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Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts¹

Institute of Biochemistry and
^* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, BIL Biomedical Research Center, CH-1066 Epalinges, Switzerland

³Correspondence: Institute of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland. E-mail: daniel.legler{at}ib.unil.ch

SPECIFIC AIM

Palmitoylation of Lck, Fyn, LAT, CD4, and CD8 is essential for their partitioning in lipid rafts and functional integrity. We investigate whether an exogenous palmitoyl-phospholipid can interfere with the recruitment of palmitoylated signaling molecules to lipid rafts and thus compromise MHC-peptide-driven CTL activation.

PRINCIPAL FINDINGS

1. DPPE, but not DOPE, partitions in lipid rafts of cloned CTL
As T cells, we used cloned H-2K^d-restricted S14 CTL, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252–260 (SYIPSAEKI), PbCS(ABA). To investigate whether dipalmitoyl-phosphatidyl-ethanolamine (DPPE), which undergoes extensive flip-flop from the outer to the inner leaflet of the cell membrane, can affect antigen-specific CTL activation by interfering with the function of raft-associated signaling molecules, we first assessed whether DPPE partitions in lipid rafts. S14 CTL were treated with EITC-labeled DPPE, lysed in cold Triton X-100, and the fluorescence of DPPE^EITC was measured in the detergent soluble (membrane, M) and insoluble (lipid rafts, DIG) fractions. As shown in Fig. 1A , 34% of DPPE was found in the DIG fraction. A similar distribution was observed for cholesterol, which is a major constituent of lipid rafts. By contrast, less than 7% of DOPE, which differs from DPPE only by having unsaturated oleic acid in place of saturated palmitic acid, partitioned in DIG.

View larger version (40K): [in this window] [in a new window]   Figure 1. DPPE partitions in lipid rafts and blocks CTL activation. A) S14 CTL were incubated with 14C-cholesterol (Chol.), EITC-labeled DPPE, or DOPE and fractionated in membrane and DIG fractions. The lipid distribution was measured by counting the radioactivity of 14C-cholesterol or by measuring the fluorescence of DPPEEITC and DOPEEITC. Mean values and SD were calculated from four experiments. B) S14 CTL treated with DPPE (filled circles) or DOPE (open circles) or untreated (filled squares) were incubated at 37°C for 90 min with increasing concentrations of PbCS(ABA)-pulsed P815 cells. Esterase release from S14 CTL was then measured. Mean values and SD were calculated of duplicate assays from one experiment that is representative of three. C) Indo-1-labeled S14 CTL were incubated at 37°C for 90 s with P815 cells unpulsed or pulsed with 1 µM PbCS(ABA), and calcium-dependent fluorescence of indo-1 was measured by flow cytometry over a period of 250 s.

2. DPPE inhibits antigen-mediated CTL activation
Treatment of S14 CTL with DPPE completely abolished esterase release (Fig. 1B ) and intracellular calcium mobilization (Fig. 1C ) upon incubation with PbCS(ABA)-sensitized APC. By contrast, these functions were barely affected by DOPE treatment of the effector cells. Similar effects were observed when S14 CTL were incubated with soluble MHC-peptide tetramers (K^d-PbCS(ABA)-tetramers). However, DPPE treatment of S14 CTL did not affect their ability to form conjugates with specific peptide-pulsed syngeneic target cells. In addition, lipid treatment had no adverse effect on cell viability.

3. DPPE does not affect the integrity of lipid rafts
DPPE-treated S14 CTL were stained with FITC-labeled cholera toxin B, a reagent that binds to the ganglioside GM1, which is enriched in lipid rafts. DPPE-treated cells exhibited the same cell surface staining pattern as untreated or DOPE-treated cells, suggesting that DPPE insertion into the membrane had no effect on the surface distribution or expression of GM1. In addition, DPPE treatment did not alter the ability of lipid rafts to form large clusters on antibody-mediated cross-linking of GM1-cholera toxin. Similar results were obtained on cross-linking of the GPI-linked Thy-1 molecule, another lipid raft constituent, suggesting that DPPE treatment does not compromise the mobility and integrity of lipid rafts, which play a crucial role in TCR signaling. DPPE and DOPE had no apparent effect on the lipid raft localization of CD3, CD8, Lck, Fyn, or Thy-1.

4. DPPE blocks the recruitment of signaling molecules to lipid rafts
To define the molecular basis of the inhibitory effect of DPPE, we compared the distribution of essential signaling molecules in lipid rafts of DPPE or DOPE-treated and untreated S14 CTL after activation with K^d-PbCS(ABA)-tetramers. As shown in Fig. 2A , this antigen-specific TCR engagement resulted in the recruitment of substantial amounts of CD3, CD8, and Lck to lipid rafts in untreated or DOPE-treated CTL, whereas translocation of these molecules was strongly reduced in DPPE-treated cells. We demonstrate that DPPE selectively prevents T cell activation by impairing Src kinase activity (Fig. 2B ) and activation-induced recruitment of palmitoylated signaling molecules to lipid rafts.

View larger version (51K): [in this window] [in a new window]   Figure 2. DPPE blocks activation-induced recruitment of signaling molecules to lipid rafts. A) S14 CTL, untreated or pretreated with DPPE or DOPE, were stimulated for 2 min with 42 nM Kd-PbCS(ABA)-tetramers and fractionated in membrane (M) and DIG (D) fractions. Equal aliquots of these fractions were analyzed by SDS-PAGE and Western blotting, using specific antibodies. One out of three experiments is shown. B) S14 CTL, untreated or pretreated with DPPE or DOPE, were activated with Kd-PbCS(ABA)-tetramers, lysed, and phosphorylation of CD3 of immunoprecipitated TCR was assessed by measuring the incorporation of 32P ATP. Mean values and SD were calculated from duplicates of three experiments, normalized relative to untreated activated S14 CTL and corrected for background (19.5±2.2%) phosphorylation for nonactivated cells. The total amount of immunoprecipitated CD3 was assessed by Western blotting.

CONCLUSIONS AND SIGNIFICANCE

Plasma membranes of lymphocytes contain microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Acylation of key signaling molecules is essential for their partitioning in lipid rafts and their contribution to the TCR signaling process. In resting T cells, the TCR complex is excluded from these microdomains (Fig. 3 ).

View larger version (37K): [in this window] [in a new window]   Figure 3. A model for the inhibition of TCR signaling by incorporating DPPE into lipid rafts. In resting T cells, palmitoylated molecules such as the coreceptor CD8 (or CD4), the Src kinases Lck and Fyn, and the adapter protein LAT are present in lipid rafts, whereas the TCR/CD3 complex and the tyrosine phosphatase CD45 are excluded. The earliest signaling event after TCR engagement is the phosphorylation of CD3 ITAM by Lck and Fyn, followed by the translocation of the TCR/CD3 complex to lipid rafts. Antigen-mediated TCR engagement causes aggregation of lipid raft-associated proteins, leading to the formation of clusters of signaling molecules. The palmitoyl-containing lipid DPPE partitions in lipid rafts and selectively prevents T cell activation by impairing Src kinase activity and activation-induced recruitment of TCR signaling molecules to lipid rafts.

Studies of Jurkat T cells, thymocytes, and hybridomas indicated that stimulation of the cells’ anti-CD3 antibodies induced translocation of TCR/CD3 and other signaling molecules to lipid rafts. Physiological antigen-specific activation of T cells, however, is induced by MHC-peptide complexes on APC. To investigate the early events of TCR signaling in cloned H-2K^d-restricted CTL, we used soluble MHC-peptide tetramers, which, unlike anti-CD3 antibodies, engage not only the TCR complex, but also the coreceptor. Activation with K^d-PbCS(ABA)-tetramers resulted in TCR engagement, which causes aggregation of lipid raft-associated proteins such as the coreceptor CD8, the Src kinases Lck and Fyn, as well as the adaptor protein LAT. Moreover, lipid raft aggregation promotes tyrosine phosphorylation and recruitment of downstream signaling molecules. This activation-induced translocation of signaling molecules to lipid rafts was strongly reduced in DPPE-treated CTL (Fig. 3) .

It has recently been reported in a human trial and in in vitro studies that polyunsaturated fatty acids modulate immune responses as a result of inhibition of protein palmitoylation and TCR signaling by modifying the composition of lipid rafts. The same findings were obtained by mutation of the acylation sites of signaling molecules, by prolonged culture of Jurkat T cells in the presence of palmitate and myristate analogs, or by disrupting lipid rafts with methyl-ß-cyclodextrin. In contrast to these agents, DPPE does not affect the integrity and protein composition of lipid rafts in resting T cells. However, it strongly inhibits the activation-induced recruitment of palmitoylated signaling molecules to lipid rafts and thus blocks antigen-specific T cell activation. DPPE acts rapidly at low concentration and therefore is an attractive new immunomodulatory agent.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0841fje ; to cite this article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0841fje

² D.F.L. and M.-A.D. contributed equally to this work.

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