Legler, D.F., Doucey, M.A., Cerottini, J.C., Bron, C. & Luescher, I.F. Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts. FASEB J. 15, 1601-1603

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Antigen-specific T-cell activation implicates a redistribution of plasma membrane-bound molecules in lipid rafts, such as the coreceptors CD8 and CD4, the Src kinases Lck and Fyn, and the linker for activation of T cells (LAT), that results in the
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  The FASEB Journal   express article 10.1096/fj.00-0841fje. Published online May 18, 2001. Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts Daniel F. Legler,* ,1  Marie-Agnès Doucey, †,1  Jean-Charles Cerottini, †  Claude Bron,* and Immanuel F. Luescher †  *Institute of Biochemistry and † Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, BIL Biomedical Research Center, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland. 1 D. F. Legler and M.-A. Doucey contributed equally to this work. Corresponding author: Daniel F. Legler, Institute of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland. E-mail: daniel.legler@ib.unil.ch ABSTRACT Antigen-specific T-cell activation implicates a redistribution of plasma membrane-bound molecules in lipid rafts, such as the coreceptors CD8 and CD4, the Src kinases Lck and Fyn, and the linker for activation of T cells (LAT), that results in the formation of signaling complexes. These molecules partition in lipid rafts because of palmitoylation of cytoplasmic, membrane proximal cysteines, which is essential for their functional integrity in T-cell activation. Here, we show that exogenous dipalmitoyl-phosphatidylethanolamine (DPPE), but not the related unsaturated dioleoyl-phosphatidylethanolamine (DOPE), partitions in lipid rafts. DPPE inhibits activation of CD8 +  T lymphocytes by sensitized syngeneic antigen-presenting cells or specific major histocompatibility complex (MHC) peptide tetramers, as indicated by esterase release and intracellular calcium mobilization. Cytotoxic T lymphocyte (CTL)-target cell conjugate formation is not affected by DPPE, indicating that engagement of the T-cell receptor by its cognate ligand is intact in lipid-treated cells. In contrast to other agents known to block raft-dependent signaling, DPPE efficiently inhibits the MHC peptide-induced recruitment of palmitoylated signaling molecules to lipid rafts and CTL activation without affecting cell viability or lipid raft integrity. Key words: signal transduction •  dipalmitoyl-phosphatidylethanolamine •  TCR pecific T-cell activation is initiated by interaction of the T-cell receptor (TCR) with antigenic peptides presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC) (1,2). Engagement of the TCR by cognate MHC peptide results in the rearrangement of signaling molecules at the site of T-cell contact with the APC. This process is crucial for signaling, because it induces apposition of protein kinases with their substrates at the contact site (3). Specialized subdomains of the T-cell membrane participate in the initiation of this process by being recruited to the site of TCR engagement (4-6). These membrane microdomains, also called lipid rafts or detergent-insoluble glycolipid-enriched membranes (DIG) (7-9), contain various acylated proteins involved in the S  early steps of T-cell activation, such as the Src kinases Lck (10-12) and Fyn (13-15), the adaptor protein LAT (linker for activation of T cells) (4), and the coreceptors CD4 (16,17) and CD8 (18,19). Lipid rafts are preformed functional domains that serve as platforms for signal transduction and membrane trafficking (7). Palmitoylation and myristoylation of Lck, Fyn, LAT, CD4, and CD8 are essential for their partitioning in lipid rafts and for contribution to TCR signaling (20). Mutation or deletion of the acylation sites of these molecules compromises their functional integrity (4,21-23). In addition, 2-bromopalmitate and 1-hydroxymyristate, analogues of palmitate and myristate, respectively, have been shown to prevent localization of these molecules to lipid rafts and hence T-cell activation (24). Likewise, prolonged incubation of T cells in the presence of polyunsaturated fatty acids has been shown to inhibit protein acylation and TCR signaling (24-26). On the basis of these observations, we investigated whether incorporation of palmitoyl-containing lipids into T-cell membrane interferes with the molecular sorting and TCR signaling in lipid rafts, without disrupting these microdomains. As T cells, we used the H-2K d -restricted cytotoxic T lymphocyte (CTL) clone S14, which is specific for a derivative of the Plasmodium berghei  circumsporozoite (PbCS) peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on K259, PbCS(ABA) (27,28). We show here that exogenous dipalmitoyl-phosphatidylethanolamine (DPPE) partitions in lipid rafts and inhibits CTL activation induced by sensitized APC or soluble K d -PbCS(ABA) tetramers. In contrast, the closely related dioleoyl-phosphatidylethanolamine (DOPE), which contains unsaturated oleic acid in place of saturated palmitic acid, failed to partition in lipid rafts and had no effect on CTL activation. Moreover, we provide evidence that DPPE prevents activation-induced translocation of acylated molecules to lipid rafts and hence induction of TCR signaling. MATERIALS AND METHODS The H-2K d -restricted CTL clone S14 was propagated and used as previously described (27,28). Syngeneic mastocytoma P815 cells were used as APC after preincubation with graded concentrations of PbCS(ABA) (27). Antibodies and soluble K d -PbCS(ABA) tetramers Hybridomas producing anti-CD8 β  KT-112, anti-CD3 ζ  H-146, and anti-TCR H-57 monoclonal antibodies were from American Type Culture Collection (ATCC, Manassas, VA). Anti-Lck monoclonal antibody 3A5 was from Upstate Biotechnology (Lake Placid, NY). Anti-Fyn antiserum was a generous gift from Dr. White (Harvard Medical School, Boston, MA). Tetramers containing K d       2 -microglobulin were formed and isolated as described previously (18). Lipids DPPE EITC  and DOPE EITC  were obtained by reacting the lipids (Sigma, Buchs, Switzerland; Avanti Polar Lipids, Alabaster, AL) in chloroform/di-isopropylethylamine (98:2) with eosin-isothiocyanate (EITC) (Sigma) overnight at 4°C. The labeled lipids were dried, reconstituted in  PBS containing 35   -octylglucopyranoside (Fluka, Buchs, Switzerland), and purified by gel filtration on Sephadex G25 (Pharmacia, Uppsala, Sweden). The detergent was removed by extensive dialysis against PBS. Cells (5 ×  10 6  /ml) were treated with unlabeled or EITC-labeled lipids (5 µg/ml) in serum-free DMEM for 45 min at 37°C, washed twice, and directly used. The uptake of DPPE and DOPE corresponded to about 30 ng of each lipid per 10 6  cells, which represents less than 0.1% of the total lipid of a cell. For labeling of S14 CTL with 14 C-labeled cholesterol, medium was incubated for 12 h with 0.4 µCi 14 C-labeled cholesterol (51 mCi/mmol, Du Pont NEN, Boston, MA) and was then incubated with the cells for 48 h. Thereafter, the cells were washed and cultured for another 24 h and fractionated. DIG fractionation S14 CTL (10 7 ) were lysed on ice for 20 min in 200 µl of 1% Triton X-100 in MN buffer (25 mM 2-(  N  -morpholino)ethanesulfonic acid, 150 mM NaCl; pH 6.5) containing 5 µg/ml leupeptin and 5 µg/ml pepstatin. The cell lysate was homogenized with a loose-fitting Dounce homogenizer (10 strokes) and then was spun at 500 g  for 7 min at 4°C; the nuclear pellet was washed sequentially in Triton X-    -octylglucopyranoside buffer (50   -octylglucopyranoside in 20 mM Tris, 500 mM NaCl; pH 8.0) containing protease inhibitors. The postnuclear supernatant was centrifuged first at 7000 g  for 12 min and then at 100,000 g  for 50 min, at 4°C. The pellets were resuspended in 200    -octylglucopyranoside buffer and are referred to as DIG fractions. The 100,000 g  supernatant is referred to as the phospholipid membrane (M) fraction. Kinase assay S14 CTL (5 ×  10 6 ) were stimulated at 10 7  cells/ml in PBS with 10 µg/ml K d -PbCS(ABA) tetramers for 2 min at 37°C and lysed on ice for 20 min in 0.5% Triton X-100, 0.5% Brji 78, 0.2 mM NaVO 4 , 3 mM NaF, 20 mM Tris, and 150 mM NaCl; pH 7.5. The lysate was spun for 5 min at 10,000 g , and TCR or Lck was immunoprecipitated from the supernatant by using H-57 or anti-Lck antibodies (kindly provided by Drs. Acuto and Di Bartolo, Pasteur Institute, Paris, France). Washed immunoprecipitates were incubated in 30 µl of kinase buffer (1 mM Tris, 25 mM HEPES, 7.5 mM NaCl, 10 mM MnCl 2 ; pH 7.4) containing 7 µCi [ γ  - 32 P]ATP (3000 Ci/mmol, Du Pont NEN) for 20 min at 30°C and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphorylated CD3 ζ  was analyzed by using a PhosphoImager and the ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Intracellular calcium mobilization S14 CTL were incubated with 5 µM of indo-1/AM (Sigma) for 45 min at 37°C in the presence or absence of the lipids. CTL were then mixed with P815 cells sensitized with PbCS(ABA) at a ratio of effector to target cell (E/T) of 1/3, and calcium mobilization-related fluorescence changes of indo-1 were measured by FACStar (Becton Dickinson, Erembodegen, Belgium). Esterase release   PbCS(ABA)-sensitized P815 cells were incubated with S14 CTL (E/T of 1/3) for 90 min at 37°C, and esterase release was determined with carbobenzoxy-CBZ- L -lysine thiobenzyl ester hydrochloride and 5,5'-dithiobis(2-nitrobenzoic acid) (Sigma) as substrate (29). Alternatively, 96-well plates were coated overnight at 4°C with 2.5 µg/well of streptavidin (Molecular Probes, Eugene, OR) in 0.1 M NaCO 3 , pH 8.3, followed by incubation with graded concentrations of biotinylated K d -PbCS(ABA) for 2 h at room temperature. The specific lysis was determined as 100 ×  (experimental         was below 10% in all experiments. Conjugate formation S14 CTL were labeled with 1 µM carboxyfluorescein succinimidyl ester (CFSE, Sigma) for 20 min. PbCS(ABA)-sensitized P815 cells were labeled with Cell Tracer Orange (Molecular Probes). For conjugate formation, S14 CTL and P815 cells were mixed at different E/T ratios, spun and incubated for 8 min at 37°C, vortexed, and analyzed by a fluorescence-activated cell sorter (FACS). Cross-linking of cholera toxin and confocal microscopy S14 CTL were labeled with 10 µg/ml fluorescein isothiocyanate-labeled cholera toxin B (cholera toxin B FITC ) (Sigma) for 30 min at 4°C and laid on slides for 45 min at 4°C in the presence or absence of anti-cholera toxin antibodies (Sigma). Alternatively, cells were labeled with rabbit anti-Thy-1 polyclonal antibody and cross-linked or not with a goat-anti-rabbit FITC  antibody (Jackson ImmunoResearch, West Grove, PA). Cells were fixed with PBS containing 3% paraformaldehyde for 10 min, and the samples were mounted in Fluorosave (Calbiochem, San Diego, CA) and examined with a confocal microscope (LSM410, Carl Zeiss, Jena, Germany). RESULTS AND DISCUSSION DPPE, but not DOPE, partitioned in lipid rafts and inhibited activation of S14 CTL  To examine whether exogenous palmitoyl lipid inhibits antigen-specific CTL activation, we first assessed whether DPPE partitions in lipid rafts. As palmitoyl lipid we chose DPPE because it undergoes extensive flip-flop from the outer to the inner leaflet of cell membranes (30). To this end, we treated the H-2K d -restricted CTL clone S14 with EITC-labeled DPPE, lysed the cells in cold Triton X-100, and measured the fluorescence of DPPE EITC  in the detergent-soluble (M) and detergent-insoluble (DIG) fractions. As shown in Fig. 1  A , approximately 34% of DPPE was found in the DIG fraction. A similar distribution was observed for cholesterol, which is a major constituent of lipid rafts (8). In contrast, less than 7% of the unsaturated DOPE partitioned in DIG. A comparable distribution of these phospholipids was observed when sucrose density gradients were used to isolate detergent light fractions of several T-lymphoma cell lines or T-cell hybridomas (data not shown). These results agree with a study of the distribution of dimyristoyl-phosphatidylethanolamine and DOPE in muscle cells via single-molecule microscopy (31). The distribution of DPPE and DOPE on S14 CTL did not change significantly when the lipid  concentration was 10-fold lower or 3-fold higher. Under these conditions, both lipids did not affect cell viability (data not shown). We next examined whether lipid treatment of S14 CTL affected their ability to recognize P815 cells pulsed with the cognate peptide PbCS(ABA). As shown in Fig. 1  B , S14 CTL treated with DPPE failed to release esterases upon incubation with sensitized P815 cells. In contrast, DOPE-treated S14 CTL released esterases in the same peptide dose-dependent manner as untreated cells (Fig. 1  B ). Lipid rafts on APC have been shown to be critical for antigen presentation by providing a local concentration of the MHC-peptide complex in the immunological synapse (32). When S14 CTL were exposed to PbCS(ABA)-pulsed P815 cells treated or not with DPPE, no difference in the efficiency of esterase release was observed, suggesting that, in contrast to agents disturbing lipid rafts such as nystatin and methyl-  -clodextrin (MCD), DPPE did not affect antigen recognition (data not shown). Esterase release is a relatively late event during T-cell activation. To determine whether DPPE affected earlier events, we assessed its effect on intracellular calcium mobilization. As shown in Fig. 1 C  , the intracellular calcium level in S14 CTL increased about fivefold above the background level after incubation with PbCS(ABA)-pulsed P815 cells. Pretreatment of S14 CTL with DPPE profoundly inhibited calcium mobilization in a dose-dependent manner, whereas DOPE had no effect. The same inhibition was observed with treatment of S14 CTL with MCD (data not shown), which has been demonstrated to disrupt the association of signaling molecules with lipid rafts (32,33). To test whether pretreatment of S14 CTL with DPPE affected their ability to form conjugates with sensitized P815 cells, we enumerated by flow cytometry the number of conjugates. After incubation of S14 CTL with P815 cells at an E/T ratio of 1/2, the efficiency of conjugate formation was 90% in untreated S14 CTL and DPPE- or DOPE-treated S14 CTL (Fig. 2). Similar results were obtained for an E/T ratio of 1/1, indicating that DPPE had no effect on conjugate formation and hence cell viability. Similar findings were obtained by assessing conjugate formation by confocal microscopy (data not shown). Taken together, these results demonstrate that exogenous DPPE, but not DOPE, efficiently partitions in CTL lipid rafts and specifically inhibits intracellular calcium mobilization and esterase release upon incubation with sensitized target cells. DPPE did not affect the integrity of lipid rafts Because lipid rafts are known to play a crucial role in TCR-mediated T-cell activation (4-6,23,33,34), we investigated whether DPPE affected lipid raft integrity. To this end, DPPE-treated S14 CTL were stained with cholera toxin B FITC . Because this reagent binds to ganglioside G M1 , which is highly enriched in lipid rafts, it is frequently used to stain these membrane microdomains (8,33,35). DPPE-treated cells exhibited the same cell surface staining pattern as untreated or DOPE-treated cells (Fig. 3  A ), indicating that DPPE insertion into the membrane had no apparent effect on the surface distribution or expression of G M1 , which is in contrast to the selective internalization reported after treatment with ganglioside lipids (6,36,37). In addition, DPPE treatment did not alter formation of large clusters upon antibody-mediated cross-linking of
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