1 The Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Queensland 4006, Australia
2 Department of Pathology, VU Medical Center, Free University, Amsterdam, The Netherlands
3 Department of Pathology, University of Queensland, St Lucia, Brisbane, Queensland 6067, Australia
Correspondence
James Flanagan
jamesF{at}qimr.edu.au
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ABSTRACT |
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INTRODUCTION |
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LMP 1 protein has been shown to associate with the cytoskeleton and aggregates to form patches on the cell membrane, a function that, like other cellular receptors, is necessary for its activation of NF-B (Gires et al., 1997
; Mann et al., 1985
). The domains involved in forming these aggregated patches have been defined as the 25 aa cytoplasmic amino terminus and the first two membrane-spanning domains (Bloss et al., 1999
). A significant proportion of LMP 1 is detergent insoluble, and as such, was originally defined as cytoskeleton associated (Liebowitz et al., 1986
). More specifically, it binds to the cytoskeleton protein vimentin and can up-regulate expression of this protein (Liebowitz & Kieff, 1989
; Liebowitz et al., 1987
). Glycosphingolipid-rich domains or lipid rafts are discrete domains within the plasma membrane that are detergent insoluble and provide a clustering point for signal transduction proteins such as G-proteins as well as LMP 1 and its interacting protein, TRAF3 (Ardila-Osorio et al., 1999
; Clausse et al., 1997
). One study has proposed that LMP 1 may also accumulate in the lysosomal compartment (Laszlo et al., 1991
). Lysosomes are thought to form by fusion of pinocytotic vesicles to yield endosomes which, in turn, transform first into multivesicular bodies (MVB) and then into mature lysosomes (Morales et al., 1999
). Haematopoietic cells have been shown to use lysosomal compartments to store and release their secretory products. The direct fusion of lysosome-related compartments with the plasma membrane in haematopoietic cells has been associated with the release of exosomes (Andrews, 2000
).
Non-structured intracellular aggregation of LMP 1 in a fraction of lymphoblastoid cells of different origin was noted by others (Boos et al., 1987; Rowe et al., 1987
) and was suggested to relate to the cell cycle stage of these cells, but this was not explored any further up till now. LMP 1 expression levels vary considerably among different EBV-carrying cell lines (Meij et al., 2000
). Recently, LMP1 was shown to be released by lymphoblastoid cells into the extracellular space, possibly associated with exosomes (Dukers et al., 2000
). Exosomes are internal vesicles of MVBs formed by inward budding of the vesicular membrane creating a membrane-enclosed compartment containing cytoplasm. Exocytosis of MVBs and the release of 40100 nm exosomes has been described for reticulocytes, B- and T-lymphocytes, mast cells, platelets and macrophages (reviewed in Denzer et al., 2000
).
Exosomes derived from antigen-presenting cells contain MHC class I and II (antigen-presenting proteins), CD86 (co-stimulatory protein) and ICAM-1 (adhesion molecule), as well as various tetraspan proteins such as CD9, CD37, CD53, CD63, CD81 and CD82, which are all involved in signal transduction, adhesion and complex formation with MHC. Furthermore, peptide-loaded MHC II-carrying exosomes can stimulate CD4+ T-lymphocyte proliferation, dendritic cell-derived MHC I-carrying exosomes can activate specific CD8+ CTL responses and mast cell-derived exosomes can stimulate both B- and T-lymphocyte proliferation (Raposo et al., 1996; Skokos et al., 2001
; Zitvogel et al., 1998
). This suggests an important role for exosomes in the immune system for antigen presentation and B- and T-lymphocyte activation. One previous report has suggested that LMP 1 may be secreted in exosomes and that an immunosuppressive domain within the conserved first transmembrane helix might influence T-cell activation and proliferation leading to T-cell anergy (Dukers et al., 2000
). In this study we have used immunofluorescence and immunoelectron microscopy to demonstrate that LMP 1 localizes to an intracellular compartment and that LMP 1 can be found in extracellular exosomes. We propose that LMP 1-containing exosomes may be involved in suppression of the immune response against LMP 1-expressing EBV-associated tumours.
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METHODS |
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Antibodies.
The LMP 1 antibodies used in this study were: mouse monoclonal antibody OT21C, specific for aa 290318; mouse monoclonal antibody OT22CN, specific for aa 113; rabbit polyclonal antisera 96-121, specific for the carboxy terminus of LMP 1 aa 186386; the rabbit polyclonal antiserum 97-48, specific for two epitopes in the carboxy terminus of LMP 1 aa 365386; and the commercially available CS1-4 antibodies, which contain a cocktail of four monoclonal antibodies specific for the carboxy terminus of LMP 1 aa 186386 (Dako). Other antibodies used in this study included rabbit polyclonal antisera GS15 (Xu et al., 1997), raised against a Golgi-specific protein (a kind gift from Professor David James, University of Queensland, Australia) and the mouse monoclonal anti-HLA DR antibody (kind gift from Rajiv Khanna, Queensland Institute of Medical Research, Australia). Secondary antibodies used were HRP-conjugated anti-mouse, FITC-conjugated anti-rabbit and PE-conjugated anti-mouse (Silenus). 10 nm gold-labelled Protein A (PAG; a gift from Jan Slot, Utrecht, The Netherlands) and 15 nm gold-labelled anti-mouse IgG (British Biocell International) were used for immunogold labelling.
Colorimetric (MTT) proliferation assay.
For the T-lymphocyte proliferation assay, PBMCs were cultured in a 96-well round-bottom culture plate at a concentration of 5x104 cells per well in RPMI 1640 medium supplemented with 10 % FCS. The mitogen phytohaemagglutinin (PHA) was added to a final concentration of 20 µg ml-1. Cells were cultured for 3 days at 37 °C in a 5 % CO2 atmosphere (volume 200 µl per well). MTT was added to a final concentration of 1 mg ml-1 and the cells were cultured for a further 4 h while formazan crystals formed. The crystals were resuspended in DMSO and the absorbance was read at 560 nm with a Tecan Spectra ELISA plate reader using Biolise software. The percentage proliferation was calculated by the following formula: [TEST(Abs. 560 nm)/PHA positive control(Abs. 560 nm)] x100.
DNA cell cycle analysis.
Cell cycle profiles were obtained by washing cells once in PBS and then resuspending the cells in a nuclear DNA stain (50 µg propidium iodide ml-1, 1 mg RNase A ml-1 and 0·02 % Triton X-100 in PBS). Cells were analysed on a FACSCalibur (Becton Dickson) using CellQuest and Modfit data analysis software. For cell cycle analyses the cell lines QIMR NB-B95-8 LCL and DG75 were incubated for 48 h with drugs that induce cell cycle checkpoints. The drugs used were Amphidicolin (5 µg ml-1; Calbiochem), Mimosine (100 µM; Sigma), Colcemid (100 ng ml-1; Gibco-BRL), Nocodazole (0·5 µg ml-1; Calbiochem) and ICRF (0·5 µg ml-1; Sigma). Cells were harvested following drug treatment and were analysed by flow cytometry for cell cycle profiles and by immunofluorescence for LMP 1 localization.
Immunofluorescence assay and confocal microscopy.
Cells were washed in PBS and spotted onto a 12-well multitest slide (ICN Biomedicals); they were then air-dried, fixed in 100 % acetone and blocked for 30 min in 20 % FCS in PBS at room temperature. They were then incubated with primary antibody (CS1-4, diluted 1 : 5 in 20 % FCS/PBS) for 1 h at room temperature. Slides were then washed three times in PBS and incubated with PE-conjugated anti-mouse antibody (diluted 1 : 160 in 20 % FCS/PBS). Co-localization of LMP with the Golgi protein GS15 was examined using immunofluorescence, as above, except slides were sequentially incubated with antibodies: firstly with CS1-4 anti-LMP (1 : 50), then PE-conjugated anti-mouse (1 : 160), then anti-GS15 antibody (1 : 30), and finally FITC anti-rabbit (1 : 100), with three washes in PBS after each incubation. The slides were then mounted in Vectashield mounting medium and were scanned using a Bio-Rad MRC 600 confocal microscope. Images were acquired using COMOS (Confocal Microscope Operating Software V6.03) and were analysed using CAS (Confocal Assistant Software V3.10).
Immunoelectron microscopy.
LMP 1 was detected by electron microscopy using the following method. Cells were washed in PBS, fixed in 4 % paraformaldehyde/PBS for 30 min, and then further washed with PBS. The cells were enrobed in 10 % gelatin, cooled to 4 °C and cut into small gelatin blocks that were infiltrated with polyvinylpyrrolidone/sucrose overnight at 4 °C. The cells were mounted on copper pins and frozen in liquid nitrogen. Cyrosectioning was done with a Leica Ultracut S ultramicrotome fitted with the Leica FCS cryosectioning system. Ultrathin sections were collected using 1·3 M sucrose and attached to Formvar-coated copper grids. For immunolabelling, the grids were blocked for 5 min in blocking buffer (20 mM glycine, 0·2 % fish skin gelatin, 0·02 % BSA in PBS), incubated for 30 min with rabbit polyclonal anti-LMP1 96-121 antiserum diluted 1 : 20 in blocking buffer, and then washed four times in blocking buffer. The grids were incubated for 30 min with 10 nm PAG diluted 1 : 85 and were washed four times in blocking buffer. For double labelling with MHC class II the grids were sequentially incubated with antibodies: firstly with mouse anti-HLA DR (1 : 20), then 15 nm-gold-labelled anti-mouse IgG, then rabbit anti-LMP 96-121 antiserum (1 : 20) and finally 10 nm PAG (1 : 85). Each grid was fixed in 1 % glutaraldehyde in PBS and then washed twice in distilled water and negatively stained for 5 min with ice-cold 0·4 % uranyl acetate in 1·8 % methylcellulose. The grids were looped and reduced to a thin film of methylcellulose and air dried for 10 min. The sections were examined under a JEOL 1010 transmission electron microscope. Images were captured on Kodak SO-163 film and digitized using a Leafscan 45 scanner.
Isolation of exosomes.
Approximately 5x107 DG75 and QIMR NB-B95-8 cells were washed in RPMI 1640 supplemented with 10 % FCS and were re-cultured for 48 h. Exosomes were then purified from 35 ml of the cell culture medium using differential centrifugation (Raposo et al., 1996). Firstly, the cells were removed by centrifugation at 300 g for 10 min. The supernatant was then sequentially centrifuged at 300 g for 10 min, 500 g for 10 min (twice), 2000 g for 30 min (twice), 10 000 g for 30 min and finally 70 000 g for 60 min. For immunoelectron microscopy the 70 000 g pellet (containing the exosomes) was resuspended in 2050 µl of growth medium and then spotted onto Formvar-coated copper grids and processed for immunolabelling as described above. For the T-lymphocyte proliferation assay exosomes were resuspended in 180 µl of growth medium.
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RESULTS |
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Intracellular LMP 1 levels are not cell cycle dependent
Since only 30 % of cells contained intracellular LMP 1 we investigated the possibility that the intracellular LMP 1 may be produced in a cell cycle-dependent manner. The LCL QIMR NB-B95-8 was cultured for 48 h in the presence of the drugs Amphidicolin or Mimosine, which block the cells in the G1 phase of the cell cycle, ICRF or Nocodazole, which block cells in the G2 phase, or Colcemid, which blocks cells in the M phase of the cell cycle. Following drug treatment the cells were harvested, dried onto slides and LMP 1 expression was detected using immunofluorescence and confocal microscopy. After treatment with each of the drugs the percentage of cells containing an intracellular portion of LMP 1 was counted over four to six fields, and remained at between 1939 % following each of the treatments (Table 1). The cell cycle profile demonstrated that the drugs blocked the cells at the appropriate stage of the cell cycle (values in bold type in Table 1
) and this treatment did not significantly affect the percentage of cells containing intracellular LMP 1. These results suggest that the intracellular localization of LMP 1 was not cell cycle dependent.
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Exosomes derived from an LCL inhibit proliferation of T cells
Previous work from Dukers et al. (2000) has shown that peptides derived from LMP 1 were capable of inhibiting the proliferation of T cells. Since we have shown that LMP 1 is present on exosomes the possibility that LMP 1 containing exosomes might be able to inhibit T cells was evaluated. To assess this an in vitro T cell proliferation assay was performed in the presence of exosomes derived from an LCL. PBMCs were isolated from three healthy donors and were seeded at 5x104 cells per well in triplicate and proliferation was stimulated with the mitogen PHA. Cells were treated with or without PHA as controls while test cells were incubated with exosomes derived from either the EBV-negative cell line DG75 or the EBV-positive LCL QIMR NB-B95-8. Cells were incubated for 3 days and proliferation was assessed using the MTT assay. The results presented in Fig. 5(C)
are an average of three experiments. The proliferation of the cells treated with DG75 exosomes was reduced somewhat (80 % of that observed with the positive control) whereas cells incubated with QIMR NB-B95-8-derived exosomes showed a significant reduction in proliferation (40 % of that observed with the positive control). These results show a significant reduction in the T-lymphocyte proliferation when cultured in the presence of LCL-derived exosomes compared to the DG75 derived exosomes (P=0·00046) and indicate that LMP 1-containing exosomes may be capable of reducing T cell proliferation.
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DISCUSSION |
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Immunoelectron microscopy was used to examine the ultrastructure of the regions that contained intracellular LMP 1 foci. The new LMP 1 rabbit polyclonal antibodies are an excellent tool for analysis of LMP 1 expression by immunoelectron microscopy. The only previous studies of LMP 1 by electron microscopy used monoclonal antibodies that were specific for a single epitope and employed fixed Araldite-embedded cells, which dramatically decreases the antigenicity of epitopes available for antibody binding (Laszlo et al., 1991). Using rabbit polyclonal LMP 1 antibodies and cryosectioning we have been able to detect far more LMP 1 in each cell, which has allowed a more detailed analysis of the intracellular staining pattern. Cryosections from the QIMR NB-B95-8 LCL immunolabelled with the rabbit polyclonal LMP 1 antibody revealed that in approximately two-thirds (9/15) of the cells LMP 1 localized to an intracellular compartment. The higher percentage of cells with intracellular LMP 1 detected using immunoelectron microscopy than was observed by immunofluorescence was likely due to the increased sensitivity of immunoelectron microscopy.
One possibility for intracellular localization of LMP 1 is as a reservoir. The intracellular LMP 1 aggregate resembles an intracellular reservoir previously described for the MHC co-stimulatory molecules CD80 and CD86 (Smyth et al., 1998). As LMP 1 mimics a constitutively active signalling molecule a reservoir of LMP 1 might allow it to be shuttled to the cell surface membrane when LMP 1 signalling is required in high amounts and then transported back to the reservoir when not required. Overexpression of LMP 1 in the DG75-EBO-EBNA 3,4,6 cell line resulted in an apparent increase in the percentage of cells containing intracellular LMP 1, supporting the possibility of the intracellular LMP 1 being a reservoir. Alternative to acting as a storage reservoir, LMP1 aggregates may represent ubiquinated clusters of proteins awaiting proteasomal digestion (Dantuma & Massucci, 2002
; Lelouard et al., 2002
) or just a clustering of overproduced improperly folded protein aggregates, collectively referred to as aggresomes (Kopito, 2000
)
The intracellular LMP 1 foci also closely resemble, in size and structure, the intracellular localization of MHC class I and class II in the endocytic pathway (Benaroch et al., 1995; Chiu et al., 1999
). MHC class II localizes to a specialized multivesicular body known as MHC class II compartments (MIICs). MIICs closely resemble lysosomes in that they contain lysosomal markers (LAMP1, CD63,
-hexosaminidase and cathepsin D), are positioned in the late endocytic pathway and are acidic (Sanderson et al., 1994
). Previous data that suggested that LMP 1 may localize to lysosomes could in fact have detected MIICs. It is from these MIICs that MHC II-loaded exosomes are produced and on fusion of the MIIC with the plasma membrane these exosomes are released (Raposo et al., 1996
). Thus there is the possibility that the intracellular LMP 1 pool may traffic together with the MHC molecules to these MIICs to be packaged and secreted in exosomes. Analysis of MHC II expression in the LCL showed co-localization with LMP 1 in 3/10 LMP 1 foci, which suggested that some of the intracellular LMP 1 localized to MIICs. The fact that not all LMP 1 foci co-localized with MHC class II could be due to either differences in the efficiency of binding of the monoclonal HLA DR antibody compared to the rabbit polyclonal LMP 1 antibody or differences in expression levels of LMP 1 and MHC class II.
LMP 1 has been shown to activate the Rho GTPase cdc42 (Puls et al., 1999). While this function has been suggested to be involved in the cytostatic effect of LMP 1 it may also be involved in the secretion of exosomes, as cdc42 has been shown to regulate the generation of different populations of transport vesicles from the trans-Golgi network (Musch et al., 2001
). Thus, LMP 1 may activate cdc42 resulting in upregulation of the production of vesicles allowing LMP 1 to be exported.
In addition, LMP 1 was found to be associated with extracellular vesicles in cryosections and to be present in preparations of purified exosomes. Utilizing the serial dissection function of the confocal microscope, we demonstrated that a large proportion of LMP 1 localized to an intracellular membrane-bound compartment that extends through the cytoplasm and partially co-localizes with the Golgi network. Previous work utilizing Western blot analysis of differentially centrifuged cell supernatant suggested that LMP 1 may be associated with exosomes (Dukers et al., 2000). Electron microscopy data showed the presence of extracellular LMP 1-positive vesicles attached to the cell surface in cryosections of an EBV-positive LCL, and LMP 1 was found to be present in exosomes isolated from the culture fluid of an LCL.
Exosomes derived from antigen-presenting cells have been shown to stimulate the proliferation of both T- and B-lymphocytes while peptides derived from LMP 1 have been shown to inhibit the proliferation of T-lymphocytes (Dukers et al., 2000). EBV-positive cells may release exosomes, containing LMP 1, which could then inhibit the proliferation of T-lymphocytes in the surrounding microenvironment. Indeed exosomes, which were shown by electron microscopy to contain LMP 1, were isolated from the LCL QIMR NB-B95-8 and were capable of inhibiting the mitogen-stimulated proliferation of the PBMCs from three donors by 40 % compared to exosomes derived from the DG75 cell line.
EBV-infected cells need to evade the immune system and remain viable for latent persistence or lytic replication. The data presented indicate that LMP 1 may be excreted from cells in exosomes and these exosomes could be involved in suppressing the immune system to allow the virus to survive. This might be relevant in the case of EBV-positive Hodgkin's disease and nasopharyngeal carcinoma where tumour cells express LMP 1 and while LMP 1 encodes CTL epitopes these tumour cells are not killed by the infiltrating T-lymphocytes (Oudejans et al., 1997, 2002
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ardila-Osorio, H., Clausse, B., Mishal, Z., Wiels, J., Tursz, T. & Busson, P. (1999). Evidence of LMP1TRAF3 interactions in glycosphingolipid-rich complexes of lymphoblastoid and nasopharyngeal carcinoma cells. Int J Cancer 81, 645649.[CrossRef][Medline]
Baichwal, V. R. & Sugden, B. (1987). Posttranslational processing of an EpsteinBarr virus-encoded membrane protein expressed in cells transformed by EpsteinBarr virus. J Virol 61, 866875.[Medline]
Benaroch, P., Yilla, M., Raposo, G., Ito, K., Miwa, K., Geuze, H. J. & Ploegh, H. L. (1995). How MHC class II molecules reach the endocytic pathway. EMBO J 14, 3749.[Abstract]
Bloss, T., Kaykas, A. & Sugden, B. (1999). Dissociation of patching by latent membrane protein-1 of EpsteinBarr virus from its stimulation of NF-B activity. J Gen Virol 80, 32273232.
Boos, H., Berger, R., Kuklik-Roos, C., Iftner, T. & Mueller-Lantzsch, N. (1987). Enhancement of EpsteinBarr virus membrane protein (LMP) expression by serum, TPA, or n-butyrate in latently infected Raji cells. Virology 159, 161165.[CrossRef][Medline]
Chiu, I., Davis, D. M. & Strominger, J. L. (1999). Trafficking of spontaneously endocytosed MHC proteins. Proc Natl Acad Sci U S A 96, 1394413949.
Clausse, B., Fizazi, K., Walczak, V., Tetaud, C., Wiels, J., Tursz, T. & Busson, P. (1997). High concentration of the EBV latent membrane protein 1 in glycosphingolipid-rich complexes from both epithelial and lymphoid cells. Virology 228, 285293.[CrossRef][Medline]
Dantuma, N. & Massucci, M. (2003). The ubiquitin/proteasome system in EpsteinBarr virus infection and related malignancies. Semin Cancer Biol (in press).
Denzer, K., Kleijmeer, M. J., Heijnen, H. F., Stoorvogel, W. & Geuze, H. J. (2000). Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J Cell Sci 113, 33653374.
Dukers, D. F., Meij, P., Vervoort, M. B., Vos, W., Scheper, R. J., Meijer, C. J., Bloemena, E. & Middeldorp, J. M. (2000). Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. J Immunol 165, 663670.
Gires, O., Zimber-Strobl, U., Gonnella, R., Ueffing, M., Marschall, G., Zeidler, R., Pich, D. & Hammerschmidt, W. (1997). Latent membrane protein 1 of EpsteinBarr virus mimics a constitutively active receptor molecule. EMBO J 16, 61316140.
Hudson, G. S., Bankier, A. T., Satchwell, S. C. & Barrell, B. G. (1985). The short unique region of the B95-8 EpsteinBarr virus genome. Virology 147, 8198.[CrossRef][Medline]
Kopito, R. R. (2000). Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10, 524530.[CrossRef][Medline]
Laszlo, L., Tuckwell, J., Self, T., Lowe, J., Landon, M., Smith, S., Hawthorne, J. N. & Mayer, R. J. (1991). The latent membrane protein-1 in EpsteinBarr virus-transformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre. J Pathol 164, 203214.[Medline]
Lelouard, H., Gatti, E., Cappello, F., Gresser, O., Camosseto, V. & Pierre, P. (2002). Transient aggregation of ubiquitinated proteins during dendritic cell maturation. Nature 417, 177182.[Medline]
Liebowitz, D. & Kieff, E. (1989). EpsteinBarr virus latent membrane protein: induction of B-cell activation antigens and membrane patch formation does not require vimentin. J Virol 63, 40514054.[Medline]
Liebowitz, D., Wang, D. & Kieff, E. (1986). Orientation and patching of the latent infection membrane protein encoded by EpsteinBarr virus. J Virol 58, 233237.[Medline]
Liebowitz, D., Kopan, R., Fuchs, E., Sample, J. & Kieff, E. (1987). An EpsteinBarr virus transforming protein associates with vimentin in lymphocytes. Mol Cell Biol 7, 22992308.[Medline]
Mann, K. P., Staunton, D. & Thorley-Lawson, D. A. (1985). EpsteinBarr virus-encoded protein found in plasma membranes of transformed cells. J Virol 55, 710720.[Medline]
Meij, P., Vervoort, M. B. H. J., Meijer, C. J. L. M., Bloemena, E. & Middeldorp, J. M. (2000). Production monitoring and purification of EBV encoded latent membrane protein 1 expressed and secreted by recombinant baculovirus infected insect cells. J Virol Methods 90, 193204.[CrossRef][Medline]
Morales, C. R., Zhao, Q. & Lefrancois, S. (1999). Biogenesis of lysosomes by endocytic flow of plasma membrane. Biocell 23, 149160.[Medline]
Musch, A., Cohen, D., Kreitzer, G. & Rodriguez-Boulan, E. (2001). cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network. EMBO J 20, 21712179.
Oudejans, J. J., Jiwa, N. M., Kummer, J. A. & 7 other authors (1997). In situ detection of activated cytotoxic cells in Hodgkin's disease biopsies: recognition of cases with poor clinical outcome. Blood 89, 13761382.
Oudejans, J. J., Harijadi, A., Kummer, J. A. & 7 other authors (2003). High numbers of granzyme B/CD8 positive tumour infiltrating lymphocytes in nasopharyngeal carcinoma biopsies predict rapid fatal outcome in patients treated with curative intent. J Pathol (in press).
Puls, A., Eliopoulos, A. G., Nobes, C. D., Bridges, T., Young, L. S. & Hall, A. (1999). Activation of the small GTPase Cdc42 by the inflammatory cytokines TNF(alpha) and IL-1, and by the EpsteinBarr virus transforming protein LMP1. J Cell Sci 112, 29832992.
Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding, C. V., Melief, C. J. & Geuze, H. J. (1996). B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183, 11611172.[Abstract]
Rowe, M., Evans, H. S., Young, L. S., Hennessy, K., Kieff, E. & Rickinson, A. B. (1987). Monoclonal antibodies to the latent membrane protein of EpsteinBarr virus reveal heterogeneity of the protein and inducible expression in virus-transformed cells. J Gen Virol 68, 15751586.[Abstract]
Sanderson, F., Kleijmeer, M. J., Kelly, A., Verwoerd, D., Tulp, A., Neefjes, J. J., Geuze, H. J. & Trowsdale, J. (1994). Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments. Science 266, 15661569.[Medline]
Skokos, D., Le-Panse, S., Villa, I., Rousselle, J. C., Peronet, R., David, B., Namane, A. & Mecheri, S. (2001). Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes. J Immunol 166, 868876.
Smyth, C., Logan, G., Weinberger, R. P., Rowe, P. B., Alexander, I. E. & Smythe, J. A. (1998). Identification of a dynamic intracellular reservoir of CD86 protein in peripheral blood monocytes that is not associated with the Golgi complex. J Immunol 160, 53905396.
Wang, D., Liebowitz, D. & Kieff, E. (1985). An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43, 831840.[Medline]
Wang, D., Liebowitz, D., Wang, F., Gregory, C., Rickinson, A., Larson, R., Springer, T. & Kieff, E. (1988). EpsteinBarr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity. J Virol 62, 41734184.[Medline]
Xu, Y., Wong, S. H., Zhang, T., Subramaniam, V. N. & Hong, W. (1997). GS15, a 15-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) homologous to rbet1. J Biol Chem 272, 2016220166.
Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D., Ricciardi-Castagnoli, P., Raposo, G. & Amigorena, S. (1998). Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 4, 594600.[Medline]
Received 4 November 2002;
accepted 21 February 2003.