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Molecular and Cellular Biology, August 2005, p. 6811-6820, Vol. 25, No. 15
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.15.6811-6820.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Laboratoire CIRID, CNRS UMR 5164, Université de Bordeaux 2, 146 rue Léo Saignat, Bordeaux 33076, France,1 Laboratoire de Biogenèse Membranaire, CNRS UMR 5200, Université de Bordeaux 2, 146 rue Léo Saignat, Bordeaux 33076, France,2 Centre de Lutte Contre le Cancer Bergonié et Institut Européen de Chimie et Biologie, 33600 Pessac, France3
Received 4 February 2005/ Returned for modification 21 March 2005/ Accepted 26 April 2005
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Recently, Muppidi and Siegel reported that Fas is localized in detergent-resistant plasma membrane microdomains termed lipid rafts or microdomains in type I cells and excluded from these domains in type II cells. In type I but not in type II cells, Fas-induced apoptosis depends on the engagement of the microdomains. The microdomains are detergent-resistant cholesterol-rich structures enriched in glycosylphosphatidyl-inositol (GPI)-linked proteins and signal-transducing molecules, e.g., the kinases of the src family (4). Following the recognition of the peptide-major histocompatibility complex, T lymphocyte activation requires the recruitment of microdomains to form the "immunological synapse" at the contact area with the antigen-presenting cell (17, 30, 32). This step is mediated by CD28, which triggers the recruitment of the microdomains at the contact to the T-cell receptor (TCR) molecules.
In a previous work, we reported that an engineered GPI-linked Fas (Fas-GPI), localized into the microdomains, was capable of enhancing Fas-mediated apoptosis in type II cells constitutively expressing functional wild-type Fas (15). By analogy to the TCR activation model, other ligand/receptor interactions are expected to occur concomitantly to the engagement of Fas by the FasL present on the killer cell. We hypothesized that in type II cells, the recruitment of the microdomains could occur via a coreceptor and as a consequence enhance the sensitivity of these cells to Fas-mediated apoptosis. Therefore, we investigated whether type II cells, such as the Jurkat cell line or activated T lymphocytes from peripheral human blood, can be sensitized to Fas engagement through the involvement of the lipid rafts.
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Antibodies and other reagents.
The anti-human Fas MAb 5D7 and anti-human FasL MAb 14C2 (15) used in flow cytometry and the isotype-matched negative control MAbs 1F10 (immunoglobulin G [IgG]) and 10C9 (IgM) (27) were all generated in the laboratory. For functional studies, the anti-human Fas agonistic MAb 7C11 (IgM) and blocking MAb ZB4 (IgG) and the anti-CD28 MAb (clone CD28.2) were from Immunotech. For the immunoblots, anti-CD28, anti-caspase 8, and anti-BID polyclonal antisera were from R&D Systems (Oxon, United Kingdom), antitransferrin MAb was from Zymed (San Francisco, CA), anti-p56Lck MAb was from Transduction Laboratories (Lexington, KY), anti-caspase 3 and anti-Bcl-2 were from Pharmingen, antiactin was from Sigma, and anti-c-FLIP and anti-Fas antiserum (C-20) were from Santa Cruz Biotechnology (Tebu, Le-Perray-En-Yvelines, France). The J558L hybridoma cell line producing CTLA4-Ig with CTLA4 fragment of mouse origin was obtained from P. Lane (Basel Institute for Immunology, Switzerland). The Fas-Fc chimera was produced in COS cells transfected with a construct generated in the laboratory. As an irrelevant control, we used a homemade COS-derived vIL6-Fc chimeric protein, with vIL6 being the human herpesvirus 8-derived IL-6 homolog. These chimeras were purified on a protein A affinity column. Phorbol myristate acetate,
-cyclodextrin (
-CD), and methyl-ß-cyclodextrin (MßCD) were from Sigma. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Leu-Glu-His-Asp(Ome)-fluoromethylketone (zLEHD-fmk) were from Bachem (Voisins-le-Bretonneux, France).
Establishment of stably transfected cell lines.
The pEGFP-Bcl-2 plasmid (31) was kindly provided by R. Youle (National Institutes of Health, Bethesda, MD). The pEGFP empty vector (Clontech, Palo Alto, CA) was used as a control. The CD28-truncated cDNA lacking CD28 intracellular region (CD28
) (25) was kindly provided by A. Weiss (UCSF, San Francisco, CA) in the pAW-neo3 plasmid. Jurkat and H9 cells (5 x 106 cells in 0.8 ml) were transfected with 5 µg of plasmid by electroporation at 280 and 290 V, respectively, and 900 µF using an Easyject+ electroporator (Eurogentec, Seraing, Belgium) and resuspended in culture medium. Selection was performed with G418 (Life Technologies, Cergy-Pontoise, France) at 1.9 mg/ml for Jurkat cells and 1 mg/ml for H9 cells for 15 days. Then the G418-resistant cells were sorted using a Coulter Elite cell sorter (Coultronics, Hialeah, FL) on the basis of green fluorescent protein (GFP) fluorescence and used as polyclonal populations, or cells were cloned by limiting dilutions. The B7-1 cDNA was kindly provided by L. Lanier (UCSF, San Francisco, CA) and subcloned in the puromycin resistance vector pLXSP. The cells were transfected with the pLXSP-B7-1 or the pLXSP plasmids at 290 V and 900 µF, selected with puromycin at 2 µg/ml for 15 days, and then subcloned by limiting dilution.
Flow cytometry staining of cells. Cells were washed, stained, and analyzed exactly as described previously (15). For the experiments using MßCD, the 1A12 cells were washed in serum-free medium, incubated in serum-free medium for 20 min at 37°C in the presence of the indicated concentration of MßCD, washed again, and finally stained.
Cell cytotoxicity assays.
The 4-h 51Cr release cytotoxicity assays using FasL-expressing cells as effectors were performed as described by Legembre et al. (15). Cytotoxicity assays using beads as effectors were performed as follows. Polystyrene beads with 6-µm diameters (Polybead Polystyrene Microsphere, Polysciences, Eppelheim, Germany) were washed three times with phosphate-buffered saline (PBS) and incubated overnight at room temperature at a ratio of 6 x 105 beads per 50 µl of antibody solution in PBS, with the IgM MAb (anti-Fas or negative control 10C9) at the indicated concentration and the IgG MAb (anti-CD28, anti-CD71, or negative control 1F10). After three washes with PBS, the beads were mixed with the 51Cr-labeled target cells at the indicated ratios, and the chromium released was measured. In experiments with phorbol myristate acetate (25 ng/ml), the cells were first labeled and then incubated for 30 min with the indicated dose of the chemical before adding the beads. In experiments with MßCD, the Jurkat cells and activated CD4+ T lymphocytes were incubated for 20 min in serum-free medium with 2 mM and 5 mM MßCD, respectively, or with
-CD at the same concentration as a control at 37°C and then washed before the addition of the effector cells or beads in medium containing 4% fetal calf serum.
Detergent lysis experiments. The cells were incubated for the indicated times with MAb-coated beads at a 10:1 (beads/cell) ratio. The cells were lysed in lysis buffer (25 mM HEPES, 1% Triton X-100, 150 mM NaCl, pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonidefluoride, 5 µg/ml aprotinin, 10 µM leupeptin) for 30 min on ice. After centrifugation (10 min, 4°C, 15,000 rpm), the supernatant was harvested. The microdomains were isolated by ultracentrifugation on a sucrose gradient using the method of Ko et al. (13). To analyze the redistribution of Fas and caspase 8 into the microdomains, Jurkat 77 cells were incubated with 1A12 or 1A12-B7-1 cells for the indicated times and lysed with the lysis buffer. Time zero corresponds to a mix of separately lysed target and killer cells.
Immunoprecipitation of the DISC. Jurkat 77 cells (7 x 106) were incubated for 25 min at 37°C with magnetic beads (Dynabeads M-450) coated with the anti-Fas MAb at 1 µg/ml and the indicated antibody at a 2:1 (beads/cell) ratio and then lysed for 20 min at 4°C in lysis buffer (25-min activation condition) or were lysed before the beads were added (0-min activation condition). The beads were separated with a magnet and washed three times with 1 ml of lysis buffer. The immunoprecipitate corresponding to 7 x 106 cells was loaded for each condition and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Western blot analysis. Protein concentration in cellular extracts was determined using the bicinchoninic acid method (Sigma) according to the manufacturer's protocol. Proteins (10 µg per lane unless otherwise stated) were separated by SDS-PAGE on 12% gels in reducing conditions and transferred to a polyvinyldifluoride membrane (Amersham, Buckinghamshire, England), as described previously (15). The peroxidase-labeled secondary antisera used were anti-mouse (Amersham), anti-goat (Vector Laboratories, Burlingame, CA), or anti-rabbit (Zymed).
DNA fragmentation assay. The target Jurkat cells (3 x 106 per condition) were incubated for 2 h with 30 x 106 coated beads (10:1 ratio). Then the cells were lysed and the DNA was precipitated and analyzed on a 1.5% agarose gel as described previously (28).
Immunofluorescence and imaging. Jurkat cells were treated with the nonblocking and nonagonistic anti-Fas MAb (5D7) and fluorescein isothiocyanate (FITC)-labeled cholera toxin B for 30 min at 4°C and then mixed with 1A12 or 1A12-B7-1 at a 1:2 ratio. The mixed cells were incubated for 30 min at 37°C and adhered for 5 min at room temperature to poly-L-lysine-coated slides (ESCO, VWR, Strasbourg, France). Cells were then fixed in PBS containing 2% formaldehyde for 15 min, washed twice in PBS-1% bovine serum albumin and stained with the secondary antibody Alexa-594-conjugated goat anti-mouse antibody in PBS-1% bovine serum albumin. Slides were washed with PBS, dried, and mounted with Fluoroprep (Biomerieux, Marcy l'Etoile, France). Images were acquired and processed on a confocal microscope (LSM 510, Carl Zeiss, Jena, Germany) with a 63x objective.
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FIG. 1. Fas-mediated cell death is amplified upon CD28 costimulation. (A) The membrane expression of B7-1 (upper panel) and of FasL (lower panel) on the 1A12 cell line (thin line) and on the 1A12-B7-1 cell line (thick line) was analyzed by flow cytometry. The dotted line depicts an isotype control MAb. (B) 1A12 ( and ) or 1A12-B7-1 ( and ) cell lines were incubated with 51Cr-labeled Jurkat 77 target cells at the indicated ratios in the presence of ( and ) soluble blocking anti-CD28 MAb (upper panel) or CTLA4-Ig (lower panel) or of ( and ) the negative control MAb or the control vIL6-Fc. (C) The 1A12-B7-1 cell line was incubated with the 51Cr-labeled Jurkat 77 target cells at an 8:1 ratio in the presence of Fas-Fc or vIL6-Fc (upper panel) or of the blocking anti-Fas MAb ZB4 or the control MAb (lower panel) at the indicated concentrations. (D) Polystyrene beads were coated with antibodies and mixed for 4 h with 51Cr-labeled Jurkat 77 target cells at the indicated ratios before cell death measurement. In the upper panel, anti-Fas MAb 7C11 (0.08 µg/ml) was coated together with the negative IgG control MAb ( ), the anti-CD28 MAb ( ), or the anti-CD71 MAb ( ) at 10 µg/ml. To quantify the maximum value of Fas-mediated apoptosis in this assay, beads were coated with 7C11 at high concentration (10 µg/ml) ( ). In the lower panel, beads were coated with either the anti-Fas MAb 7C11 (0.08 µg/ml) and the anti-CD28 MAb ( ) or the negative IgM control MAb (0.08 µg/ml) and the anti-CD28 MAb ( ). Results represent the means and errors of three independently repeated experiments.
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FIG. 2. CD28 cosignal increases the Fas-mediated apoptotic pathway. Beads were coated with the anti-Fas (7C11) and the indicated antibody and then incubated with target Jurkat 77 cells at a 10:1 (beads/cell) ratio for 2 h before the cells were lysed. (A) DNA fragmentation was analyzed by agarose gel electrophoresis as described in Materials and Methods. (B) Activation of caspase 8 and caspase 3 was analyzed by immunoblotting. The results shown are representative of three independent experiments.
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) which is unable to transduce any cosignal for T-cell activation (25) or the control empty vector as a control. The cell populations raised expressed identical levels of membrane Fas (Fig. 3A), and a large amount of truncated CD28 was expressed on the cell surface in the Jurkat CD28
cell line (Fig. 3A). The control and CD28
-transfected cell lines displayed identical sensitivities to Fas stimulation (Fig. 3B). Due to residual expression of wild-type CD28 on around 20% of the control cell line (Fig. 3A), its sensitivity to killing after Fas/CD28 coligation slightly increased. However, the CD28
cell line was again much more sensitive to Fas/CD28 cotriggering (Fig. 3B). This result demonstrated that CD28
was capable of amplifying Fas-mediated cell death and did not inhibit the endogenous residual wild-type CD28 by acting as a dominant negative decoy receptor as could have been expected. Therefore, the proapoptotic role of CD28 on Fas-induced apoptosis was completely independent of its signal-transducing intracellular region.
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FIG. 3. CD28 intracellular region is not required for enhancing Fas-mediated apoptosis. (A) Fas and CD28 expression were analyzed by flow cytometry analysis on the bulk populations of sorted CD28-negative Jurkat cells stably transfected with the CD28 construct (thick line) or the control empty vector (thin line). The dashed line is the negative IgG control MAb. (B) CD28 (filled bars) or control (open bars) Jurkat cells were incubated with beads coated with the anti-Fas (0.08 µg/ml) and the negative IgG control MAb (upper panel) or the anti-CD28 MAb (lower panel) at the indicated ratios. Cell death was measured using the 51Cr release assay. Results represent the means and errors of three independently repeated experiments.
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were exclusively localized in the microdomains.
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FIG. 4. Fas is redistributed in the lipid rafts upon CD28 cosignal. (A) Triton X-100 cell lysates prepared from the Jurkat 77 (upper part) or the Jurkat CD28 (lower part) cell lines were separated by ultracentrifugation on a sucrose gradient. Ten micrograms of proteins per lane was loaded, separated by SDS-PAGE, and analyzed by immunoblotting for their content in the indicated molecules. (B) Jurkat cells (50 x 106) were incubated at the indicated time with the 1A12 or 1A12-B7-1 cell lines as indicated (ratio, 1:2) and lysed. The lipid rafts were isolated by ultracentrifugation on a sucrose gradient, and Fas or caspase 8 was analyzed by immunoblotting. (Upper panel) Time-dependent relocation of Fas into the microdomains in response to 1A12-B7-1 cells. (Lower panel) Relocation of Fas and caspase 8 in response to 1A12 or 1A12-B7-1 cells after an incubation of 30 min. (C) Densitometry analysis of Fas and caspase 8 distribution. Values correspond to ratios of the intensity of specific bands at 30 min versus 0 min (see Materials and Methods) with 1A12 ( ) or 1A12-B7-1 ( ) cells.
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FIG. 5. CD28 cosignal induces the relocation of Fas into the lipid rafts. Jurkat 77 cells were stained with anti-Fas MAb (5D7) and FITC-labeled cholera toxin B (green) for 30 min at 4°C, washed twice, and then stimulated for 30 min with (A) the 1A12 or (B) the 1A12-B7-1 cell line at 37°C. Anti-Fas was detected with an Alexa-594-conjugated goat anti-mouse antibody (red). For the bottom of panels A and B, the red and green fluorescence intensities of the plasma membrane were measured around the perimeter of the cell and plotted in the corresponding graphs. The y axis represents relative fluorescence intensity of the pixels, and the x axis represents the distance around the periphery of the cell.
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-CD cannot (32). Treatment of the Jurkat cell line with MßCD did not alter the membrane expression of CD28 and of Fas (Fig. 6A). When Jurkat cells were preincubated with MßCD, the CD28-mediated amplification of Fas apoptosis was abrogated (Fig. 6B, compare open and filled triangles), whereas
-CD had no effect (Fig. 6B, compare open and filled squares). To analyze the consequence of microdomain disruption on Fas signaling, the cells were incubated with or without MßCD, washed to remove the excess of chemical, and mixed with the MAb-coated beads for 45 min before cell lysis and analysis of caspase 3 cleavage. As previously described (2, 18), the disruption of lipid rafts did not modify the Fas-mediated apoptosis pathway in our type II cell line (Fig. 6C, compare lanes 5 and 6). In contrast, the pretreatment with MßCD completely abrogated the CD28-mediated amplification of caspase 3 cleavage (Fig. 6C, compare lanes 2 and 4), leading to the conclusion that lipid raft recruitment can enhance Fas-induced cell death in a type II cell line.
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FIG. 6. Disruption of the microdomains inhibits the CD28 cosignal. (A) Stability of membrane receptor expression level following MßCD treatment. Jurkat 77 cells were incubated for 20 min with (thick line) or without (thin line) MßCD (2 mM), and then the surface level of CD28 and Fas was analyzed by flow cytometry. The dashed line is the negative IgG control MAb. (B) Jurkat 77 cells were treated for 20 min with 2 mM MßCD or -CD and incubated with either 1A12 or 1A12-B7-1 effector cells, and a 51Cr release assay was performed. (C) Jurkat cells were treated as in panel B, incubated for 45 min with beads coated with the indicated antibodies, and then lysed. Caspase 3 activation was analyzed by immunoblotting. The data are representative of three independent experiments.
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As already reported (18), we observed that a large amount of Fas was concentrated in the lipid rafts, in contrast to the type II Jurkat cell line (Fig. 7A). Because CD28 expression in H9 cells was very faint (Fig. 7B), and given the dispensability of CD28 intracellular region for raft recruitment, we overexpressed CD28
or the empty control vector in H9 cells. The bulks of stably transfected H9 cells were analyzed by flow cytometry to estimate Fas and CD28 expression (Fig. 7B). H9 CD28
and control transfectants displayed comparable levels of Fas (Fig. 7B). To analyze whether the microdomain recruitment could increase Fas signaling in the type I cell line, we performed 51chromium release cytotoxicity assays with the effector cells 1A12 and 1A12-B7-1 (Fig. 7C) and with polystyrene beads coated with a concentration of anti-Fas MAb triggering minimal cell death (1 µg/ml) and either the anti-CD28 MAb or the negative control MAb (Fig. 7D). CD28 recruitment in the H9 CD28
cell line did not alter the sensitivity to Fas-induced apoptosis. This observation is consistent with the demonstration that the type I cells already use efficiently the microdomains in response to Fas (18) and suggests that type I cells cannot be modulated by the indirect recruitment of CD28-associated lipid rafts.
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FIG. 7. CD28-mediated microdomain recruitment in a type I cell line does not improve the Fas-dependent apoptotic signal. (A) H9 (type I) and Jurkat 77 (type II) cells were lysed, and lipid rafts were separated from the rest of the membrane by ultracentrifugation on a sucrose gradient. The distribution of the indicated proteins was analyzed by immunoblotting. (B) Fas and CD28 expression in the CD28-deficient H9 cell line transfected with empty vector (thick line) or with the CD28 -containing plasmid (dashed line). The thin line represents a negative control MAb. (C) Control H9 cells or CD28 -expressing H9 cell lines were incubated with the 1A12 or 1A12-B7-1 cell line, and apoptosis was measured with the 51Cr release assay. (D) H9 and CD28 -expressing H9 cells were incubated in the presence of beads coated with the anti-Fas MAb 7C11 (1 µg/ml) and either the anti-CD28 MAb or the negative IgG control MAb (10 µg/ml), and a 51Cr release assay was performed. For all of the 51Cr release assays, results represent the means and errors of three independently repeated experiments.
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FIG. 8. CD28 cosignal does not alter DISC formation but enhances the mitochondrion-dependent apoptotic pathway. (A) Magnetic beads were coated with the anti-Fas MAb (1 µg/ml) and the indicated antibody (10 µg/ml) and then incubated with 7 x 106 Jurkat 77 cells for 25 min at a 2:1 (beads/cell) ratio before the cells were lysed. As a control, cells were lysed before the beads were added (time zero). The DISC was immunoprecipitated, and its components c-FLIPL, FADD, procaspase 8, and Fas were analyzed by immunoblotting. A total cell lysate was also analyzed as a control. (B) Beads coated with anti-Fas and the indicated MAb were incubated with target Jurkat cells for 2 h at a 10:1 (beads/cell) ratio. The cells were lysed, and the uncleaved p22 BID was detected by immunoblotting. The cleaved fragments are not detected with the antibody used. Detection of ß-actin is used as a control for protein loading. (C) 51Cr-labeled Jurkat 77 cells were incubated for 30 min at 37°C with the pan-caspase inhibitor zVAD-fmk or the specific caspase 9 inhibitor zLEHD-fmk (10 and 50 µM, respectively) and then mixed with beads coated with a high concentration of anti-Fas MAb alone (1 µg/ml, white bars) or anti-Fas MAb (0.08 µg/ml) with the anti-CD28 MAb (black bars) at a 10:1 (beads/cell) ratio for 4 h. Apoptosis was measured by 51Cr release assay. (D) Jurkat 77 cells were transfected with GFP-Bcl-2 or GFP, and protein expression was analyzed in cell lysates by performing a Bcl-2 immunoblot. The sensitivity of Jurkat GFP ( ) and Jurkat GFP-Bcl-2 ( ) cells to Fas-mediated apoptosis was analyzed in a 51Cr release assay using beads coated with a high concentration of 7C11 (1 µg/ml). (E) Jurkat GFP (filled symbols) or Jurkat GFP-Bcl-2 (open symbols) cells were incubated with beads coated with anti-Fas (0.08 µg/ml) and anti-CD28 (triangles) or the negative IgG control (squares). For all of the 51Cr release assays, results represent the means and errors of three independently repeated experiments, and graphs are representative of three independent experiments.
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Second, we analyzed the caspase 9 activity, which is induced via the mitochondrial release of cytochrome c. Caspase 9 activity can be specifically blocked with the peptidic inhibitor zLEHD-fmk, whereas the inhibitor zVAD-fmk blocks all caspases. As such, zVAD-fmk completely inhibited the cell death triggered by beads covered with a high anti-Fas concentration (1 µg/ml of 7C11) or with the anti-Fas/anti-CD28 beads (Fig. 8C, 7C11/anti-CD28), showing that apoptosis triggered by Fas/CD28 coligation is totally dependent on caspase activation. In contrast, zLEHD-fmk strongly impaired the death signal triggered by the Fas/CD28 coligation while having a weaker effect on the signal mediated by Fas alone (75% versus 34% inhibition, respectively), indicating that the effect of CD28 preferentially involved caspase 9.
Bcl-2 inhibits the mitochondrial cell death pathway. We generated Jurkat cells overexpressing a GFP-Bcl-2 chimera or GFP alone as a control (Fig. 8D, insert) and displaying comparable amounts of surface CD28 and Fas as detected by flow cytometry (result not shown). The Jurkat GFP-Bcl-2 cells were slightly less sensitive than the control cells to killing via Fas, as shown with beads coated with anti-Fas MAb at a high concentration (1 µg/ml) (Fig. 8D). In contrast, Bcl-2 overexpression totally abrogated the enhancing effect of CD28 (Fig. 8E, compare filled and empty triangles). Taken together, these experiments demonstrated that in a type II cell line the microdomain recruitment selectively acted through the mitochondrion-dependent pathway of Fas-mediated apoptosis.
CD28-mediated microdomain recruitment enhances Fas-mediated apoptosis of primary activated CD4+ T lymphocytes from peripheral blood. Primary activated CD4+ T cells are refractory to bivalent Fas stimulus but become sensitive after TCR restimulation (18). Fas is excluded from the microdomains in these primary activated T cells and is redistributed in these structures after TCR restimulation. Therefore, primary activated T cells behaved as type II cells (18). To extend our findings to primary immune cells, we analyzed whether the primary activated CD4+ T lymphocytes could be sensitized to Fas-mediated apoptosis pathway via the recruitment of CD28-associated microdomains. CD4+ T lymphocytes were enriched by negative selection from PBMC of healthy donors and polyclonally activated with phytohemagglutinin and IL-2. As observed with the Jurkat cells, the 1A12-B7-1 cell line killed more efficiently the activated CD4+ lymphocytes than the 1A12 cell line, and blocking the CD28/B7-1 interaction with the neutralizing anti-CD28 MAb inhibited the killing triggered by the 1A12-B7-1 cell line but not by the 1A12 cell line (Fig. 9A). When using MAb-coated beads as effectors with a concentration of anti-Fas MAb triggering minimal apoptosis, the coligation of CD28 also enhanced Fas-mediated cell death, whereas CD28 did not trigger cell death by itself (Fig. 9B). Fas/CD28 coligation via antibody-coated polystyrene beads efficiently increased the cleavage of caspase 3 and BID compared to the cleavage triggered by the anti-Fas MAb (Fig. 9C). Moreover, pretreatment of the CD4+ T lymphocytes with MßCD abrogated the enhancing effect of CD28 on Fas-induced cell death (Fig. 9D), as well as on caspase 3 cleavage (Fig. 9E). Therefore, as observed with Jurkat cell line, the Fas-mediated apoptosis in the primary activated T lymphocytes can be efficiently increased by microdomain recruitment.
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FIG. 9. CD28 corecruitment increases Fas-mediated apoptosis in primary activated CD4+ T lymphocytes from peripheral blood. (A) 51Cr-labeled activated CD4+ T lymphocytes were incubated with 1A12 or 1A12-B7-1 cells in the absence ( ) or in the presence ( ) of the blocking anti-CD28 MAb. (B) 51Cr-labeled activated CD4+ T lymphocytes were incubated with beads coated with the anti-Fas MAb (2 µg/ml) and either the negative IgG control MAb () or the anti-CD28 MAb ( ). As a control, beads were coated with the anti-CD28 MAb and the negative IgM control MAb ( ). (C) Primary activated CD4+ T lymphocytes were incubated for 2 h with beads coated with the anti-Fas MAb at 2 µg/ml and either the anti-CD28 MAb or an irrelevant IgG MAb. The cells were lysed, and total proteins (10 µg/lane) were separated by SDS-PAGE before immunoblotting for caspase 3 and BID. (D) Primary activated CD4+ T lymphocytes were treated for 20 min with 5 mM MßCD or -CD and incubated with either 1A12 effector cells or 1A12-B7-1 cells, and a 51Cr release assay was performed. (E) Primary activated CD4+ T lymphocytes were treated for 20 min with 5 mM MßCD or left untreated. After washing, the cells were incubated for 45 min with beads coated with the indicated antibodies and then lysed, and total proteins (10 µg/lane) were separated by SDS-PAGE before immunoblotting for caspase 3. For all of the 51Cr-release assays, results represent the means and errors of three independently repeated experiments.
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Upon CD28 engagement, its intracellular region recruits and activates the PI3-K signaling pathway, which is involved in the protection of T lymphocytes from Fas-mediated apoptosis (12). Although our conclusions seem at first glance contradictory to these findings, we actually do not challenge these results in the present study for the following two reasons. First, we focused on the effect on Fas stimulation of CD28-mediated microdomain recruitment and not on the consequences on Fas sensitivity of the costimulation of CD28 with TCR. Second, we demonstrated that CD28 amplifies the Fas-mediated cell death pathway independently of its intracellular region, which is known to be absolutely required for activating the PI3-K pathway (25). It is noteworthy that in the same way as for caspase 8, which plays a major role in both T-cell activation and T-cell elimination (5, 20), the costimulatory molecule CD28 could be used in the two opposite processes. Therefore, the T-lymphocyte activation process would require for its completion that pathways involved in the delayed elimination of the T cells are functional, which may decrease the risk of uncontrolled T-cell proliferation.
We demonstrate that the recruitment of microdomains can improve Fas-mediated apoptosis in so-called type II cells. As activated lymphocytes are described as type II cells, with Fas being excluded from microdomains (18), our findings suggest that target cells could use this mechanism to eliminate the surrounding T lymphocytes for evading the immune system, e.g., in the case of transformed or virus-infected cells. Indeed, it has been reported that Hodgkin's lymphoma cells express FasL (7) and also overexpress the CD28 ligands CD80 and CD86 (21). Similarly, cytomegalovirus-infected dendritic cells coexpress FasL and CD28 ligands. These antigen-presenting cells can eliminate activated T lymphocytes via a Fas-dependent mechanism, while these T cells were resistant to FasL alone. Therefore, the infected dendritic cells could play the role of trojan horses which, instead of stimulating T-cell activation via antigen presentation, can enhance T-cell deletion, allowing the cytomegalovirus to evade the immune response (19).
We recently demonstrated that a microdomain-concentrated GPI-linked chimeric Fas protein was able to amplify the Fas signaling pathway. Therefore, the costimulation of Fas-mediated apoptosis through microdomain recruitment may involve membrane coreceptors distinct from CD28. It has been reported that cells from squamous head-and-neck carcinoma, one of the most immunosuppressive human cancers, express high levels of membrane FasL. Strikingly, these cells selectively enhanced the mitochondrial pathway of apoptosis in Jurkat target cells compared to the stimulation of Fas alone (10). Although this mechanism remains unsolved to date, we suggest the possible involvement of a microdomain-recruiting coreceptor in this immune evasion phenomenon. More generally, we propose that the recruitment of the microdomains to Fas through nonapoptotic ligand/receptor pairs during cell-to-cell contacts represents an as-yet-unraveled general pathway for modulating Fas-induced cell death.
P.L. is supported by a grant from the Association pour la Recherche sur le Cancer. This work was supported by grants from the Association pour la Recherche sur le Cancer and from the Ligue Contre le Cancer des Landes, de la Charente, de la Dordogne, de la Gironde, des Pyrénées-Atlantiques.
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