (PMA) and calcium response (ionomycin).

In addition, this study provides several lines of evidence that teriflunomide strongly affects costimulatory outside-in signaling provided by LFA-1, which is crucial for T cell activation, including effective T cell adhesion and Th1 differentiation (20, 22, 45). Mg2+ plus EGTA induces a high-affinity state of LFA-1, which allows increased binding of LFA-1 to ICAM-1 (21). However, T cell adhesion to immobilized ICAM-1 induced by Mg2+ plus EGTA requires cytoskeletal remodeling and therefore depends on active signaling rather than being a passive process (20). Hence, together with the suppression of ICAM-1–induced augmentation of surface marker and IL-2 expression, impaired T cell adhesion induced by Mg2+ plus EGTA in the presence of unaltered LFA-1 cell surface expression indicates disturbed LFA-1–mediated signaling. Interestingly, our results are consistent with those of other studies on the effects of leflunomide on CD43-induced actin cytoskeleton remodeling (46) and homotypic cell aggregation (47), suggesting that leflunomide generally affects cytoskeletal-driven events. Recently, a molecule that is pivotal for cytoskeletal-driven events such as integrin activation and is also involved in integrin signaling, termed RAPL (regulator of adhesion and polarization enriched in lymphocytes), has been described (48). Hence, future investigations might reveal an interaction of teriflunomide with the activation of this regulator of T cell–adhesive events.

Another putative target of teriflunomide action in T cells is cyclooxygenase 2 (COX-2), which has been shown to be inhibited by A77 1726 in fibroblast-like synoviocytes (49). Importantly, because our experiments analyzed very early events of T cell activation and only already-activated T cells express COX-2, it is conceivable that modulation of COX-2 or its products is not causally involved in the observed effects of teriflunomide. However, inhibition of COX-2 activity at later stages of the effector T cell response might contribute to the complete mode of action of teriflunomide. Collectively, our data indicate that teriflunomide hampers T cell responsiveness beyond the inhibition of critical signaling steps such as LCK activity, which might be reflected by impaired calcium mobilization. Teriflunomide also critically affects integrin functions, which results in impaired adhesion and integrin-mediated costimulation.

Integrin functions are crucial for T cell activation by APCs through the formation of the mature IS (19). Notably, teriflunomide affects not only LFA-1 avidity and outside-in signaling, but also its relocalization to the IS. Inhibited clustering of CD3 within the IS indicates that only an immature type of IS is formed in the presence of teriflunomide, which is insufficient for the formation of stable T cell/APC conjugates (12). Since stable conjugates are indispensable for induction of immune responses (32), the effects of teriflunomide on IS formation could therefore underlie hampered T cell responsiveness in vivo.

In conclusion, we have shown that the active leflunomide metabolite teriflunomide impairs integrin avidity and integrin-mediated signals, which leads to the abrogation of successful T cell/APC interactions as expressed by the formation of the mature IS and antigen-specific conjugates. Furthermore, suppressed adhesion to extracellular matrix proteins may be crucial for the effects of teriflunomide in modifying exaggerated immune responses by interfering with immune cell invasion of inflamed tissue, such as the synovial membrane in RA. These data therefore reveal a novel mode of action of teriflunomide during the induction of cellular immune responses, which may contribute to its clinical effectiveness in diseases involving exaggerated immune responses.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Bianca Weissenhorn for expert technical assistance and Aventis for kindly providing teriflunomide.

REFERENCES

  1. Top of page
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  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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