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*
Department of Cell Biology, Institute of Biomembranes and Centre for Biomedical Genetics, University Medical Center, Utrecht University, Utrecht, The Netherlands;
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and
Mabtech AB, Nacka, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In B cells, MHC class II molecules predominantly reside in MHC class II compartments (MIICs) (10), which are the subcellular site of peptide loading onto MHC class II molecules (11). MIICs are part of the endosomal/lysosomal pathway in APC and often have a multivesicular phenotype (12, 13). The internal vesicles of multivesicular MIICs are probably formed by inward budding from the limiting membrane (14), a process that seems to require phosphatidylinositol 3-kinase activity (15). Multivesicular MIICs can fuse with the plasma membrane in an exocytic manner, thereby releasing their internal vesicles, which have been shown to carry MHC class II molecules at their surface and to be capable of specifically stimulating CD4-positive T cells in vitro. The released internal vesicles of MIICs have been called exosomes (16), a term that was introduced for exocytosed vesicles in reticulocytes (17). Exocytosis of MIICs or multivesicular bodies has also been found in other APC, such as dendritic cells (18, 19), tonsil B cells (M.J.K. and H.J.G., unpublished data), and other hemopoietic cells, such as mast cells (20) and platelets (21).
We have previously shown that the internal vesicles of MIICs and exosomes are enriched in members of the tetraspan protein superfamily, such as CD37, CD53, CD63, CD81, and CD82 (22). Tetraspan proteins have been shown to be involved in Ag presentation, T cell signaling, T cell activation, motility, and adhesion. They are able to form oligomeric complexes with other tetraspan proteins (23, 24, 25), HLA-DR (23, 26), HLA-DM and -DO (26) and interact with integrins (25, 27) and the T cell coreceptors CD4 and CD8 (28). By immunoelectron microscopy (IEM), tetraspan proteins are confined to the internal vesicles of MIICs and exosomes, whereas other MIIC markers, such as Lamp-1, Lamp-2, and HLA-DM, reside primarily in the MIIC-limiting membrane (22) and are absent from exosomes.
In this paper, we present an IEM analysis of the microvesicles attached to the FDC surface in situ and show that these vesicles represent exosomes, possibly derived from B cells. The specific accumulation of exosomes on FDCs might play a role in fine tuning of the immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EBV-transformed B cell line RN was maintained as described (16). Commercial mouse mAbs were anti-CD37 (S-B3; Sanofi, Montpellier, France), anti-CD107b/Lamp-2 (H4B4; PharMingen, San Diego, CA), anti-CD63/Lamp-3 (CLB-gran1/2, 435), anti-CD53 (MEM-53), and anti-LFA-1 (CLB-LFA-1/1, CD18) from CLB (Amsterdam, The Netherlands). The mAbs against CD81 (M38) and CD82 (C33) were a gift of Dr. O. Yoshie (Osaka, Japan); mAb 5C1 (HLA-DM) was obtained from Dr. J. Trowsdale (Cambridge, U.K.). The mAb 6C4 (against lysobisphosphatidic acid (LBPA)) was provided by Jean Gruenberg (Geneva, Switzerland). The rabbit polyclonal Abs used in this study were: anti-MHC class II from Dr. H. L. Ploegh (Massachusetts Institute of Technology, Cambridge, MA), anti-biotin (Rockland, Gilbertsville, PA), anti-CD35/CR1 (3D9) obtained from J. A. Schifferli (Basel, Switzerland) (29), polyclonal anti-Ii (ICN-1 and ICC5) from Dr. P. A. Morton (Monsanto Co., St. Louis, MO) (30), and anti-mouse IgG from Dako (Glostrup, Denmark).
Coisolation of FDCs with in vivo activated B lymphocytes
Fresh human tonsils were obtained from patients undergoing routine tonsillectomy. B cells were purified as previously described (31, 32). In short, T cells were removed by two cycles of rosetting with neuraminidase-treated sheep erythrocytes. The nonrosetting cells were further depleted of adherent cells by incubation in tissue culture flasks in HEPES-buffered RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 50% heat-inactivated FCS for 1 h at 37°C. The nonadherent cells were recovered and subjected to centrifugation through a discontinuous 65, 52.5, and 42.5% Percoll gradient (Amersham Pharmacia Biotech, Uppsala, Sweden). At the interface of 42.5%–52.5% Percoll, the low density fraction was harvested which contained in vivo activated B cells and several FDCs. Cells were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in phosphate buffer and processed for ultracryotomy as described below.
Isolation of FDC-B cell clusters
FDCs were isolated from tonsils as described by Parmentier et al. (33). Tonsils were cut into pieces and treated with a collagenase (200 U/ml collagenase IV, Worthington Biochemical, Lakewood, NJ)-DNase (10 U/ml DNase I, Boehringer Mannheim, Mannheim, Germany) solution in IMDM (Life Technologies), followed by density sedimentation on a cold discontinuous BSA gradient (Path-o-cyte 4, bovine albumin, Instruchemie, The Netherlands) in HBSS consisting of layers of 1.5, 2.5, and 5%. The cells at the 2.5%–5% interface were harvested and washed in HBSS and IMDM. After incubating the cells with a mAb against human LFA-1 for 30 min at 4°C, LFA-1 expressing B cells were depleted by sheep anti-mouse Ig-coated Dynabeads (Dynal, Oslo, Norway). The resulting FDC-enriched cell suspension was cultured for 12–14 h at 37°C in IMDM supplemented with 10% FCS. After culture, it contained FDC/B cell clusters and other cell types as small lymphocytes, plasma cells, macrophages, dendritic cells, erythrocytes, and exceptionally mast cells and basophils.
Isolation of exosomes
Exosomes were isolated by differential centrifugation as previously described (16). Briefly, RN cells were washed by centrifugation and recultured in fresh medium for 18 h. Cell culture media (35 ml) containing about 5 x 107 cells were centrifuged once for 10 min at 200 x g (pellet P1), twice for 10 min at 500 x g (pellet P2), twice for 15 min at 2,000 x g (pellet P3), once for 30 min at 10,000 x g (pellet P4), and finally once for 60 min at 70,000 x g (pellet P5), using a rotor SW27 (Beckman Instruments, Fullerton, CA). To remove the excess of serum proteins, P5 was washed once in 35 ml PBS and centrifuged again for 60 min at 70,000 x g. For in vitro binding studies with isolated human FDCs, this pellet was resuspended in 1 ml PBS and biotinylated by adding EZ-Link sulfo-NHS-biotin (Pierce, Rockford, IL) at a final concentration of 1 mg/ml for 30 min at room temperature. The reaction was blocked by resuspension in 34 ml PBS/15 mM ammonium chloride and sedimented for 60 min at 70,000 x g. The pellet was washed once in 35 ml PBS and resuspended in 100 µl PBS, 1% BSA, 5x penicillin/streptomycin. The resulting homogenous suspension of small vesicles contained only small amounts of plasma membrane and is referred to as biotinylated exosomes. Biotinylation efficiency was >95% as checked by whole mount immunoelectron microscopic analysis (22).
In vitro attachment of exosomes
Isolated FDC/B cell clusters were allowed to attach overnight in a 35-mm petri dish. After the cells were washed three times with 1 ml HBSS at 37°C, the removal of cells in suspension was checked microscopically and 900 µl HBSS, 10% FCS was added. Parallel plates were incubated for 1 h at 37°C with 100 µl PBS, 1% BSA, 5x penicillin/streptomycin with or without biotinylated exosomes. Cells were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in phosphate buffer and processed as described below.
Immunoelectron microscopy
Cells were processed for ultrathin cryosectioning and immunolabeling as described previously (34, 35, 36, 37). Briefly, cells were fixed in a mixture of 2% paraformaldehyde and 0.2% glutaraldehyde in phosphate buffer. After being washed with PBS-glycine and PBS, cell pellets were embedded in 10 or 12% gelatin, cut into small blocks, which were infiltrated with 2.3 M sucrose overnight at 4°C. Finally, the blocks were frozen in liquid nitrogen for cryosectioning. Ultrathin cryosections were picked up from the knife with a mixture of 2.3 M sucrose and 2% methylcellulose. The sections were indirectly immunolabeled with 10-nm protein A-gold particles in single labelings or 10- and 15-nm particles in double labelings. Finally, the ultrathin sections were embedded in a mixture of 2% methylcellulose and 0.4% uranyl.
Quantitative analysis
Ultrathin cryosections of cells from the in vitro binding experiment were immunolabeled by use of a polyclonal rabbit Ab against biotin and subsequently visualized by 10 nm protein A-gold. Electron micrographs were randomly taken from FDC-B cell clusters and various other cell types at magnifications of x10,000 and x5,000–10,000, respectively. The plasma membrane length of FDCs forming a cluster and solitary other cells was determined according to the method of Weibel (38). The number of biotin-positive vesicles attached to it was determined by counting vesicles with a distance of <40 nm from the plasma membrane. Finally, a Student t test was performed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FDCs coisolated with in vivo activated B lymphocytes were
morphologically characterized by the presence of long and branched
dendrites with desmosomes at contact sites between neighboring
dendrites. Immunogold labeling showed that FDCs were heavily labeled
for CD35 (complement receptor 1), whereas different Abs against other
FDC-specific markers such as the long isoform of CR2/CD21
(39) failed to react in IEM. Interestingly, numerous 40-
to 70-nm membrane vesicles were seen attached to the surface of FDCs
(Fig. 1
), but not those of B cells. These
microvesicles were different from the iccosomes described by Szakal et
al. (40), which are 
5 times larger. Microvesicles were
found on FDCs from several donors and with three different isolation
procedures.
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FDCs have been shown to exhibit MHC class II at their cell surface
(8) but are unable to synthesize it themselves
(5). Therefore cells must acquire MHC class II passively
from other sources (5), a process that has not yet been
clarified. As shown in Fig. 1
B, MHC class II molecules at
the FDC surface are confined to the microvesicles attached to their
plasma membrane. In accordance with the functional data of Gray et al.
(5), the FDC plasma membrane as well as the intracellular
compartments were devoid of MHC class II by IEM. These observations
suggest that the microvesicles represent MHC class II carrier vesicles
derived from other cells that provide the FDCs with exogenous MHC class
II at their surface.
The composition of microvesicles resembles that of exosomes
The size of the microvesicles, as well as their labeling for MHC
class II, prompted us to investigate whether these structures represent
exosomes. We have previously shown that B cells secrete 50-nm MHC
class II-expressing exosomes during exocytosis of multivesicular
endosomes or MIICs (16). B cell-derived exosomes are
enriched in members of the tetraspan family of proteins, expose MHC
class I and II at their surface, contain the costimulatory molecule
CD86, but are poor in the lysosomal membrane proteins Lamp-1 and
Lamp-2, as well as HLA-DM (22). Microvesicles attached to
FDCs exhibit a remarkably similar marker profile as characterized by
IEM (Table I). They contain MHC class I
and class II molecules. In addition, they are positive for the
tetraspan proteins CD37, CD53, CD63, CD81, and CD82 (Fig. 2), but are poor or negative for Lamp-1
and Lamp-2 and proteins associated with Ag presentation such as Ii and
HLA-DM. Importantly, the microvesicles contained lysobisphosphatidic
acid (Fig. 3A), a lipid of the
internal vesicles of multivesicular endosomes (Fig. 3B)
(41). Taken together, these marker patterns strongly
suggest that the microvesicles are exosomes secreted by APC and that
they adhere but not fuse with the plasma membrane of FDCs.
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Because FDC/B cell clusters isolated with an enzymatic
treatment (as described in Materials and Methods) did not
show many FDC-attached microvesicles, we decided to use these
FDC-enriched cell populations for an in vitro binding assay with
exogenous biotinylated exosomes derived from human B cells. Exosomes
were isolated as described before (16). Binding of
exosomes to the various cell types present in the FDC-enriched cell
population was expressed as the number of biotin-labeled exosomes per
unit plasma membrane length (Table II).
Biotin-positive microvesicles were found attached to the FDC surface
(Fig. 4). On average, the FDC plasma
membrane showed 39 attached biotin-positive vesicles per 100-µm
plasma membrane. This was in marked contrast to other cell types
present in the FDC preparation like lymphocytes, plasma cells,
macrophages, dendritic cells, erythrocytes, and occasional mast cells
and basophils. For these cells, which served as internal controls, the
number of biotinylated microvesicles attached to their surface did not
exceed 7 per 100-µm plasma membrane. We conclude that B cell-derived
exosomes exhibit a high preference to bind to FDCs.
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To exclude the possibility that binding of exosomes to FDCs is
merely an in vitro artifact, we analyzed FDCs in situ in germinal
centers of human tonsils by cryoimmunogold electron microscopy. In
their physiological state, FDCs form a follicular reticulum together
with B lymphocytes. A typical example of such a binucleated FDC
surrounded by several B lymphocytes is shown in Fig. 5A. We frequently observed
microvesicles labeled for the exosomal marker CD82 and MHC class II
attached to the plasma membrane of FDCs in situ (Fig. 5B).
This labeling pattern could be followed along the branching processes
of FDCs (not shown). These observations indicate that exosome binding
to the surface of FDCs is not due to experimental manipulations, but is
a physiological phenomenon.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FDCs have been reported to passively acquire MHC class II molecules
(5), and the authors suggested that shed material from
surrounding B cells (42) might bind to the FDC surface.
However, the morphological basis for this phenomenon was not further
investigated. We now show that FDCs bear MHC class II-containing
microvesicles with a size of 40–70 nm at their surface. These
microvesicles were different from FDC-derived iccosomes
(40). Iccosomes are pinched off from the dendritic
processes of FDCs during the early phase of a germinal center reaction
and therefore should contain the same proteins as the FDC plasma
membrane. This is not the case for the microvesicles. Furthermore, the
diameter of iccosomes is 0.25–0.4 µm, which is 5 times larger
than the microvesicles attached to FDCs described in this study. The
presence of MHC class II molecules at the cell surface of mouse FDCs
has been demonstrated before at the ultrastructural level using the
immunoperoxidase technique (43). This technique resulted
in a diffuse staining at the FDC surface and did not allow the
discrimination between peroxidase deposits on the FDC plasma membrane
and adjacent vesicles. On the basis of our findings that microvesicles
attached to the FDC surface contain MHC class II, we suggest that these
earlier data show staining of exosomes rather than surface staining of
FDCs. This interpretation is more consistent with bone marrow
transplantation experiments published by Gray et al. (5),
showing that FDCs passively acquire MHC class II molecules from the
donor.
B cell apoptosis is an abundant event in lymphoid follicles and may result in membrane fragments that in some way or another might bind to FDCs. However, the FDC attached microvesicles do not represent B cell remnants. They are of rather uniform size and are enriched in lysosomal markers and LBPA, a lipid highly enriched in internal membranes of multivesicular endosomes (41).
We have previously demonstrated that B cells secrete microvesicles
called exosomes (16) that are characterized by the
presence of MHC class II, the costimulatory molecule CD86, and several
tetraspan proteins (CD37, CD53, CD63, CD81, CD82) (22).
The protein composition of microvesicles attached to FDCs turned out to
be identical with that of B cell-derived exosomes (22)
(Table I). All tetraspan proteins present on exosomes were also found
on the microvesicles. Together, these data strongly suggest that the
microvesicles on FDCs represent exosomes. Because FDCs are in close
contact with B cells, these latter are likely candidates for exosome
delivery to FDCs. Although the molecular basis of this interaction has
not yet been elucidated, we found that exosomes bind specifically to
FDCs as shown in vitro (Table II). Tetraspan proteins are enriched on
exosomes and microvesicles attached to FDCs. Their precise function is
largely unknown, and it is tempting to speculate that they might play a
role in the attachment of microvesicles to FDCs. Tetraspan proteins are
able to form oligomeric complexes with other tetraspan proteins
(23, 24, 25), HLA-DR (23, 26) and to interact
with integrins (25, 27) and the T cell coreceptors CD4 and
CD8 (28). CD81 is part of the CD19/CD21/CD81-signaling
complex, which is proposed to lower the threshold for B cell responses
(44). CD81 acts as a costimulatory molecule, which is
important for the optimal stimulation of Ab production
(45). CD82 and CD63 have been reported to form complexes
with MHC class II, HLA-DM, and HLA-DO, which reside in MIICs,
suggesting an important role for these tetraspan proteins in peptide
loading of MHC class II (26). HLA-DM is restricted to the
limiting membrane of MIICs and is not present on the internal vesicles
of MIICs and their secreted equivalent, the exosomes (22, 26). The same holds true for the lysosomal membrane proteins
Lamp-1 and Lamp-2. How internal vesicles of MIICs are formed and
how proteins are sorted into or excluded from the vesicles is not
known, but recent data showed that multivesicular body morphogenesis
seems to require phosphatidylinositol 3-kinase activity
(15).
Exosomes have been discussed earlier to have a costimulatory function in vitro (16), and in previous studies we have shown that the MHC class II molecules on exosomes are in a SDS-stable configuration, implying that they are peptide loaded (16). Indeed, they can specifically stimulate CD4-positive T cells in vitro (16), but their physiological target has remained undetermined. We have shown that FDCs display microvesicles at their surface, which strongly resemble exosomes. What could be the function of exosomes attached to FDCs? Ready-to-use peptide-loaded MHC class II molecules on exosomes might play a role in Th cell recruitment into the apical light zone of the germinal center. In this microenvironment, the last check for Ag specificity of B cells during germinal center reaction takes place, namely, the isotype switching and differentiation into either plasma cells or memory B cells (4), which also involves T cell help. It has been demonstrated that CD4-positive memory T cells are present in the outer zone of germinal centers (46) and that coculture of these T cells with germinal center B cells rapidly induces a memory phenotype in B cells (47). However, it is unclear how Ag-specific T cells manage to find the selected candidate memory B lymphocytes in the three-dimensional structure of the germinal center. We hypothesize that exosomes, which display peptide-loaded MHC class II molecules, attach to the highly mobile processes of FDCs, which could provide guidance for Ag-specific T cells from the outer zone of the follicle into the apical light zone. These exosomes could be derived from B cells in the germinal center, but might also originate from DCs (18, 19). If exosomes on FDCs have the function to guide Ag-specific T cells for costimulation of B cell differentiation to the place of action, one should assume that this step must be tightly regulated to avoid constant stimulation. Clearance of exosomes and their half-life in vivo are therefore interesting subjects of further investigation. According to observations in our in vitro binding experiment, macrophages might play a role in removing exosomes, because we found biotin-labeled microvesicles throughout their endocytic system (not shown). Due to the fact that half-life determination in humans is impossible, further functional experiments are crucially dependent on the availability of a murine B cell line that generates sufficient exosomes. Future experiments in the mouse system will probably give more insight in the role of exosomes in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hans J. Geuze, Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht, AZU, Room G 02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
3 Abbreviations used in this paper: FDC, follicular dendritic cell; IEM, immunoelectron microscopy; LBPA, lysobisphosphatidic acid; MIIC, MHC class II-enriched compartment.
Received for publication July 26, 1999. Accepted for publication May 16, 2000.
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References |
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-granules. Blood 94:3191.
IIbß3 integrins in haemopoietic cell lines and modulates cell-cell adhesion. Biochem. J. 338:61.
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F. Andre, N. Chaput, N. E. C. Schartz, C. Flament, N. Aubert, J. Bernard, F. Lemonnier, G. Raposo, B. Escudier, D.-H. Hsu, et al. Exosomes as Potent Cell-Free Peptide-Based Vaccine. I. Dendritic Cell-Derived Exosomes Transfer Functional MHC Class I/Peptide Complexes to Dendritic Cells J. Immunol., February 15, 2004; 172(4): 2126 - 2136. [Abstract] [Full Text] [PDF]
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 G Van Niel, J Mallegol, C Bevilacqua, C Candalh, S Brugiere, E Tomaskovic-Crook, J K Heath, N Cerf-Bensussan, and M Heyman Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice Gut, December 1, 2003; 52(12): 1690 - 1697. [Abstract] [Full Text] [PDF]
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 C. Admyre, J. Grunewald, J. Thyberg, S. Gripenback, G. Tornling, A. Eklund, A. Scheynius, and S. Gabrielsson Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid Eur. Respir. J., October 1, 2003; 22(4): 578 - 583. [Abstract] [Full Text] [PDF]
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S. J. Gould, A. M. Booth, and J. E. K. Hildreth The Trojan exosome hypothesis PNAS, September 16, 2003; 100(19): 10592 - 10597. [Abstract] [Full Text] [PDF]
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 N. M. McKechnie, D. Copland, and G. Braun Hr44 Secreted with Exosomes: Loss from Ciliary Epithelium in Response to Inflammation Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2650 - 2656. [Abstract] [Full Text] [PDF]
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 R. Wubbolts, R. S. Leckie, P. T. M. Veenhuizen, G. Schwarzmann, W. Mobius, J. Hoernschemeyer, J.-W. Slot, H. J. Geuze, and W. Stoorvogel Proteomic and Biochemical Analyses of Human B Cell-derived Exosomes. POTENTIAL IMPLICATIONS FOR THEIR FUNCTION AND MULTIVESICULAR BODY FORMATION J. Biol. Chem., March 21, 2003; 278(13): 10963 - 10972. [Abstract] [Full Text] [PDF]
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D. Skokos, H. G. Botros, C. Demeure, J. Morin, R. Peronet, G. Birkenmeier, S. Boudaly, and S. Mecheri Mast Cell-Derived Exosomes Induce Phenotypic and Functional Maturation of Dendritic Cells and Elicit Specific Immune Responses In Vivo J. Immunol., March 15, 2003; 170(6): 3037 - 3045. [Abstract] [Full Text] [PDF]
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B. Fritzsching, B. Schwer, J. Kartenbeck, A. Pedal, V. Horejsi, and M. Ott Release and Intercellular Transfer of Cell Surface CD81 Via Microparticles J. Immunol., November 15, 2002; 169(10): 5531 - 5537. [Abstract] [Full Text] [PDF]
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 G. van Niel and M. Heyman The Epithelial Cell Cytoskeleton and Intracellular Trafficking: II. Intestinal epithelial cell exosomes: perspectives on their structure and function Am J Physiol Gastrointest Liver Physiol, August 1, 2002; 283(2): G251 - G255. [Abstract] [Full Text] [PDF]
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H. Vincent-Schneider, P. Stumptner-Cuvelette, D. Lankar, S. Pain, G. Raposo, P. Benaroch, and C. Bonnerot Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells Int. Immunol., July 1, 2002; 14(7): 713 - 722. [Abstract] [Full Text] [PDF]
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N. Blanchard, D. Lankar, F. Faure, A. Regnault, C. Dumont, G. Raposo, and C. Hivroz TCR Activation of Human T Cells Induces the Production of Exosomes Bearing the TCR/CD3/{zeta} Complex J. Immunol., April 1, 2002; 168(7): 3235 - 3241. [Abstract] [Full Text] [PDF]
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 J. Colino, Y. Shen, and C. M. Snapper Dendritic Cells Pulsed with Intact Streptococcus pneumoniae Elicit both Protein- and Polysaccharide-specific Immunoglobulin Isotype Responses In Vivo through Distinct Mechanisms J. Exp. Med., December 31, 2001; 195(1): 1 - 14. [Abstract] [Full Text] [PDF]
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C. Thery, M. Boussac, P. Veron, P. Ricciardi-Castagnoli, G. Raposo, J. Garin, and S. Amigorena Proteomic Analysis of Dendritic Cell-Derived Exosomes: A Secreted Subcellular Compartment Distinct from Apoptotic Vesicles J. Immunol., June 15, 2001; 166(12): 7309 - 7318. [Abstract] [Full Text] [PDF]
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D. Skokos, S. Le Panse, I. Villa, J.-C. Rousselle, R. Peronet, B. David, A. Namane, and S. Mecheri Mast Cell-Dependent B and T Lymphocyte Activation Is Mediated by the Secretion of Immunologically Active Exosomes J. Immunol., January 15, 2001; 166(2): 868 - 876. [Abstract] [Full Text] [PDF]
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 K Denzer, M. Kleijmeer, H. Heijnen, W Stoorvogel, and H. Geuze Exosome: from internal vesicle of the multivesicular body to intercellular signaling device J. Cell Sci., January 10, 2000; 113(19): 3365 - 3374. [Abstract] [PDF]
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