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Originally published online as doi:10.1189/jlb.1104687 on February 9, 2005

Published online before print February 9, 2005
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(Journal of Leukocyte Biology. 2005;77:680-688.)
© 2005 by Society for Leukocyte Biology

Polyunsaturated fatty acids interfere with formation of the immunological synapse

René Geyeregger*, Maximilian Zeyda*, Gerhard J. Zlabinger{dagger}, Werner Waldhäusl*,{ddagger} and Thomas M. Stulnig*,{ddagger},1

* Department of Internal Medicine III and
{dagger} Institute of Immunology, Medical University of Vienna, Austria; and
{ddagger} CeMM-Center of Molecular Medicine of the Austrian Academy of Sciences, Vienna

1 Correspondence: Department of Internal Medicine III, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: thomas.stulnig{at}meduniwien.ac.at


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyunsaturated fatty acids (PUFAs) exert inhibitory effects on T cell-mediated immune responses. Activation of T cells in vivo depends on formation of an immunological synapse (IS) at the T cell/antigen-presenting cell (APC) interface. Here, we analyzed effects of PUFA treatment on the formation of the IS and APC-induced human T cell activation. In T cells treated with the PUFA eicosapentaenoic (EPA; 20:5,n-3) and arachidonic acid (20:4,n-6), stimulated by superantigen-presenting cells or APCs, relocalization to the IS of distinct molecules [F-actin, talin, leukocyte functional antigen-1{alpha}, clusters of differentiation (CD)3{varepsilon}] was inhibited markedly compared with cells treated with saturated fatty acid, whereas relocalization of protein kinase C{theta} to the IS remained unaffected. CD3-induced, sustained phosphorylation of nucleotide exchange factor Vav, which controls cytoskeletal rearrangements underlying IS formation, was significantly reduced in EPA-treated Jurkat and peripheral blood T cells. In addition, T cell raft disruption by methyl-ß-cyclodextrin treatment and experiments with a chimeric linker for activation of T cell proteins, which is resistant to PUFA effects on lipid rafts, revealed modifications of lipid rafts as a crucial factor for PUFA-mediated inhibition of APC-stimulated cytoskeletal rearrangements. Furthermore, the efficiency of T cell/APC conjugate formation was significantly reduced with EPA-treated T cells, as was stimulation of CD69 expression, which is not altered following antibody-mediated T cell activation. In conclusion, PUFA treatment of T cells qualitatively and quantitatively alters IS formation, thereby extending T cell signaling defects to pathways that are not intrinsically altered in PUFA-treated T cells when stimulated by antibodies.

Key Words: antigen presentation • human • immunosuppression • signal transduction • T lymphocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyunsaturated fatty acids (PUFAs) exert immunomodulatory effects, particularly by interfering with T cell activation [1 ]. Treatment with PUFAs provides clinical benefits in various inflammatory diseases, e.g., rheumatoid arthritis and ulcerative colitis, and following organ transplantation [1 2 3 4 ]. Particularly, the two major n-3 PUFAs occurring in fish oils, namely eicosapentaenoic (EPA; 20:5,n-3) and docosahexaenoic acid (22:6,n-3), appear effective with varying relative potency. PUFA-mediated alterations of T lymphocyte function and cytokine production appear to be independent of altered prostaglandin and leukotriene synthesis [5 6 7 8 ]. PUFAs diminish T cell receptor (TCR)/clusters of differentiation (CD)3-induced calcium response and downstream events, such as nuclear factor of activated T cell activation, cytokine secretion, interleukin-2 receptor expression, and proliferation in human T cells [9 , 10 ]. Although PUFA-mediated alterations on T cell signaling have been investigated previously using monoclonal antibodies (mAb) against CD3 and costimulatory molecules, a possible impact of PUFAs on T cell/antigen-presenting cell (APC) interactions remains obscure.

Activation of T cells by APCs in vivo depends on formation of the immunological synapse (IS) [11 ]. During IS formation, T cell adhesion molecules and signaling molecules are relocalized to the T cell/APC interface to facilitate stable interactions of T cells with APCs and sustained signaling, which is necessary for full T cell activation [12 , 13 ]. IS formation is an active and regulated process that requires TCR-mediated cytoskeletal rearrangements [14 , 15 ]. Upon stimulation, polymerized F-actin and its membrane-anchoring protein talin cluster at the T cell/APC interface [16 , 17 ]. These cytoskeletal rearrangements are regulated by Vav, a 95-kDa nucleotide exchange factor that is activated by tyrosine phosphorylation [17 , 18 ]. By its potential to recruit signaling and adaptor molecules including phospholipase C{gamma}1 (PLC{gamma}1) and Vav, the linker for activation of T cells (LAT) is involved in activation of the calcium pathway as well as in induction of cytoskeletal rearrangements [19 , 20 ]. Previous studies have revealed that loss of LAT from rafts underlies inhibition of CD3-induced PLC{gamma}1/calcium signaling in PUFA-treated T cells [21 , 22 ], but a possible alteration of Vav activation has not yet been elucidated.

In this study, we investigated effects of PUFAs on T cell/APC interactions including formation of the IS and preceding signaling events. Relocalization of selected molecules to the IS between T cells and superantigen or antigen-presenting B cells was significantly inhibited when T cells have been treated with PUFAs of the n-3 (EPA) and the n-6 series [arachidonic acid (AA), 20:4,n-6]. To elucidate underlying mechanisms, we show that T cell EPA treatment inhibits CD3-induced Vav phosphorylation and that modifications of lipid rafts are a crucial factor for PUFA-mediated inhibition of stimulated cytoskeletal rearrangements. To emphasize functional consequences of PUFA-mediated defects in T cell/APC interactions, we demonstrate that EPA treatment of T cells leads to reduced antigen-induced conjugate formation, which results in spreading of T cell activation defects to pathways that are not intrinsically altered in PUFA-mediated T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Antibodies were obtained as follows: anti-CD3{varepsilon} (OKT3) from Ortho Pharmaceuticals (Raritan, NJ); anti-CD3{varepsilon} (UCH-T1) from Santa Cruz Biotechnology (CA); anti-protein kinase C (anti-PKC){theta} and anti-leukocyte functional antigen (anti-LFA)-1{alpha} from BD Transduction Laboratories (San Diego, CA); phycoerythrin (PE)-labeled anti-CD25, PE-labeled anti-CD69, and fluorescein isothiocyanate (FITC)-labeled anti-CD19 from BD PharMingen (San Jose, CA); anti-human Vav, anti-LAT, and horseradish peroxidase (HRP)-labeled antiphosphotyrosine (4G10) from Upstate Biotechnology (Lake Placid, NY); antitalin and goat anti-mouse (GAM) immunoglobulin G (IgG) from Sigma-Aldrich (St. Louis, MO); Alexa Fluor® 488-labeled F(ab')2 fragments of GAM or goat anti-rabbit IgG from Molecular Probes (Eugene, OR); biotinylated anti-mouse IgG from Vector Laboratories (Burlingame, CA); HRP-labeled GAM IgG from Bio-Rad (Hercules, CA).

Cells and fatty acid treatment
Human Jurkat T cell line E6-1 was cultivated in RPMI-1640 medium (Invitrogen, Groningen, The Netherlands) containing 10% bovine calf serum (BCS; Hyclone Laboratories, Logan, UT). The LAT-deficient Jurkat line ANJ3 [23] was generously provided by Lawrence E. Samelson (National Institutes of Health, Bethesda, MD) and stably reconstituted with wild-type LAT or phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG)-LAT chimeric protein, as described [22 ]. Lucy R. Wedderburn (Institute of Child Health and Great Ormond Street Hospital for Children NHS Trust, London, UK) generously provided Jurkat clone CH7C17, transfected with influenza hemagglutinin (HA) peptide (HA 307–319, PKYVKQNTLKLAT)-specific TCR [24, 25]. The human leukocyte antigen-matching (DRB1*0101-positive) Epstein-Barr-Virus-transformed B cell line HOM-2 as well as the B cell line Raji were cultivated in RPMI-1640 medium supplemented with 10% BCS. Peripheral blood T lymphocytes (PBTLs) were purified from buffy coats obtained from healthy volunteers using Ficoll-Paque (Amersham, Uppsala, Sweden) density gradient centrifugation, followed by rosetting with neuraminidase-treated sheep erythrocytes (Dade Behring, Marburg, Germany). The obtained population contained >90% CD3+ cells, which were treated with fatty acids as described in detail elsewhere [9 , 10 , 21 , 22 ]. Briefly, Jurkat cells and PBTLs were treated for 2 and 3 days, respectively, in serum-free Iscove’s modified Dulbecco’s medium (Invitrogen) containing 0.4% (w/v) bovine serum albumin (fraction V, Sigma-Aldrich) and indicated concentrations of EPA (20:5,n-3), AA (20:4,n-6), and stearic acid (18:0; all Sigma-Aldrich) or methyl-ß-cyclodextrin (MCD; Sigma-Aldrich). PUFA treatment did not essentially affect viability of T cells as assessed by staining with a 0.25% trypan blue solution (Sigma-Aldrich). The effects on T cells of stearic acid treatment were previously found indistinguishable from vehicle alone [ethanol, ≤0.5% (v/v)] concerning fatty acid composition, membrane subdomain distribution of proteins, calcium signaling, and downstream T cell activation (refs. [10 , 21 , 22 ] and data not shown). In some experiments, 10 µM butylated hydroxy-toluene (BHT; Sigma-Aldrich) was included.

Superantigen or antigen-induced IS and conjugate formation
For superantigen stimulation, Raji cells were labeled with CellTrackerTM Orange (5-(and -6)-(((4-chloromethyl) benzoyl)amino)tetramethylrhodamine (CMTMR), Molecular Probes] and pulsed with 5 µg/ml staphylococcal enterotoxin E (SEE; Toxin Technology, Saratosa, FL) in Hank’s balanced salt solution (HBSS) at 37°C for 20 min. After washing, Raji cells were incubated with Jurkat E6-1 T cells at a ratio of 1:1 at 37°C for 1 or 15 min. Reaction was stopped by adding ice-cold HBSS. For antigen-specific stimulation, 5 x 106/ml HOM-2 cells were labeled at 37°C for 3 h with CMTMR and 200 µg/ml HA 307–319 peptide, 200 µg/ml inactive peptide (HA K316E), and 20 µg/ml staphylococcal enterotoxin B (SEB; Sigma-Aldrich) or were left unpulsed, respectively. After washing, HOM-2 cells were incubated with CH7C17 T cells at a ratio of 1:1 at 37°C for 1 or 15 min. Cells were plated on poly-L-lysine-coated slides (Marienfeld, Lauda-Koenigshofen, Germany) and allowed to settle for 15 min on ice. For staining of F-actin, CD3{varepsilon}, LAT, and PKC{theta}, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature (except for staining of CD3{varepsilon}), and treated with the appropriate antibodies or Alexa Fluor® 488-labeled phalloidin (for staining of F-actin; Molecular Probes) for 30 min at room temperature. For staining of talin and LFA-1{alpha}, cells were air-dried for 1 h, fixed with ice-cold (–20°C) methanol, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature, and treated with the appropriate antibodies for 30 min at room temperature. The percentage of T cells forming conjugates with APCs was determined by counting at least 300 T cells per blinded sample by two individuals.

CD3 capping
EPA or stearic acid (18:0)-treated Jurkat E6-1 T cells and ANJ3 T cell-derived clones (1x106/ml) were incubated in HBSS (INVITROGEN, Groningen, The Netherlands), supplemented with 10 mM Hepes and 10% BCS, including 1 µg/ml anti-CD3{varepsilon} antibody at 37°C for 10 min. PBTLs (1x106/ml) were incubated with anti-CD3{varepsilon} antibody on ice for 30 min, washed, and cross-linked by adding (10 µg/ml) biotinylated GAM at 37°C for 10 min. Receptor clustering was stopped by adding ice-cold medium, including 0.2% sodium azide. After washing, cells were cytospun with 100 g for 3 min onto poly-L-lysine-coated slides and fixed with cold (–20°C) acetone for 15 s and cold (–20°C) methanol for 90 s. Jurkat and ANJ3 T cells were stained for CD3{varepsilon} with biotinylated GAM followed by Alexa Fluor® 488-labeled avidin (Molecular Probes), whereas PBTLs were stained with Alexa Fluor® 488-labeled avidin alone. Fluorescence microscopy (40x objectives, Aristoplan, Leica, Knowlhill, UK) was used to determine the percentage of cells with capped CD3{varepsilon} by counting of at least 100 cells per blinded sample by two individuals.

Vav phosphorylation
Immunoprecipitation was applied as described previously [22 ]. Briefly, Jurkat and peripheral blood T cells were washed with RPMI 1640 and stimulated via CD3 by incubation with 10 µg/ml OKT3 for 1 min followed by cross-linking with 20 µg/ml GAM IgG (Fc-specific) at 37°C for 1 or 15 min. After stopping the reaction by addition of ice-cold washing buffer, cells (2x107 Jurkat cells/ml; 2.5x107 PBTLs/ml) were lysed on ice for 30 min in Tris-buffered saline (pH 7.4) containing 1% Nonidet P-40 (Pierce, Rockford, IL) and phosphatase and protease inhibitors as described [22 ]. Vav was immunoprecipated from postnuclear supernatant using GammaBindTM Plus beads (Pharmacia Biotech, Uppsala, Sweden), preincubated with anti-Vav antibody. Standard Western blotting procedures were applied, and proteins were detected by chemiluminescence on a Lumi-Imager (Roche Molecular Biochemicals, Indianapolis, IN).

T cell activation markers
Fatty acid-treated Jurkat E6-1 T cells or CH7C17 (106/ml) were stimulated in 24-well plates by Raji cells, pulsed with 5 µg/ml superantigen or HOM-2 cells (106/ml), 2, 20, or 200 µg/ml HA peptide, or inactive peptide HA K316E at 37°C for 18 h. To distinguish between APCs and T cells, APCs were stained with the B cell marker anti-CD19 FITC, and T cells were analyzed for CD25 and CD69 surface expression by using respective PE-labeled antibodies and flow cytometry.

Statistics
Data are presented as means ± SEM. Comparisons were performed by two-tail unpaired Student’s t-test with correction for multiple comparisons according to Bonferroni-Holm when appropriate. Testing against a common control (as seen in Figure 1G ) was perfomed by one-way ANOVA followed by Dunnett’s t-test. A P ≤ 0.05 was considered to be statistically significant.



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  		Figure 1. PUFA treatment of T cells inhibits superantigen-induced IS formation. Jurkat T cells were incubated with 50 µM stearic (18:0) acid, EPA (20:5,n-3), or AA (20:4,n-6) for 2 days and stimulated for 1 or 15 min with superantigen-pulsed APCs or 15 min with unpulsed APCs (–), as indicated. Indicated proteins were visualized by indirect immunofluorescence. Typical examples of conjugates, positive (A, C) or negative (B, D) for relocalization of indicated proteins, are shown. (E) The diagram shows the percentage of conjugates counted positive for protein relocalization in means ± SEM of eight independent experiments. Corrected significance versus 18:0-treated samples: o, P = 0.06; *, P ≤ 0.05; **, P ≤ 0.01. (F) The percentage of conjugates counted positive for F-actin relocalization in means ± SEM of three independent experiments in the presence or absence of BHT after 15 min of stimulation is shown. n.s, Nonsignificant. (G) The percentage of conjugates counted positive for protein relocalization in means ± SEM of three independent experiments treated with different PUFAs and stimulated for 15 min. Significance versus 18:0-treated samples according to Dunnett’s t-test: *, P≤ 0.05, with whole model P ≤ 0.01 in all parameters except PKC{theta}, for which P was nonsignificant.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PUFA treatment of T cells inhibits superantigen- and antigen-induced relocalization of molecules to the IS
PUFA effects on APC-mediated T cell activation were first studied by evaluating relocalization of molecules to the IS in a superantigen-specific model system. To this end, Jurkat T cells treated with EPA (20:5,n-3), a marine long-chain PUFA, which turned out to be most effective in our previous studies on T cells [10 ], or saturated stearic acid (18:0), which served as a control, were stimulated by coincubation with Raji B cells as APCs pulsed with superantigen SEE. IS formation was assessed by analyzing the relocalization of cytoskeletal (F-actin, talin), adhesion (LFA-1{alpha}), and signaling molecules (CD3{varepsilon}, LAT, PKC{theta}) of T cells to the T cell/APC interface. Approximately 35% of conjugates relocalized F-actin and talin to the IS within 1 min of SEE stimulation, independently of fatty acid treatment. There was no relocalization of CD3 and LAT at 1 min after stimulation, indicating an early stage of IS formation [16 , 26 ], whereas 30–40% of LAT and CD3, respectively, were relocalized after 15 min, revealing formation of mature IS. The number of conjugates with relocalized F-actin, talin, and LFA-1 was markedly diminished in EPA (20:5,n-3)- compared with stearic acid (18:0)-treated T cells after 15 min of stimulation. Also, the percentages of conjugates with relocalized signaling molecules CD3 and LAT were significantly lower at this time-point, whereas PKC{theta} relocalization to the IS was not affected by EPA treatment (Fig. 1E) . As shown in Figure 1F , addition of the antioxidant BHT [27 ] did not interfere with the inhibitory effect of EPA, ruling out that lipid peroxidation plays a significant role. To investigate whether the PUFA effects were limited to n-3 PUFAs such as EPA (20:5,n-3), we also treated T cells with the n-6 PUFA AA (20:4,n-6) before stimulating with superantigen-presenting B cells. Figure 1G shows that in EPA-treated cells, defects in IS formation were similar to those in AA-treated cells. Thus, PUFA inhibited IS formation independent of lipid peroxidation and irrespective of whether n-3 or n-6 PUFAs were used. We hence focused on the effects of EPA in further experiments.

To investigate effects of T cell EPA treatment on antigen-specific IS formation, we used Jurkat T cells transfected with a TCR specific for the HA 307–319 (clone CH7C17), which were activated with a B cell line (HOM-2) pulsed with the active (HA) or a mutated peptide (HA K316E) or with the superantigen SEB, respectively. As shown in Figure 2 , with HA and SEB, ~20% and 10% of conjugates showed F-actin relocalization after 1 min of stimulation, respectively, with no apparent influence of EPA treatment. After 15 min of stimulation by peptide-presenting APCs, the number of conjugates with relocalized F-actin was significantly reduced (16±2% vs. 30±5%) when T cells were treated with EPA compared with stearic acid (18:0)-treated T cells. Comparable results were obtained after SEB stimulation (9±1% vs. 26±8%). No influence of EPA treatment on the percentage of conjugates showing PKC{theta} relocalization was seen after 15 min of stimulation with HA or SEB. Thus, PUFA treatment of T cells qualitatively alters superantigen- and antigen-induced IS formation.



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  		Figure 2. EPA treatment inhibits antigen-induced IS formation. CH7C17 T cells were treated with 50 µM saturated stearic acid (18:0) or polyunsaturated EPA (20:5,n-3) for 2 days and stimulated for 1 or 15 min APCs pulsed with antigen peptide (HA 307–319) or superantigen (SEB) as indicated. Cells were left unstimulated by incubation for 15 min with APCs pulsed with inactive peptide (HA K316E) or unpulsed APCs (SEB: –). Relocalization of indicated proteins was analyzed by indirect immunofluorescence. Typical examples of conjugates stained for indicated proteins are shown. The diagrams show the percentage of conjugates positive for relocalization of indicated proteins in means ± SEM of five independent experiments. Corrected significance versus 18:0-treated samples: *, P ≤ 0.05.

 
PUFA treatment diminishes CD3-induced Vav phosphorylation and CD3 capping
Cytoskeletal rearrangement processes in T cells that are necessary for IS formation are controlled by the guanosine 5'-triphosphate exchange factor Vav, which is activated by tyrosine phosphorylation [17 , 18 , 28 , 29 ]. Vav was tyrosine-phosphorylated significantly less after 15 min in EPA-treated Jurkat T cells than stearic acid (18:0)-treated cells (Fig. 3A ), whereas no significant difference between EPA and 18:0-treated cells could be determined after 1 min of stimulation. Comparable results of EPA-mediated Vav inhibition after 15 min of stimulation were seen in PBTLs (Fig. 3B) . When T cells are activated via anti-CD3 antibodies, cytoskeletal rearrangements are induced, resulting in formation of so-called "caps," i.e., large clusters of CD3 that can be detected easily by immunofluorescence [17 , 28 ]. As shown in Figure 4 , treatment of Jurkat T cells as well as PBTLs with EPA significantly inhibited CD3 cap formation compared with treatment with saturated stearic acid (18:0). Thus, EPA inhibits cytoskeletal rearrangements, irrespective of whether T cells were stimulated by APC or CD3 antibodies.



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  		Figure 3. CD3-induced Vav phosphorylation is inhibited in EPA-treated T cells. Jurkat T cells (A) or peripheral blood T cells (B) were incubated with 50 µM saturated stearic acid (18:0; control) or polyunsaturated EPA (20:5,n-3) for 2 days. Cells were left unstimulated or activated by cross-linking CD3{varepsilon} for 1 or 15 min, as indicated. Vav was immunoprecipitated (IP: Vav) from postnuclear supernatants, and tyrosine phosphorylation (pY) of the precipitated protein was determined by Western blotting (WB). The diagram shows means ± SEM of chemiluminescence intensities of pY bands related to those of the respective Vav protein bands blotted on stripped membranes expressed in percent of control of four independent experiments. Corrected significance versus 18:0-treated samples: ***, P ≤ 0.001.

 


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  		Figure 4. CD3 cap formation is diminished in EPA-enriched T cells. (A) Jurkat T cells (E6-1) were incubated with 50 µM stearic acid (18:0) or polyunsaturated EPA (20:5,n-3) for 2 days and stimulated for 10 min with anti-CD3 mAb. CD3 was visualized by indirect immunofluorescence, and the percentage of cells showing caps was quantified. Example images for (18:0)- and (20:5,n-3)-treated cells are shown. Arrowheads indicate CD3 caps. Diagrams show means ± SEM of four independent experiments. (B) Peripheral blood T cells were incubated for 3 days with indicated fatty acids and stimulated for 10 min by CD3 cross-linking. CD3 was visualized by indirect immunofluorescence, and the percentage of cells with caps was quantified. Significance versus 18:0-treated samples: *, P < 0.1; **, P ≤ 0.01.

 
PUFAs inhibit cytoskeletal rearrangements by modifications of lipid rafts
To test the requirement of intact lipid rafts on superantigen-induced relocalization of molecules to the IS in this system, we disrupted rafts by MCD treatment. Whereas moderate lipid raft alterations by PUFA particularly affected formation of the mature IS (Fig. 1) , highly efficient raft disruption by MCD inhibited IS formation already at a very early stage (Fig. 5 ). The number of conjugates with relocalized F-actin and LAT after 15 min of stimulation was significantly reduced by ~90% and 50% in MCD-treated compared with control-treated T cells, whereas MCD treatment failed to inhibit relocalization of PKC{theta} to the IS (Fig. 5) . Thus, both raft-disrupting treatments of T cells affected IS formation by inhibiting relocalization of F-actin and LAT but not PKC{theta}.



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  		Figure 5. Effects of MCD treatment on IS formation. Jurkat T cells were treated with 10 mM MCD (solid bars) or left untreated (open bars) before being stimulated for 1 or 15 min with superantigen-pulsed APCs or for 15 min with unpulsed APCs (–), as indicated. Indicated proteins were visualized by indirect immunofluorescence. The diagrams show the percentage of conjugates counted positive for protein relocalization in means ± SEM of five independent experiments. Corrected significance versus untreated samples: o, P= 0.07; ***, P ≤ 0.001.

 
PUFAs have been shown to affect early T cell calcium response, mainly by displacing LAT from membrane lipid rafts [22 ]. To test whether the displacement of LAT from rafts also underlies PUFA-mediated inhibition of CD3 cap formation, we used a LAT-deficient cell line (ANJ3) reconstituted with wild-type LAT (ANJ3/LAT) or with a "PUFA-resistant", genetically modified LAT (ANJ3/PAG-LAT). The PAG-LAT protein has been shown previously not to be displaced from rafts by PUFA treatment [22 ]. As in parental Jurkat T cells or PBTLs, CD3 cap formation was significantly inhibited by EPA treatment in ANJ3/LAT T cells (Fig. 6 ). In contrast, no inhibition of CD3 capping occurred in EPA-treated ANJ3/PAG-LAT cells. No CD3 cap formation was seen in control (vector)-transfected cells (not shown). These results show that lipid raft localization and particularly, displacement of LAT from lipid rafts underlie diminished CD3 cap formation in EPA-treated T cells.



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  		Figure 6. CD3 capping of PUFA-resistant PAG-LAT-transfected cells is not affected by EPA treatment. LAT-deficient Jurkat T cells (ANJ3) stably transfected with wild-type LAT or PAG-LAT were incubated with 30 µM saturated stearic acid (18:0) or polyunsaturated EPA (20:5,n-3) for 2 days and stimulated for 10 min via anti-CD3. CD3{varepsilon} was visualized by indirect immunofluorescence. (A) Typical examples of conjugates, positive or negative for CD3 capping, are shown; caps are indicated by arrowheads. (B) The diagram shows the percentage of cells containing caps of five independent experiments. Significance versus 18:0-treated samples: **, P ≤ 0.01.

 
PUFA-mediated interference with T cell/APC interactions results in diminished T cell activation
In addition to qualitative alterations of conjugate formation (Fig. 2) , we quantified antigen-induced T cell/APC conjugate formation following T cell fatty acid treatment (Fig. 7 ). After 15 min of coincubation, less than 25% of EPA-treated T cells formed conjugates with HOM-2 B cells presenting HA peptide or SEB compared with ~40% of 18:0-treated cells. T cell/APC conjugate formation could not be determined in the model system involving Raji cells as a result of strong, spontaneous conjugate formation even in unstimulated cells (data not shown). Previous studies with anti-CD3 mAb-stimulated T cells revealed that PUFA treatment inhibits surface expression of the activation marker CD25 but not of CD69 [9 ]. To investigate effects of EPA treatment on T cell downstream activation induced by APCs, we stimulated T cells via antigen- or superantigen-pulsed APCs and analyzed expression of activation markers. In both model systems, EPA-treated T cells showed diminished CD25 expression compared with control-treated T cells when stimulated via APC (Fig. 8A and 8B ). It is important that expression of CD69 was only inhibited by EPA in the CH7C17/HOM-2 model system (Fig. 8A) , which also showed decreased T cell/APC conjugate formation (Figs. 7 and Fig. 8A) but not in the culture containing Raji cells, which form conjugates, irrespective of PUFA treatment (Fig. 8B) . These data reveal that diminished conjugate formation leads to spreading of PUFA-mediated T cell inhibition to pathways that are not intrinsically altered in PUFA-treated T cells.



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  		Figure 7. EPA treatment inhibits induced T cell/APC conjugate formation. T cells CH7C17 were treated with 50 µM stearic acid (18:0) or EPA (20:5,n-3) for 2 days and stimulated for 1 or 15 min with APCs (HOM-2) pulsed with antigen peptide (HA 307–319) or superantigen (SEB), as indicated. Unstimulated cells were incubated for 15 min with APCs pulsed with inactive peptide (HA K316E) or unpulsed APCs (SEB: –). The percentage of T cells forming conjugates with APCs is shown in diagrams in means ± SEM of four independent experiments. Significance versus 18:0-treated control: *, P ≤ 0.05.

 


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  		Figure 8. Effects of EPA treatment on APC-induced surface activation marker expression. Jurkat T cells CH7C17 (A) and E6-1 (B), respectively, were modified by incubation with 50 µM saturated stearic acid (18:0) or polyunsaturated EPA (20:5,n-3) for 2 days. (A) Fatty acid-treated CH7C17 Jurkat T cells were stimulated overnight by HOM-2 cells pulsed with HA peptide (HA 307–319) or 200 µg/ml inactive peptide (HA K316E). (B) Fatty acid-treated Jurkat E6-1 T cells were stimulated overnight by Raji cells pulsed with superantigen (SEE) or left unpulsed (–) as indicated. Cell-surface expression of CD25 and CD69 was analyzed by two-color flow cytometry, in which the B cell marker CD19 was used to discriminate between T cell (CD19–, black) and APC (CD19+, gray). Dot blots show typical results of at least three independent experiments. Diagrams show geometric means of fluorescence intensities of T cells related to those obtained from stimulated control, which was set to 100% in mean ± SEM. Corrected significance versus respective 18:0-treated controls. *, P≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Formation of the IS between APCs and T cells is the initiating step of T cell-driven immune responses. Here, we show that PUFA treatment of T cells induces specific alterations in IS formation, which occurs via two maturation stages. During the first minutes of T cell/APC interaction, an immature IS is formed, which is characterized by absence or low amounts of CD3. The T cell/APC contact develops to a mature IS, which is characterized by central localization of CD3 [16 , 26 ]. PUFA treatment affects formation of the mature IS particularly, as revealed by inhibition of cytoskeletal protein relocalization (F-actin, talin) after 15 min of stimulation but not during the early phase (Fig. 1E) . The selectivity for inhibition of the mature but not the immature IS may be related to the fact that sustained Vav phosphorylation following TCR/CD3 stimulation was predominantly inhibited in EPA-treated T cells (Fig. 3) .

Beyond qualitative changes in IS formation, EPA treatment also markedly reduced the efficiency of forming conjugates between T cells and APCs (Fig. 7) . Integrins such as LFA-1{alpha} are crucial for the formation of high-affinity T cell/APC interaction [30 ]. LFA-1{alpha} relocalization to the IS, which depends on Vav and cytoskeletal rearrangements [31 , 32 ], was significantly diminished in EPA-treated T cells (Fig. 1E) . Thus, reduced amounts of LFA-1{alpha} and other integrins at the IS could underlie impaired conjugate formation of PUFA-treated T cells. As a consequence, altered IS formation could extend PUFA defects in T cell activation to signaling pathways that are not intrinsically altered in PUFA-treated T cells by conventional antibody stimulation. In accordance to this notion, stimulated expression of CD69 was inhibited in EPA-treated T cells stimulated with HOM-2 cells as APCs but not when stimulated via Raji cells that spontaneously formed conjugates in a highly efficient manner (Fig. 7 and ref. [9 ]). Diminished conjugate formation may also support data from in vivo PUFA treatment such as reduced interferon-{gamma} secretion, which is not found in vitro by antibody-mediated stimulation of PUFA-treated T cells [9 , 33 ].

In contrast to CD3 and LAT, relocalization of PKC{theta} to the IS was not influenced by PUFA treatment (Figs. 1 and 2) . This is surprising, as data obtained from Vav-deficient T cells suggest that PKC{theta} recruitment depends on Vav-induced actin polymerization [34 ]. Apparently, partial inhibition of Vav phosphorylation by PUFA treatment leaves enough Vav activity for recruitment of some (e.g., PKC{theta}) but not all molecules to the IS. This is in accordance with previous findings that CD3-induced activation of the inhibitor {kappa}B/nuclear factor-{kappa}B pathway, which depends on PKC{theta}, is not affected in PUFA-treated T cells [22 ] and may be related to the fact that membrane recruitment and activation of PKC{theta} are mediated by a nonconventional, Vav-dependent pathway [35 ]. It is remarkable that our results are comparable with those obtained from T cells deficient in DOCK2, a protein regulating TCR/CD3-induced rearrangement of the actin cytoskeleton [36 ]. In DOCK2-deficient T cells, relocalization of CD3 but not PKC{theta} toward the T cell/APC interface was abolished, similar to our data with PUFA-treated cells. However, the exact mechanisms beyond Vav and DOCK2, which are responsible for the relocalization of distinct molecules to the IS, remain to be elucidated.

PUFAs are well known to modify lipid rafts [10 , 21 , 37 ]. As T cell lipid rafts are probably not relocalized to the IS (refs. [38 , 39 ] and unpublished data), modifications of lipid rafts may disturb signals inducing IS formation rather than affecting molecule translocation directly. The adaptor LAT resides in lipid rafts under normal conditions, and the LAT signalosome, i.e., adaptor and signaling proteins associated with LAT [19 , 20 ], is of central interest with respect to effects of PUFAs on T cell signal transduction [22 ]. To test whether PUFA modification of lipid rafts in general and LAT displacement from lipid rafts in particular also underlie defective Vav downstream signaling, as is the case for defective PLC{gamma}1/calcium signaling [22 ], we applied two experimental approaches. First, raft disruption by MCD treatment caused a similar pattern of disturbed molecule recruitment to the IS in our experiments, with diminished relocalization of F-actin and LAT but undisturbed clustering of PKC{theta}, although the latter is in contrast to results obtained in a different experimental system [40 ]. The different extent of inhibition of LAT and F-actin relocalization by MCD and PUFA treatment (Fig. 1 , cf., Fig. 5 ) could support the proposed existence of lipid raft heterogeneity [41 ], with different rafts modified primarily by PUFAs and MCD, respectively. Second, we could demonstrate that LAT displacement from lipid rafts also underlies PUFA-mediated inhibition of CD3 capping by using LAT-deficient cells reconstituted with wild-type LAT or a chimeric LAT protein that retains its localization within lipid rafts after EPA treatment (Fig. 6 ; ref. [22 ]). A further hint for the critical involvement of lipid rafts for PUFA effects on IS formation is that EPA treatment of Jurkat T cells primarily affected the second wave of Vav phosphorylation, which was shown to depend on lipid rafts (Fig. 3 ; ref. [29 ]). Hence, inhibition of Vav downstream signaling leading to defective IS formation probably originates from modification of T cell lipid rafts resulting in displacement of LAT from rafts.

According to results of this and previous studies, PUFA treatment of T cells leads to diminished signaling events such as calcium response and Vav phosphorylation. Consequently, cytoskeletal rearrangements are blocked, leading to incomplete formation of the IS and stable T cell/APC conjugates. As is the case for PUFA-mediated inhibition of T cell calcium signaling and events further downstream, LAT displacement from lipid rafts again appears to be a key molecular alteration underlying PUFA effects on T cell activation. Disturbed IS and conjugate formation could extend PUFA-mediated defects to activation pathways that are not intrinsically altered in PUFA-treated T cells when directly stimulated by antibodies, thereby adding a novel aspect to explain PUFA-mediated immunomodulation.


   ACKNOWLEDGEMENTS
 
This work was supported by the Austrian Science Fund (P16788-B13 to T. M. S.) and CeMM-Center of Molecular Medicine, a basic research institute within the companies of the Austrian Acadamy of Sciences (to T. M. S. and W. W.). We are grateful to Lawrence E. Samelson for generously providing LAT-deficient ANJ3 Jurkat cells; Lucy R. Wedderburn for providing Jurkat clone CH7C17 and B cell line HOM-2; and Margarete Mario for technical assistance.

Received November 25, 2004; revised December 27, 2004; accepted January 16, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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