Complement fixation diluent
B lymphoblastoid cell line
Exosomes are secreted nanometer-sized vesicles derived from antigen-presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell-free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen-presenting cell-derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)-anchored regulators CD55 and CD59,but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI-anchored complement regulators in order to permit their survival in the extracellular environment.
Exosomes are nanometer-sized vesicles, formed within MHC class II-enriched endosomal compartments of antigen-presenting cells (APC) through inward budding of the limiting membrane, forming multivesicular bodies (MVB). The subsequent fusion of MVB with the plasma membrane transports MHC class II molecules with newly acquired antigenic peptides to the cell surface and also results in the release of exosomes into the extracellular space 1–3.
Exosome-like vesicles have been described in a variety of cells including reticulocytes 4, platelets 5, T cells 6, 7, intestinal epithelia 8, 9 B lymphocytes 10, dendritic cells 11, and some tumor cells 12. Although likely to have a diverse range of functions related to their cell of origin, there is mounting evidence for an important role for exosomes in the regulation of immunological responses. For example, exosomes of APC were demonstrated to be effective in stimulating antigen-specific CD4+10 and CD8+11 T cell responses, both in vitro and inanimal models. Exosomes derived from other cells, such as intestinal epithelia, may also be involved in regulating immunological responses, possibly by inducing tolerance 8. Although the mechanism by which exosomes regulate immunity requires clarification, it is likely that this involves cross-priming T cell responses via dendritic cells 12. Exosomes have therefore been proposed as a basis for cell-free immunotherapeutic vaccines for cancer.
Structurally, APC-derived exosomes are 40–90 nm diameter vesicles composed of a phospholipid/cholesterol membrane. Numerous proteins, including MHC class II, MHC class I and costimulatory molecules 10, 11, tetraspanin family members such as CD81 13, and a host of others 14, 15, have been identifiedin the exosome membrane. The exosome lumen is less well characterized, but may contain annexins, heat shock proteins, viral proteins and esterases 15, 16, cytoskeletal proteins, and proteins implicated in intracellular transportation, signaling, and apoptosis 17. Exosomes are structurally and compositionally distinct from microvesicles released from apoptotic cells 17 and other vesicles released from budding of the plasma membrane 5. They represent a discrete population of secreted vesicles whose physiological roles are likely to be determined by factors such as their molecular composition, their rate and site of secretion, and the nature of the extracellular milieu into which they are shed.
The extracellular environment is naturally hostile towards vesicular structures, due principally to the propensity of membrane vesicles to activate the complement system, resulting in their rapid opsonization or lysis 18, 19. Complement activation is well described for man-made liposomes, utilized therapeutically as drug-transportation vehicles 20–23. Although these simple lipid membranes may not be directly antigenic, complement may be activated in an antibody-independent manner through electrostatic interactions, for example, between C1q and the liposomal membrane 24, and the degree of activation is dependent on lipid composition 25, 26 and vesicle size 27, 28. Exosomes, derived from antigen-processing compartments, are likely to be associated with antigenic proteins, and may be particularly prone to binding serum immunoglobulins and activating complement.
It is well established that cells protect themselves from homologous complement lysis through the activity of membrane-bound molecules capable of inhibiting the complement pathway at several points. The principal molecules include CD46 (membrane cofactor protein) and CD55 (decay accelerating factor), which together regulate the C3 and C5 convertases 29, 30, and CD59, which inhibits the terminal pathway of complement preventing the formation of the membrane attack complex 31, 32.
We have investigated the expression and function of membrane complement regulators on exosomes from human monocyte-derived dendritic cells and cells of B lymphocyte origin. We show that both CD55 and CD59 are expressed by APC-derived exosomes and function in their protection from complement attack. Our findings suggest that exosomes are unlikely secreted as a degradatory route for redundant molecules 4; rather they are equipped to survive extracellularly to perform their function(s). This has clear implications for the physiological roles of exosomes and for their future therapeutic application in the context of immune modulation.
2.1 Flow cytometric characterization of exosome-bead complexes
Exosome-coated beads were prepared from human APC including monocyte-derived dendritic cells, EBV-transformed B cells, and untransfected C1r-un and HLA-A*0201-transfected (C1r-A2) B cell lines as previously described 14. Beads were stained for MHC class II, MHC class I, CD59, CD55, and CD46, and analyzed by flow cytometry. Exosome-coated beads stained strongly positively for MHC class II for all cell lines examined.
Altering bead-exosome coupling conditions such as increasing the amount of exosomes added or increasing the bead-coupling time did not further increase staining, indicating that beads were saturated under the conditions used (not shown). Exosome-beads from all cell types except the C1r-un line (which is class I-deficient) were strongly positive for MHC class I. Exosome-beads from all sources stained weakly but reproducibly for CD59 (Fig. 1). There was no detectable (Fig. 1) or minimal (see Fig. 7a) staining for CD55, and CD46 was not detected. CD59, CD55 and CD46 were strongly expressed on the cell surface (not shown).
2.2 APC-derived exosomes express CD55 and CD59 but not CD46
Exosome lysates were prepared from several B lymphocyte and dendritic cell lines, and the presence of CD55, CD59 and CD46 was investigated by Western blotting. CD59 was present on exosomes from all APC sources; results shown are for dendritic cells and C1r-A2 cells (Fig. 2a). For each, several discrete bands were apparent in the molecular mass range 18–22 kDa, representing CD59 glycoforms 32, 33. Beads incubated with exosomes derived from the class II-deficient but CD59-positive cell line K562 were not positive for CD59 by Western analysis, confirming that a positive result required co-localization of class II and CD59, and was not due to binding of soluble CD59 33 to the bead.
Exosomes from all APC sources were also positive by Western blotting for CD55, displaying a single band of ∼70 kDa (Fig. 2b). Western blotting failed to demonstrate reactivity for CD46 in any exosome lysate examined (not shown). Exosomes from B-LCL(LL), an EBV-transformed B lymphoblastoid cell line (B-LCL) were also prepared by differential ultracentrifugation as previously described 10, which allowed us to directly compare the relative levels of exosomal CD59, CD55 and CD46 expression with that in cell lysate preparations. There was some enrichment of CD59 in exosomes compared to cell lysates (Fig. 2c), and to a lesser degree this was true also for CD55 expression (Fig. 2d). Bands for CD46 were only present in the cell lysate preparations (Fig. 2e).
2.3 Exosomal CD55 inhibits complement C3b deposition
We investigated whether exosomal CD55 was functional in protecting exosomal membranes from complement deposition. Exosome-bead complexes were sensitized to complement by labeling with anti-class I (W6/32), and CD55 blocked with two blocking antibodies (BRIC216 and BRIC110, both IgG1). An isotype-matched antibody (specific for exosomally expressed CD18) was used as a control. Exosome-beads were exposed to various dilutions of human serum, depleted of complement C8 to prevent exosome destruction by the membrane attack complex. The level of exosome-bound C3b was measured by flow cytometry. There was significantly increased C3b binding in samples that had been incubated with CD55-blocking antibodies (Fig. 3). At a serum dilution of 1:40, this increase was highly significant (p<0.001) when compared to isotype-matched controls. Exosomally expressed CD55 is therefore functional in protecting the membrane from the deposition of C3.
2.4 Exosomal CD59 and CD55 confer resistance to complement-mediated lysis
In order to measure complement lysis of the exosomal vesicle, bead-associated exosomes were loaded with the fluorescent dye Calcein-AM. Upon uptake the dye is de-esterified and becomes trapped in the exosome lumen. Calcein loading was optimized using B –lymphocyte-derived exosomes, and effective loading was achieved at 37°C. The degree of exosome loading was dependent on the dose of Calcein-AM added and the length of the incubation (Fig. 4a, b). Optimal loading was achieved by incubating with 25 μM Calcein-AM for 20 h at 37°C (Fig. 4b). Once loaded, fluorescence was well retained, with only a 16.67% decrease over a subsequent 24-h period for exosomes loaded as described above. Treating exosomes with 5% Ttriton X-100 in complement fixation diluent (CFD) lysed the exosome membrane, leading to a drop in fluorescence to background levels.
The role of CD59 in protecting exosomes from lysis was assessed by measuring the loss of fluorescence from Calcein-loaded exosome-bead complexes under various conditions. B –lymphocyte-derived exosomes were exposed to various dilutions of fresh human serum from three different donors, in the presence of sensitizing antibody (W6/32), anti-CD59 blocking antibody (MEM-43), or both antibodies. In the absence of sensitizing antibody, exosomes were not significantly lysed at any serum dose, even in the presence of anti-CD59. In contrast, exosomes sensitized with W6/32 demonstrated a dose-dependent susceptibility to complement lysis. This complement lysis was potentiated by the presence of CD59-blocking antibody (Fig. 5). Similar results were obtained for exosomes of dendritic cell origin (not shown).
The importance of complement fixation in this system was confirmed by utilizing exosomes from the class I-deficient line C1r-un and the class I-transfected line C1r-A2 (see Fig. 1). These exosomes were incubated with W6/32 (0–10 μg/ml) in the presence of MEM-43 (0–1 μg/ ml). Class I-deficient exosomes were resistant to complement-mediated lysis, even in the presence of high doses of both W6/32 and MEM-43 (Fig. 6a). In contrast, the class I-expressing C1r-A2 exosomes were lysed in a sensitizing antibody dose-dependent manner. Lysis was further enhanced by CD59 blockade (Fig. 6b).
To investigate the role of CD55 and the combined effects of the two regulators on resisting lysis, Calcein-loaded exosomes were sensitized with W6/32, and blocking antibodies against CD59 (MEM-43) and/or CD55 (BRIC216 and BRIC110), or isotype-matched control antibodies were added. R-PE-conjugated W6/32 was used in order to permit assessment of exosome retention on the beads in the presence of serum, and isotype-control antibodies specific for CD20 and CD18, which are expressed at similar levels to CD55 and CD59, respectively (Fig. 7a), were included to demonstrate the specificity of the blocking effect. Exosome-beads were then exposed to fresh serum. Blockade of either CD59 or CD55 alone resulted in a significant exosome lysis, CD59 having the greatest effect (Fig. 7b). The combined blockade of CD55 and CD59 further enhanced exosome lysis (Fig. 7b). There was no loss of exosomes complexed to beads under these conditions, as indicated by consistently strong staining with R-PE-conjugated (-R-PE) anti-MHC class I (Fig. 7c).
Exosomes may play important roles in the regulation of immune responses in vivo that require a capacity to survive in the periphery 10–12. The degradation of exosomes in vivo has not been investigated to date, but a likely mechanism involves the activation of the complement system, resulting in their opsonization or lysis, as this mechanism is efficient in degrading other membranous vesicles 18–28. Exosomes derived from APC, by virtue of their association with antigens, may be particularly prone to complement damage or destruction. Although several proteomic studies of APC exosome composition have been published 15, 17, no evidence for complement protective molecules on these exosomes has previously been described.
Here we extend the molecular characterization of human APC-derived exosomes, and demonstrate that MHC class II-positive exosomes express the glycosylphosphatidylinositol (GPI)-linked complement regulatory proteins CD59 and CD55. Exosomes, captured on beads through MHC class II, were positive for CD59 by flow cytometric analysis and Western blotting. Due to the relative low levels of expression, exosomal CD55 was not consistently detected by flow cytometry, but was detected by Western analysis. Although CD46 was abundant in lysates of parent cells (Fig. 2c), it was not detected on exosomes by either method. This may suggest selective exclusion of CD46 from exosomes. Exosomes derived from reticulocytes are enriched in GPI-linked proteins 16, suggesting a possible role for the GPI link in the recruitment of molecules into exosomes, but this has not been reported for APC exosomes. Although the mechanism for CD46 exclusion is not known, nor is the functional importance of this clear, we are not surprised to find some differences in the phenotype of exosomes compared to the cell surface. For example, dendritic cell-derived exosomes lack molecules such as FcγRII/III 17 and CD40 17 found in abundance at the cell surface, while the endosomal marker LAMP2 is absent from the cell surface, but expressed by exosomes 17.
Analysis of exosomes has been complicated by their small size. By capturing exosomes on large beads (4,500 nm), however, a method for analyzing exosome surface markers and exosome lysis by flow cytometry was developed. Exosomes in complex with beads and incubated in vitro with human serum were not spontaneously lysed. In order to address whether expressed CD55 and CD59 were playing a protective role, exosomes were first sensitized, targeting the abundantly expressed MHC class I with antibody (W6/32), well characterized for complement-fixing ability 34. Incubation of sensitized exosomes with serum caused complement activation (assessed by C3b deposition) and lysis (assessed by Calcein release from the exosome lumen). The expressed complement regulators were then blocked, singly or in combination, and the effects of the blockade on C3b deposition and exosome membrane lysis assessed.
CD55 plays an important role in binding the C3 convertases of the classical and alternative complement pathways 35, and in accelerating the dissociation of the catalytic subunits. Blocking CD55 in our system, using two blocking monoclonal antibodies 36, caused a significant increase in exosome-associated C3b. Exosomal CD55, therefore, is capable of inhibiting the initial deposition of C3b. CD59 inhibits the assembly of the membrane attack complex, and blocking either CD55 or CD59 caused increased lysis of exosomes as assessed by Calcein release, and the combined blockade of both inhibitors had an additive effect on lysis, demonstrating that these GPI-linked complement inhibitors work in concert to provide resistance to complement lysis.
The essential role of sensitization was further demonstrated using the B-LCL C1r-A2 and C1r-un, which secrete identical exosomes except that the parent C1r-un line lack MHC class I. This line was resistant to complement lysis at all doses of sensitizing antibody and in the face of blocking antibody against CD59, whereas the class I-transfected C1r-A2 line was susceptible to lysis following sensitization, and lysis was enhanced by the presence of the blocking antibodies. These data also demonstrate that the alternative pathway of complement activation is not subject to initiation bythe exosome membrane.
The secretion of exosomes by reticulocytes has been proposed as a mechanism for shedding redundant cellular components (e.g. CD71), making these exosomes a waste product in erythrocyte development 4. Nevertheless, such exosomes have also been shown to express the complement regulatory components CD55 and CD59 16. Together with our data, thisstrongly suggests that exosomes from multiple sources are secreted into the extracellular environment fully equipped to withstand degradation by the complement system, in order to accomplish their extracellular functions. For APC, this may involve the regulation of immunological responses, through T cell stimulation/inhibition 10, 11, or through cross-priming dendritic cells 12, roles that likely require stability in the circulation. Extracellular persistence of exosomes is thus important for their physiological roles, and the expression of functional complement regulators on their membranes is likely to be a central factor in their success. For APC-derived exosomes, likely to have immunogenic antigens associated with the membrane, there is a particular risk of being rapidly neutralized by antibody binding and complement lysis. In the current study we have mimicked this situation by binding antibody to MHC class I in order to obtain significant complement lysis and investigate the roles of membrane regulators. However, it should be possible to utilize a more physiological system with peptide-loaded exosomes as target and anti-peptide antibodies as the trigger to complement activation.
Our data demonstrate a hitherto unrecognized role of complement regulators in protecting exosomes shed from cells in order to permit survival in the circulation. Parallels can be drawn with our earlier studies in prostasomes, vesicular bodies shed from cells of the genital tract and present in abundance in seminal plasma 37. Here too, expression of the GPI-anchored complement regulators CD55 and CD59 on these vesicles is essential for survival and function in vivo.
4 Materials and methods
4.1 Cell lines
B-LCL were prepared as previously described 38. The MHC class I-deficient B-LCL C1r-un, and C1r-A2 (the parent line transfected with HLA-A*0201) 39, and amyelogenous leukemia line (K562 ECACC no. 89121407) deficient in MHC class II were obtained from the European Collection of Animal Cell Cultures (Porton Down, Salisbury, GB) and maintained in 10% FCS in RPMI 1640 containing penicillin/streptomycin (100 IU/ml) and 10 mM Hepes (from Gibco/Life Technologies). Monocyte-derived dendritic cells were prepared as previously described 40.
4.2 Preparation of human serum
Serum was prepared freshly for each lysis experiment. Blood from healthy volunteers was collected in silica-coated vaccutainers (Becton Dickinson). After 40 min, serum was isolated from coagulated blood and contaminating cells by centrifugation. Serum was diluted (1:200 to 1:10) in CFD buffer (Oxoid, GB) for subsequent experiments. For studies of complement C3 deposition, serum was depleted of C8 as previously described 41.
4.3 Exosome isolation
Exosomes were isolated as previously described 14. In brief, cell suspensions (24 h or longer conditioning) were centrifuged twice at 100×g for 10 min, to remove cells.The cell-free supernatant was further centrifuged at 400×g for 10 min, and then 2,000g for 15 min, to eliminate cellular fragments/debris. Finally, the supernatants were filtered (0.2 μm) prior to the addition of pre-washed Dyna-beads, ready-coated with anti-HLA-DP, -DQ, -DR antibodies (Dynal, Oslo, Norway) under conditions which yield beads completely saturated with MHC class II-positive exosomes. Typically, 0.5×106–1×106 beads were added to 10–15 ml of conditioned medium, and incubated overnight at 4°C.
Exosomes were also prepared by differential ultracentrifugation methods, as described 10. Briefly, cell-free conditioned media was centrifuged at 20,000×g for 35 min, then at 70,000×g for 1 h. The exosome pellet was washed in large volume of PBS. Finally the exosome pellet was re-suspended in lysis buffer, and analyzed by Western blotting.
4.4 Exosome characterization by flow cytometry
Exosome-bead complexes (5×104) were incubated with conjugated antibody for 1 h at 4°C, washed and analyzed by flow cytometry as described 14. Only single beadswere gated for fluorescence analysis. A bead-only control was included for each antibody of interest, and the fluorescence intensity was normalized to ∼100 fluorescence units for eachantibody used. The conjugated antibodies included FITC-conjugated (-FITC) anti-HLA-DP-, -DQ-, -DR (clone CR3/43) and anti-HLA-A-, -B-, -C-R-PE (clone W6/32; Dako), anti-CD59-FITC (clone BRA-10G), anti-CD55-FITC (clone 67; Alexis Biotechnologies, London, GB) or BRIC216-FITC (IBGRL, Oxford, GB) and anti-CD46-FITC (clone169–1E4.3; Alexis Biotechnologies). As potential targets for isotype-controlantibodies in blocking studies of CD59 and CD55, exosome-bead complexes were stained with anti-CD20-FITC (clone B9E9, IgG2a) and anti-CD18-FITC (clone MEM-48, IgG1), from Cymbus Biotechnologies.
4.5 Detection of complement C3 binding
Exosome-bead complexes (1×106) were sensitized with anti-MHC class I (clone W6/32) and either blocking monoclonal antibodies reactive around the active site of CD55 (BRIC216, IgG1, and BRIC110, IgG1 36; each at 10 μg/ml) or unconjugated anti-CD18 (clone MEM-48, at 20 μg/ml) as an isotype-matched control. BRIC110 and BRIC216 have been confirmed as non-complement-activating (B. Paul Morgan, unpublished data); both are required for efficient blocking of CD55 function 42. Beads were washed and incubated (7×104 each incubation) in 0.5% BSA/CFD at 37°C for 1 h with various dilutions of C8-depleted serum, washed and stained using rabbit polyclonal anti-C3 (a gift from Dr. Marc Fontaine, Rouen, France), and anti-rabbit-Ig-R-PE which was strictly not cross-reactive across species (Stratech Scientific Ltd., Soham, GB). Beads were analyzed by flow cytometry.
4.6 Fluorescent labeling of the exosome lumen
Exosome-bead complexes were loaded with the fluorescent dye, Calcein-AM (Molecular Probes Inc). Optimal loading was achieved with 106 exosome-beads in 1 ml 0.5% BSA/PBS containing 50 μM Calcein-AM. Following 24 h incubation at 37°C, beads were washed four times in 0.5% BSA in CFD buffer, to remove unincorporated dye. A proportion of the beads was incubated in 5% Triton X-100for 5 min to obtain 100% Calcein release as a positive control for each experiment. In addition, a portion of exosome-beads was not loaded with dye and used to normalize fluorescence intensity to 100, for each experiment.
4.7 Complement-mediated exosome lysis
For complement activation, Calcein-loaded exosome-bead complexes were sensitized with anti-MHC class I antibody (clone W6/32). To determine the function of CD59, a blocking antibody (clone MEM-43, IgG2a, 43, 44) was added either alone or in combination with the complement-fixing antibody (W6/32). Similarly, CD55 blocking was achieved with the blocking antibodies BRIC216 and BRIC110 (both IgG1). In some experiments isotype-matched control antibodies (anti-CD20, IgG2a, or anti-CD18, IgG1) were also included. After washing away unbound antibody, exosome-beads (105) were incubated in 100 μl of fresh human serum diluted in CFD/2% BSA, for 1 h at 37°C. Exosome-beads were analyzed by flow cytometry. The mean channel fluorescence valueof the histogram plot was taken as a measure of intensity. A measure of maximum fluorescence was obtained by measuring the fluorescence of Calcein-loaded exosome-beads in serum free CFD buffer, andthis value was used as 100% fluorescence for each experiment.
4.8 Western blotting
Exosomes for Western blotting were prepared by bead isolation or by ultracentrifugation. Bead-isolated exosomes (5×106–10×106 beads per sample) were lysed for 30 min in 200 μl of lysis buffer (containing 2% NP40, 1 mM PMSF, 1 μg/ml pepstatin and leupeptin, 10 mM EDTA). After removing beads from the lysate, protein was measured (BCA-assay kit, Pierce) and 5 μg of protein was added per well. Exosome pellets, prepared by ultracentrifugation, were resuspended in 100 μl of lysis buffer, and 6–0.6 μg of protein added per well. For the preparation of cell lysates, 2 million cells were washed twice in PBS and finally resuspended in 800 μl of lysis buffer. After incubation at room temperature for 1 h, samples were briefly centrifugedat 2,000×g, and protein concentration in supernatants determined. To each well 6–0.6 μg of protein was added. Samples were electrophoresed on 10% or 4–20%-gradient polyacrylamide gels (ReadyGels, Bio-Rad, GB), transferred onto polyvinylidene difluoride (PVDF) membrane (Hybond-P, Amersham Pharmacia Biotech UK Ltd.) and blocked overnight in 3% dried milk in PBS containing 0.1% Tween-20. Primary antibodies (0.25–1 μg/ml) included anti-CD59 (MEM-43), anti-CD55 (clone 143–30; gift from Dr. J. Miller, Cymbus Biotechnologies, GB) or anti-CD55 (clone BRIC216) or anti-CD46 (clone MEM-258) from Dr. Vaclav Horejsi (Prague, Czech Republic). The detection antibody was anti-mouse IgG-HRP (Dako, used at 1:25,000). Immunoblots were detected using the ECL+ method of Amersham Pharmacia. CD59 and CD55 were purified from human erythrocytes by affinity chromatography on monoclonal antibody columns and used as positive controls.
Student's t-test was used to compare antibody blocked with unblocked controls. A p value of <0.05 was taken as significant.
This work was supported by Cancer Research Wales and the European Comission Grant QLRT-2001 00093.