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Originally published online as doi:10.1189/jlb.0606378 on October 12, 2006

Published online before print October 12, 2006
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(Journal of Leukocyte Biology. 2007;81:319-327.)
© 2007 by Society for Leukocyte Biology

Impairment of T cell interactions with antigen-presenting cells by immunosuppressive drugs reveals involvement of calcineurin and NF-{kappa}B in immunological synapse formation

Maximilian Zeyda*, René Geyeregger*, Marko Poglitsch{dagger}, Thomas Weichhart{dagger}, Gerhard J. Zlabinger{ddagger}, Shigeo Koyasu§, Walter H. Hörl{dagger}, Thomas M. Stulnig*, Bruno Watschinger{dagger} and Marcus D. Saemann{dagger},1

Department of Internal Medicine III,
* Clinical Divisions of Endocrinology and Metabolism and
{dagger} Nephrology and Dialysis, and
{ddagger} Institute of Immunology, Medical University of Vienna, Vienna, Austria; and
§ Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan

1Correspondence: Department of Internal Medicine III, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: marcus.saemann{at}meduniwien.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A stable supramolecular cluster in T cells at the contact site of APCs, the immunological synapse (IS), is essential for full T cell activation. Failure of IS maturation, as determined by defective relocalization of the TCR/CD3 complex at the T cell/APC contact site, is linked with T cell hyporesponsiveness. The effects of clinically used immunosuppressants on these critical events, however, are undefined. Here, we show that treatment of T cells with cyclosporin A, FK506, and dexamethasone, which are known to inhibit calcineurin and NF-{kappa}B, respectively, but not rapamycin, the inhibitor of mammalian target of rapamycin, selectively prevented TCR/CD3 relocalization into the IS, while relocalization of adhesion and cytoskeletal proteins as well as T cell/APC conjugate formation remained unaltered. The involvement of calcineurin and NF-{kappa}B in IS maturation was confirmed by using specific inhibitors of these molecules (FR901725, gossypol, SN50). FK778, as an inhibitor of DNA replication and also TCR/CD3-activated tyrosine kinases, globally abrogated cytoskeletal, adhesion, and signaling molecule relocalization, thereby preventing formation of an IS at an earlier, immature stage along with impaired, antigen-specific T cell/APC conjugate formation. Collectively, blocking IS formation at distinct stages may mediate effects on T cell activation of currently used immunosuppressants, apart from their capacity to block gene transcription, cytokine signaling, and DNA replication. Furthermore, these data imply novel functions of calcineurin and NF-{kappa}B for successful IS maturation.

Key Words: supramolecular activation clusters • human T cell activation • immunosuppression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell activation requires the recognition of allogeneic antigens adequately presented by MHC molecules of professional APCs, such as DC and B cells to cognate TCRs and furthermore, engagement of costimulatory and adhesion receptors by their ligands [1 , 2 ]. These events lead to the activation of a complex intracellular signaling machinery that culminates in the activation of cytokine production, which is essential for subsequent T cell expansion and leads to graft destruction after allogeneic transplantation [3 ]. To control allogeneic transplant rejection, pharmacological interference with cytokine production and cytokine sensitivity by targeting the activation of critical TCR- and cytokine-induced signaling molecules, such as calcineurin, NF-{kappa}B, and the mammalian target of rapamycin (mTOR), have been used successfully in the clinic and are currently the mainstay for most immunosuppressive protocols [4 ]. The commonly used drugs cyclosporin A (CsA) and FK506 target calcineurin activation via immunophilins, thereby blocking transactivation of the NF-AT, which is essential for, e.g., IL-2 gene transcription [5 6 7 ]. Glucocorticoids exert their immunosuppressive effect by inhibiting TCR/CD3- but also cytokine receptor-mediated signaling events [8 ]. Their principle molecular mechanism resides in their ability to block the activation of the central transcription factor NF-{kappa}B via, e.g., induction of its inhibitor I{kappa}B [9 10 11 ]. Rapamycin blocks cytokine receptor-driven cell-cycle progression of T cells via inhibition of mTOR, but recently was also shown to decrease IL-2 mRNA stability upon early T cell activation [12 13 14 ]. Finally, the malononitrilamide FK778 acts in a dual way by blocking TCR/CD3-induced tyrosine kinase activity as well as de novo pyrimidine synthesis and thus, DNA replication, but the detailed mode of action of FK778 is not entirely known yet [15 16 17 ].

T cell activation requires prolonged contact with the APC by formation of stable T cell/APC conjugates and complex molecular rearrangements at the T cell/APC contact site, the so-called immunological synapse (IS) [18 , 19 ]. The IS constitutes a supramolecular surface structure initiated by dynamic remodeling of the actin cytoskeleton, which drives the congregation of specific adhesion and signaling molecules, including the ß2integrin LFA-1, the TCR/CD3 complex, and protein kinase C (PKC)-{theta}, at the contact site between T cell and APC [20 ]. IS formation is an active and highly regulated process, which is induced by the integration of signals derived from the TCR/CD3 complex and costimulatory receptors [21 22 23 ]. The resulting spatial concentration of signaling mediators facilitates effective signal transduction and is a prerequisite for full T cell activation including cytokine production and proliferation as well as further lymphocyte differentiation [24 , 25 ]. In particular, the clustering of the TCR/CD3, an indicator for the evolution of the IS from "immature" to a "mature" stage, correlates with effective T cell activation [26 ].

Interference with IS formation has previously been shown to be a potential mode of action of immunomodulatory chemokines [27 ] as well as to be a mechanism for anti-inflammatory and antirheumatic agents [28 29 30 ]. Thereby, it is suggested that preventing T cell activation by interfering with IS formation might represent a novel, therapeutic strategy for controlling T cell responsiveness such as in transplant rejection or autoimmunity. Indeed, it has been demonstrated recently that genetic deletion of DOCK2, an immune cell-restricted molecule essential for antigen-induced translocation of TCR/CD3 toward the IS [31 ], attenuates allograft rejection in mice [32 ]. This effect is mediated by impaired priming and activation of alloreactive T cells in secondary lymphoid organs, leading to the prevention of significant graft tissue infiltration [32 ].

Previous data indicate that FK778 severely interferes with IS formation [33 ], while the effects of currently used, immunosuppressive drugs on the early events occurring at the interface of T cells and professional APCs still remain unknown. Here, we demonstrate for the first time that diverse immunosuppressive drugs, currently used in clinical organ transplantation, affect discrete steps of the IS formation. The potential biological and clinical relevance of the modulation of the IS and T cell/APC interactions is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell isolation and culture
To obtain peripheral blood T lymphocytes (PBTLs), mononuclear cells were purified from buffy coats of peripheral blood given by healthy volunteers using Ficoll-Paque (Amersham, Uppsala, Sweden) density gradient centrifugation. Subsequently, PBTLs were isolated by rosetting with neuraminidase-treated sheep erythrocytes (Dade Behring, Marburg, Germany). Cells were cultivated in RPMI-1640 medium (Invitrogen, Groningen, The Netherlands) supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin, and 10% FCS (Hyclone, Logan, UT) and incubated with the indicated substances. Immunosuppressants used were CsA (1 µg/ml, Novartis AG, Basel, Switzerland), FK506 and FK778 (100 ng/ml and 200 µM, respectively, unless stated otherwise, Fujisawa Healthcare Inc., Japan), dexamethasone (1 µM, Sigma Chemical Co., St. Louis, MO), and rapamycin (200 nM, Wyeth, Madison, NJ). Other substances used were the calcineurin inhibitors FR901725 [34 ] (10 µg/ml) and Gossypol (10 µM, Sigma Chemical Co.) and the proteasome inhibitor SN50 (25 µg/m, Calbiochem, San Diego, CA), selectively inhibiting NF-{kappa}B activation, and uridine (200 µM, Sigma Chemical Co.), which was added together with FK778. Solvent alone did not affect the investigated events (data not shown). The respective treatments did not affect cell viability (>95%), as assessed by trypan blue and propidium iodide exclusion.

T cell/APC conjugate and IS formation
Formation of IS and T cell/APC conjugates was analyzed as described [28 ]. Briefly, for superantigen stimulation, Raji cells were labeled with CellTrackerTM Orange 5-(and-6)-[4-chloromethyl(benzoyl)amino]tetramethylrhodamine (CMTMR; Molecular Probes, Eugene, OR) and pulsed with 5 µg/ml Staphylococcal Enterotoxin E (Toxin Technology Inc., Saratosa, FL). After washing, Raji cells were incubated with immunosuppressant or vehicle-treated Jurkat (JE6-1, American Type Culture Collection, Manassas, VA) T cells at a ratio of 1:1 at 37°C for 15 min. Reaction was stopped by adding ice-cold HBSS. Cells were plated on poly-L-lysine-coated slides (Marienfeld, Lauda-Koenigshofen, Germany). For staining of F-actin and CD3{epsilon}, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS (not for CD3 staining), and stained with anti-CD3{epsilon} (Clone UCH-T1, Santa Cruz Biotechnology, CA) or Alexa Fluor®488-labeled phalloidin for staining of F-actin. For staining of LFA-1{alpha} and PKC-{theta}, cells were air-dried, fixed with cold (–20°C) methanol, and treated with anti-LFA-1{alpha} and anti-PKC-{theta}, respectively (both termed Clone 27, BD Transduction Lab, Franklin Lakes, NJ). Alexa Fluor®488-labeled secondary antibodies were used (Molecular Probes). The percentage of conjugates showing relocalization of the respective molecules was determined by counting at least 100 conjugates per sample by two individuals in a blinded manner.

For antigen-specific stimulation, HOM-2 cells were labeled with CellTrackerTM Orange CMTMR and pulsed with 200 µg/ml hemagglutinin (HA) 307–319 antigen peptide or inactive peptide (HA K316E), respectively. After washing, HOM-2 cells were incubated with CH7C17 T cells (transfected with HA 307–319-specific TCR [35 , 36 ], 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 15 min. Cells of different samples were plated at the same density on poly-L-lysine-coated slides and stained for F-actin using Fluor®488-labeled phalloidin (Molecular Probes). The percentage of T cells forming conjugates with APCs was determined by counting at least 300 T cells per sample by two individuals in a blinded manner.

T cell/DC conjugate formation
For analysis of T cell potency to form conjugates with allogeneic, professional APCs, mature, monocyte-derived DC were generated by incubation of CD14-positive cells isolated from buffy coats with GM-CSF and IL-4 for 6 days and subsequent stimulation with 100 ng/ml LPS for 2 days, as detailed [37 ]. PBTLs and DC were stained with 10 µM carboxyseminaphthorhodafluor-1 (Molecular Probes; green emission detected in channel FL-1) and 0.5 µM chloromethyl-fluorescein diacetate Cell Tracker (Molecular Probes; red emission detected in channel FL-3), respectively, for 30 min at 37°C. Washed PBTLs (5x105) were resuspended in 0.5 ml RPMI 1640, including 10% FCS in a 1.4-ml U-tube (Micronic B.V., Lelystat, The Netherlands). DC (5x104; 0.5 ml medium) were added in, and cells were cocultured for 2 h at 37°C. Cells were resuspended carefully by pipetting, and stable conjugates were analyzed by fluorescence microscopy and quantified by flow cytometric analysis (FACSCalibur, BD Biosciences, San Jose, CA). The conjugate formation efficiency was calculated as the percentages of DC conjugated to T cells as counted in the respective quadrants of the FL-1 versus FL-3 dot plot (see Fig. 3B ). The conjugate efficiency of untreated cells was 53.9 ± 3%.


Figure 3
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  		Figure 3. Effects of immunosuppressants on T cell conjugate formation with allogeneic DC. (A) Example of light and fluorescence microscopic photographs of PBTLs (green cells), treated or not with FK778, as indicated, and incubated for 15 min with allogeneic DC (red cells). (B) Example FACS analysis plots of PBTLs, treated or not with FK778 and incubated for 15 min with allogeneic DC. Conjugates are detected in the upper right quadrant. Numbers give conjugate formation efficiency, which was calculated as the percentages of DC conjugated to T cells. (C) Conjugate formation with allogeneic DC of T cells treated overnight with indicated immunosuppressants (CsA, FK506, dexamethasone, rapamycin, FK778). The diagram shows mean conjugate formation efficiency expressed in percent of untreated samples ± SEM of eight different T cell/DC combinations using T cells obtained from four independent donors. **, P < 0.01; ***; P < 0.001, versus untreated.

 
Homotypic T cell aggregation, integrin surface expression
PBTLs were incubated at 1 x 106 cells/ml in 24-well culture plates and stimulated with immobilized anti-CD3 (overnight incubation of 24-well plates with 0.1 µg/ml OKT3 in PBS) and anti-CD28 IgG (Leu-28, 0.5 µg/ml, BD Biosciences) for 24 h. Cell clustering was determined by light microscopy. Expression of LFA-1 was analyzed using FITC- or PE-labeled mAb against the LFA-1{alpha} chain (CD11a) and -ß chain (CD18, all Becton Dickinson). Samples were analyzed using a FACSCalibur (Becton Dickinson).

Statistics
Data are presented in means ± SEM. Comparisons were performed by one-way ANOVA and post-hoc Dunnett’s t-test (two sides), and a P ≤ 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunosuppressants interfere with formation of the IS
Formation of an IS is a key feature of T cell-mediated immune responses and a prerequisite for the regulated induction of T cell effector processes [38 ]. We aimed at investigating whether different immunosuppressants have an effect on IS formation as characterized by relocalization of cytoskeletal, adhesion, and signaling molecules toward the T cell/APC interface. To this end, we used a system involving a Jurkat T cell line and a Raji B cell line as APCs, which spontaneously and independently of superantigen form conjugates and after 15 min of conjugation form a mature IS in a superantigen-dependent manner [28 ]. Figure 1 shows that treatment of T cells with FK778 significantly reduced recruitment of CD3 molecules and strongly blocked the translocation LFA-1, as shown before [33 ], and also F-actin by more than 80% (Fig. 1) . It is surprising that also treatment of T cells with the calcineurin inhibitors CsA and FK506 as well as the corticosteroid dexamethasone resulted in a significant reduction of superantigen-induced relocalization of CD3 toward the IS to 50–60% of the untreated control (Fig. 1A and 1B) , while the mTOR inhibitor rapamycin had no effect on all analyzed events (Fig. 1B) . In contrast to FK778, the effect of calcineurin inhibitors and corticosteroids was selective for TCR/CD3 recruitment into the APC/T cell interface, resulting in an immature type of IS with normal relocalization of F-actin and the adhesion molecule LFA-1 but diminished clustering of TCR/CD3 molecules (Fig. 1) . The spontanous conjugate formation between Jurkat and Raji cells was not affected by any treatment (not shown). These results indicate that the immunosuppressive drugs CsA and FK506 and also dexamethasone are able to interfere with the hallmark of IS maturation, i.e., TCR/CD3 relocalization.


Figure 1
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  		Figure 1. Effects of immunosuppressants on the formation of the IS. Jurkat T cells were treated overnight with the indicated immunosuppressants [CsA, FK506, dexamethasone (Dex), rapamycin (Rapa), FK778] and stimulated for 15 min with superantigen-pulsed APCs (Raji B cells) or left unstimulated with unpulsed APCs. CD3, F-actin, and LFA-1{alpha} were visualized by indirect immunofluorescence (green). (A) Typical examples of conjugates with untreated (left panel) or immunosuppressant-treated (as indicated) T cells with superantigen-pulsed APCs (red cells) are shown. Arrowheads indicate conjugates positive for protein relocalization. (B) The diagrams show a summary of at least four independent experiments in mean ± SEM expressed as percent of stimulated, untreated control. Open bars, unpulsed APCs; solid bars, superantigen-pulsed APCs as stimulator cells. Significance versus superantigen-stimulated, untreated control: **, P < 0.01; ***; P < 0.001.

 
Effects of immunosuppressants on the induction of stable T cell/APC conjugates
Next, we investigated whether currently used immunosuppressants have an impact on the induction of stable T cell/APC conjugates, one of the earliest events required for T cell activation. To analyze this possibility in a system closely mimicking physiological T cell stimulation by APC, we studied the potential effects of immunosuppressants in an antigen-specific system. Therefore, we used Jurkat T cells expressing the HA 307–319 epitope-specific TCR and B cells presenting the nominal antigen HA peptide HA 307–319 as corresponding APCs. T cells formed antigen peptide-induced conjugates with APCs, whereas the inactive peptide HA K316E resulted in only minor, spontaneous conjugate formation (Fig. 2A ). Treatment of T cells with CsA, FK506, dexamethasone, and rapamycin did not significantly influence HA antigen-specific conjugate formation (Fig. 2A and 2B) . However, treatment with FK778 drastically blocked the ability of T cells to form antigen-induced, stable conjugates with APCs (Fig. 2A and 2B) .


Figure 2
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  		Figure 2. Effects of various immunosuppressants on antigen-induced T cell/APC conjugate formation. HA 307–319-specific T cells were treated overnight with the indicated immunosuppressants (CsA, FK506, dexamethasone, rapamycin, FK778) and stimulated for 15 min by incubation with HOM-2 B cells pulsed with HA 307–319 antigen peptide or left unstimulated by incubation with HA K316E inactive, peptide-pulsed APCs, as indicated. (A) Examples for untreated and CsA- and FK778-treated T cells (green) incubated with APCs (red) are shown. Arrowheads indicate conjugates. (B) The proportion of T cells forming conjugates with APCs of at least four independent experiments (300 T cells counted in each) is shown in the diagram and expressed in percent of untreated, antigen-stimulated control. Open bars, inactive, peptide-pulsed APCs; solid bars, antigen peptide-pulsed APCs as stimulator cells; ***; P < 0.001, versus untreated, antigen-stimulated control.

 
To assess the impact of immunosuppressants on conjugate formation in an allogeneic cell system, we used a novel, flow cytometric technique, which allows quantification of the initiation of an antidonor T cell response by detecting conjugate formation between freshly isolated T cells and allogeneic DC as APC. Incubation of T cells with allogeneic DC resulted in a rapid conjugate formation, as visualized by light and fluorescent microscopy (Fig. 3A ) as well as flow cytometry (Fig. 3B) . Treatment of T cells with FK778 abrogated their ability to form conjugates with allogeneic DC (Fig. 3C) in a dose-dependent manner (data not shown), confirming the results obtained with the antigen-specific system. In contrast, calcineurin or mTOR inhibitors did not affect T cell efficacy to form conjugates with DC, and dexamethasone had only a minor effect (Fig. 3C) . Collectively, these data show that FK778, which impaired the relocalization of LFA-1 to the IS (Fig. 1) , inhibited sufficient T cell/APC conjugates, and the calcineurin inhibitors and dexamethasone, which selectively interfered with CD3 relocalization to the IS, did not considerably influence the formation of stable conjugates.

Disruption of T cell/APC interactions by FK778 is independent of its antimetabolic activity
FK778 acts as an antimetabolite by inhibiting pyrimidine synthesis and inhibits the activity of tyrosine kinases [15 16 17 ]. To gain insight whether the FK778-mediated inhibition of de novo pyrimidine synthesis caused the observed abrogation of conjugate formation, we added uridine to the cultures, which is sufficient to bypass the dihydroorotate dehydrogenase blockade [29 , 39 ], and analyzed conjugate formation in various model systems. The inhibition of antigen-induced conjugates was not reversed by addition of exogenous uridine (Fig. 4A ) nor was the formation of conjugates with allogeneic DC (Fig. 4B) . Furthermore, we analyzed activation-induced, homotypic T cell aggregation, which constitutes a hallmark of T cell activation and depends on LFA-1 binding to its ligand ICAM-1, as does T cell/APC conjugate formation [40 ]. PBTLs stimulated with CD3 plus CD28 mAb formed large, dense cell clusters, which however, were reduced markedly in size and numbers when cells were treated with 100 µM FK778 (Fig. 4C) . Treatment of PBTLs with 200 µM FK778, a concentration that is exceeded up to threefold in plasmas of FK778-treated patients [41 ], completely abolished CD3/CD28 mAb-induced cell clusters (Fig. 4C) . The addition of uridine to the cultures did not prevent FK778-mediated inhibition of activation-induced T cell clustering (Fig. 4C) . It is notable that expression of LFA-1{alpha} and -ß chains was not affected by FK778 or uridine (Fig. 4D) . Thus, activation-induced cell adhesion of Jurkat as well as primary T cells was suppressed by FK778, independently of alterations of the pyrimidine pool or integrin expression. Our finding that antigen-induced conjugate formation is inhibited to the same extent when cells are only treated for 2 h with FK778 (data not shown) supported this notion further.


Figure 4
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  		Figure 4. Independency of an altered pyrimidine pool of FK778-mediated interference with conjugate formation. Human PBTLs were treated overnight with medium or with FK778 in combination with (+) or without (–) 200 µM uridine (U). (A) Antigen-induced conjugate formation was analyzed as described in the legend to Figure 1 . (B) T cell conjugate formation with allogeneic DC was analyzed as described in the legend to Figure 2 . (C) Cells were stimulated overnight via plate-bound CD3 and CD28 mAb. The photographs show typical examples for the induced cell clustering out of at least four independent experiments. (D) Surface expression of LFA-1{alpha} chain (CD11a) and -ß chain (CD18) of FK778 ± uridine-treated PBTLs was analyzed by flow cytometry. The diagram shows mean immunofluorescence intensities (MFI) of four independent experiments expressed in percent of untreated control ± SEM.

 
Selective blockade of calcineurin and NF-{kappa}B activation mimics the effects of immunosuppressants
We next investigated the signaling pathways, which are potentially involved in the selective modulation of TCR/CD3 relocalization by CsA, FK506, and dexamethasone. CsA and FK506 are well known to bind immunophilins and thus, act as specific inhibitors of the calcineurin/NF-AT signaling pathway [7 ], and dexamethasone primarily inhibits activation of NF-{kappa}B in T cells via distinct mechanisms [10 , 11 ]. To assess an involvement of calcineurin in IS formation, we used FR901725 and gossypol, which are direct inhibitors of calcineurin activity [34 ]. For further analysis of the dependence of IS formation on NF-{kappa}B activity, we used the highly specific, cell-permeable SN50 peptide, which inhibits the nuclear translocation of NF-{kappa}B [42 ]. To avoid secondary effects as a result of altered gene expression, which might occur after overnight treatment, as performed so far, we short-term-treated (2 h) the T cells with the indicated immunosuppressants and inhibitors. We observed that inhibitors of calcineurin and NF-{kappa}B prevented the relocalization of TCR/CD3 into the IS, and F-actin, LFA-1, and PKC-{theta} normally clustered at the IS (Fig. 5 ), with the exception of a minor effect of CsA on LFA-1 relocalization (Fig. 5) . Also, an overnight treatment with FR901725, gossypol, and SN50 resulted in nearly exactly the same results as the short-term treatment (data not shown). As CsA and dexamethasone are known to affect distinct T cell signaling pathways, a synergism of these substances on CD3 relocalization to the IS could have been expected. However, T cells treated with a combination of these immunosuppressants were not inhibited stronger in CD3 relocalization than by CsA or dexamethasone alone (Fig. 5) . Collectively, these data suggest that maturation of the IS critically depends on calcineurin and NF-{kappa}B signaling pathways.


Figure 5
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  		Figure 5. Effects of pharmacological inhibitors of calcineurin and NF-{kappa}B activity and combined immunosuppressants on formation of the IS. Jurkat T cells were treated for 2 h with medium, CsA, FK506, dexamethasone, rapamycin, FK778, FR901725 (FR), gossypol (Goss), SN50, or CsA + dexamethasone (C+Dex) and stimulated for 15 min with superantigen-pulsed APCs. CD3, F-actin, LFA-1{alpha}, and PKC-{theta} were visualized by indirect immunofluorescence and analyzed for relocalization to the T cell/APC contact site. The diagrams give a summary of at least four independent experiments in mean ± SEM, expressed as percent of stimulated, untreated control. Significances: *, P < 0.05; **, P < 0.01; ***; P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study demonstrate that currently used immunosuppressive drugs, such as calcineurin inhibitors or corticosteroids, but not mTOR inhibitors, affect the recruitment of the TCR/CD3 complex into the T cell/APC contact zone, which constitutes the hallmark of a mature IS. A recent report showed similar alterations, including defective TCR/CD3 clustering, resulting in an immature IS type but unaffected T cell/APC conjugate formation in orally tolerized T cells [43 ]. Another study demonstrated that human anergic T cells form an immature IS, and although capable of inducing IFN-{gamma}, these cells were unable to produce IL-2 [26 ]. Moreover, genetic deletion of DOCK2, which is essential for relocalization of TCR/CD3 but not LFA-1 and PKC-{theta} toward the IS, attenuates allograft rejection in mice [31 , 32 ]. Hence, prevention of IS maturation, as characterized by selectively blocked relocalization of the TCR/CD3 to the IS, may constitute a mechanism for the action of CsA/FK506 and corticosteroids in addition to their well known suppression of cytokine gene transcription. This observed interference with early processes of T cell activation may contribute to the clinical efficacy of these drugs. This assumption is strengthened by our use of overnight incubation periods that may allow secondary effects as a result of altered gene transcription, which could also occur in vivo during long treatment periods. Future studies undertaken with the understanding that these drugs affect IS maturation may facilitate the development of less toxic, novel immunosuppressive drugs, specifically interfering with the early stages of adaptive immune cell activation. Such efforts are warranted, as calcineurin inhibitors, although they have made human organ transplantation nearly a routine practice, exert distinct toxic effects, leading to complications such as renal dysfunction, hypertension, neurological toxicity, and diabetes mellitus [44 , 45 ]. A further contribution to their beneficial effects may be the action of these drugs on the second partner of IS formation, the professional APC. For instance, CsA has been shown to affect DC activation [46 ] and may therefore affect DC/T cell conjugate formation [47 ], while in our experimental setup only T cells have been exposed to calcineurin inhibitors, leaving conjugate formation intact. Moreover, in most experiments, we used B cells as APCs, which in contrast to DC, do not contribute actively to IS formation by rearrangement of the actin cytoskeleton [48 ].

Beyond unraveling a novel mode of action of well known, immunosuppressive drugs, the data presented herein provide new insight into the mechanisms of IS and T cell/APC conjugate formation. If APC-induced T cell signaling is blocked at an early stage, e.g., with FK778, formation of the immature IS as hallmarked by relocalized F-actin and LFA-1 is impaired along with abolished conjugation with different kinds of APCs. It is therefore assumed that distinct early signals, which activate the cytoskeleton that in turn promotes sustained early signals such as calcium response [49 ], could be essential for an immature IS to proceed to proper conjugate formation (Fig. 6 ). Such signals necessary for the initiation of synapse formation could be provided by adhesion molecules such as LFA-1 [22 ] and ICAM-3 [50 ] and costimulatory receptors [22 ]. Of note, also, the few TCR molecules initially available at the APC-contact site have been suggested to be sufficient for induction of further processes of IS formation [23 , 51 ]. Montoya et al. [52 ] showed that the number of stabilized T cell/APC conjugates correlates with the number of mature synapses, i.e., clustered TCR/CD3 in the IS, and relocalization of ICAM-3 is not sufficient for conjugate stabilization. Hence, in view of these studies, as well as our findings, it seems conceivable to suggest that an immature IS, which comprises clustered F-actin and LFA-1, but not ICAM-3 alone, is sufficient to induce distinct signals promoting stable conjugates, and further signals that can be blocked with inhibitors of TCR/CD3-induced calcineurin and NF-{kappa}B activation are essential for the formation of a mature synapse (Fig. 6) .


Figure 6
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  		Figure 6. A model for IS maturation and effects of immunosuppressants on mechanisms driving formation of the IS. (A) Initial contact providing signals for formation of an immature IS and stable conjugates. (B) Immature IS, providing signals that require calcineurin and NF-{kappa}B to induce IS maturation. (C) Mature IS, facilitating effective T cell activation. Red arrows indicate signal transduction; black arrows indicate molecule translocation. The interference by immunosuppressants is indicated by black bars.

 
The selective interference with IS maturation by CsA/FK506 and dexamethasone (Figs. 1 and 5) indicates that signaling mediators, such as calcineurin and NF-{kappa}B, are critically involved in IS maturation. This notion was confirmed by the use of specific pharmacological inhibitors (Fig. 5) , although it has to be taken into account that inhibitors could have unspecific side effects. For instance, gossypol also binds calmodulin and inhibits lipoxygenase activity. Therefore, we additionally used FR901725, a chemically distinct calcineurin inhibitor, with essentially the same results. Although the detailed molecular mechanisms that link calcineurin and NF-{kappa}B activity with the movement of distinct molecules into the IS are currently unknown, it is tempting to speculate about an "upstream" signaling potential for these molecules. In line with possible influences of calcineurin-dependent signaling pathways on early T cell activation events independent of calcineurin-mediated induction of gene transcription, it has been demonstrated that CsA and FK506 suppress the activation of JNK and p38 pathways at a level upstream of MEK kinases [34 ] and also affect the membrane molecule linker for activation of T cells [52 ]. As inhibition of calcineurin and NF-{kappa}B does not synergistically affect IS formation (Fig. 5) , it is suggested that calcineurin and NF-{kappa}B act within a common signaling pathway, which is indicated in our model depicted in Figure 6 . Indeed, various data demonstrated that NF-{kappa}B can be activated via a CsA-sensitive, calcineurin-dependent signaling pathway [53 ], which may be involved in controlling membrane-proximal molecular movements essential for the formation of a mature IS. Clearly, future studies are needed to further delineate the role of NF-{kappa}B, including its mode of action during IS maturation. Altogether, our data, particularly the requirements for stable conjugate formation as well as the involvement of calcineurin and NF-{kappa} B in IS maturation, as depicted in Figure 6 , considerably extend our current view about the molecular mechanisms of immunosuppressive drugs as well as critical elements of IS maturation [54 ].

The common surrogate model for the initiation of organ rejection and graft-versus-host disease is the MLR using DC, as these specialized cells are the most potent APCs for allogeneic T cells. Assessment of T cell proliferation is not appropriate to address the relevance of the impairment of early T cell activation events such as IS formation by immunosuppressants, as their block of distal signal transduction, in any case, affects the T cell proliferative response. In vivo, prolonged engagement of naive T cells with mature DC is necessary for T cell activation, as assessed by IL-2 gene transcription [55 ]. Hence, by applying our novel technique of flow cytometric determination of the induction of stable, allogeneic DC/T cell conjugates (Fig. 3) , we were able to assess the effects of immunosuppressive drugs on the initiating stage of an alloantigen-specific immune response. The particular effectiveness of FK778 in this respect may be an important contribution to the interference of these drugs with T cell activation at multiple effector stages, as significantly reduced numbers of allogeneically activated T cells may arise before the cell cycle-blocking effects of FK778 [56 ] become relevant. This effective dual mode of action of FK778 on T cells, together with its action on APCs, such as DC [57 ], could allow the reduction of the immunosuppressive burden, as it was shown with the related substance leflunomide in selected cases of organ transplant recipients [58 ], and might, therefore, open new views on options for immunosuppression. Our results indicate that FK778-mediated inhibition of de novo pyrimidine synthesis is not responsible for hampered IS formation, adhesion, and conjugate formation (Fig. 4) . A possible explanation for this finding could be the reported inhibition of Src family tyrosine kinase activity by malononitrilamides such as leflunomide [15 , 16 ]. However, our experiments with the related substance teriflunomide indicated that malononitrilamides do not totally block T cell signal transduction but rather selectively affect distinct signaling pathways involved in integrin activation [29 ]. Moreover, a recent study showed that APC-induced T cell signaling events, such as formation of TCR/CD3 microclusters, are insensitive to the Src family kinase inhibition, and in another study, Lck deficiency or blockade of Src family kinases leads to an enhancement of T cell stimulation by superantigen-presenting B cells [59 ]. Hence, the detailed molecular mechanism(s) accounting for the observed effects of FK778 on T cell/APC interactions are probably distinct from inhibition of Src family kinases and remain to be elucidated. Accordingly, also, the effects of dexamethasone on CD3 relocalization most probably do not relate to effects of glucocorticoids on Src family tyrosine kinase activity and their membrane compartmentalization, as shown in previous studies [60 , 61 ].

In conclusion, the currently used immunosuppressants CsA, FK506, dexamethasone, and FK778 affect the evolution of an IS at distinct stages. These data considerably extend previous findings about the mode of action of these drugs and might further help to understand how they exert their powerful clinical efficacy. Moreover, as a result of the growing understanding of the molecular mechanisms that underlie these peculiar events, novel, immune cell-selective strategies for the interference with early T cell/APC interactions and thus inhibition of adaptive immune responsiveness could represent particularly effective and less toxic options for the treatment of, e.g., autoimmune diseases and transplant rejection.


   ACKNOWLEDGEMENTS
 
This work was supported by the grants from the Austrian Jubilee Fund (ÖNB 10282 to M. D. S.) and the Austrian Science Fund (P16788-B13 and P18776-B11 to T. M. S). We thank Bianca Weissenhorn for expert technical assistance.

Received June 7, 2006; revised August 30, 2006; accepted September 11, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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