Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles that carry bioactive cargo, including proteins, lipids, and nucleic acids. Once taken up by target cells, EVs can modify the physiology of the recipient cells. In past studies, we reported that engagement of the glycophosphatidylinositol-anchored receptor CD24 on B lymphocytes (B cells) causes the release of EVs. However, a potential function for these EVs was not clear. Thus, we investigated whether EVs derived from CD24 or IgM-stimulated donor WEHI-231 murine B cells can transfer functional cargo to recipient cells. We employed a model system where donor cells expressing palmitoylated GFP (WEHI-231-GFP) were cocultured, after stimulation, with recipient cells lacking either IgM (WEHI-303 murine B cells) or CD24 (CD24 knockout mouse bone marrow B cells). Uptake of lipid-associated GFP, IgM, or CD24 by labeled recipient cells was analyzed by flow cytometry. We found that stimulation of either CD24 or IgM on the donor cells caused the transfer of lipids, CD24, and IgM to recipient cells. Importantly, we found that the transferred receptors are functional in recipient cells, thus endowing recipient cells with a second BCR or sensitivity to anti-CD24–induced apoptosis. In the case of the BCR, we found that EVs were conclusively involved in this transfer, whereas in the case in the CD24 the involvement of EVs is suggested. Overall, these data show that extracellular signals received by one cell can change the sensitivity of neighboring cells to the same or different stimuli, which may impact B cell development or activation.
Extracellular vesicles (EVs) are released from all mammalian cells that have been examined (1). These vesicles are a heterogeneous group of phospholipid bilayer-enclosed particles that are classified based on their release pathway and size (2). Exosomes are the smallest EVs at 30–100 nm in diameter and are released through exocytosis from multivesicular bodies (3, 4). Slightly larger than exosomes, microvesicles (MVs) or ectosomes are formed by outward budding from the cells’ plasma membrane and are from 100 nm to 1 µm in size (3, 4). The largest type of EVs are apoptotic bodies that are generated by cells undergoing the final stages of apoptosis and can range from 1 µm to 5 µm (5). EVs are critical mediators of cell-to-cell communication during normal and pathological physiological processes (6). EVs contain cargo that reflect the cell of origin and which can be transferred to recipient cells (7–9). These cargoes include lipids, proteins, and genetic material such as mRNA, miRNA, and DNA. Once taken up by recipient cells, EVs can alter the biological functions of target cells (10). For example, the first report of EVs found that B lymphocytes release EVs carrying peptide-MHC class II complexes that can directly activate CD4+ T cells (8).
The visualization of EVs is essential for understanding EV biology. EVs can be visualized directly via fluorescent chemical labeling using lipophilic dyes such as PKH26 or PKH67, which label cell membranes by the insertion of their aliphatic chains into the lipid bilayer. Fluorescence microscopy and flow cytometry can then be used to detect EV uptake, as has been done for breast cancer cells, macrophages, dendritic cells, endothelial cells, and myocardial cells (11). However, PKH-labeled EVs may be degraded and/or recycled in vivo. In addition, PKH dyes can form micellar structures identical in size to EVs and can be retained in association with other lipid entities for long periods. Therefore, inaccurate spatiotemporal assessment of EV fate can be inferred based on the use of these dyes (12). In contrast, fluorescent proteins such as GFP and tandem dimer Tomato (tdTomato) that are fused to a palmitoylation signal (palm-GFP and palm-tdTomato) have been reported to label all membrane-enclosed vesicles, including EVs (13). This specific labeling allows tracking of EVs in culture for long periods of time in a more accurate manner than lipophilic dyes.
B lymphocytes (B cells) are critical components of the immune system. B cells are generated in the bone marrow from multipotent hematopoietic stem cells that undergo maturation in discrete stages (14). At each stage of B cell development, different cell surface receptors are used to identify each stage using either the Hardy fraction designators (15, 16) or established independent markers (17). The primary driver of this maturation is the generation of the BCR, which is composed of the membrane-bound IgM and two accessory molecules, Igα and Igβ (CD79a and CD79b, respectively).
Of particular relevance for this study is CD24, also called heat-stable Ag, which is highly expressed at the pro– and pre–B cell stages (18). CD24 is a lipid raft-localized GPI-anchored membrane protein of 27 aa with extensive, but variable, N- and O-linked glycosylation (19, 20). Several ligands have been identified for CD24, including P-, L-, and E-selectin, sialic acid–binding Ig-like lectin-G or Siglec 10, TAG-1, contactin, L1 cell adhesion molecule, and neural cell adhesion molecule (21). However, the relevant CD24 ligand for bone marrow B cells is not known. Engaging CD24 on pro– or pre–B cells with anti-CD24 Abs leads to apoptosis and CD24 engagement in mature splenic B cell blocks CD40-induced proliferation (22, 23). We have also found that Ab-mediated engagement of CD24 induces apoptosis in the immature murine WEHI-231 B cell line, demonstrating that this cell line models the CD24-mediated effects on developing B cells (24). Importantly, we found that engagement of CD24 increases the release of EVs into the extracellular environment (24, 25). In addition, CD24-mediated stimulation promoted changes in the membrane protein composition of the secreted EVs (25).
In this study, we tested the hypothesis that EVs released from anti-CD24– or anti-IgM–stimulated B cells can transfer functional receptors to recipient cells. We analyzed EV transfer by using cells that express palmitoylated fluorescence markers, which allow us to track EVs released from both donor and recipient cells. We visualized lipid transfer by tracking the transfer of palm-GFP or palm-tdTomato from donor to recipient cells. We visualized protein transfer by tracking the transfer of IgM from BCR-positive cells (WEHI-231) to BCR-negative cells (WEHI-303) and the transfer of CD24 from WEHI-231 cells to primary B cells isolated from CD24 knockout (CD24KO) mice. We found that stimulation of either CD24 or IgM on donor cells resulted in the transfer of both CD24 and IgM to recipient cells. We found that both IgM and GFP are packaged into isolated EVs, indicating that the transfer of lipid with transmembrane protein is most likely via EVs secreted by donor cells. To determine if the transferred receptors were functional, we assessed output from the newly transferred receptor on the recipient cells. We found that the transferred CD24 and IgM on the recipient cells were functional receptors that could initiate signaling and apoptosis in the recipient cells. These data suggest that stimulation of B cells could alter the responses of bystander cells via release of EVs.
Materials and Methods
The Institutional Animal Care committee at Memorial University of Newfoundland approved all animal procedures (protocol 17-01-SC). C57BL/6N Cd24atm1Pjln homozygous mice (CD24KO) (26–28) were a gift from Dr. Yang Liu (Center for Cancer and Immunology Research, Children’s National Medical Center, Washington, DC).
Cell culture and transfection
All materials for cell culture were obtained from Life Technologies (Carlsbad, CA) unless otherwise indicated. Isolated bone marrow-derived immature B cells and cell lines were maintained in RPMI 1640 medium supplemented with 10% heat‐inactivated FBS, 1% antibiotic/antimycotic, 1% sodium pyruvate, and 0.1% mercaptoethanol (complete medium) at 37°C in a humidified 5% CO2 atmosphere. WEHI-231 cells (American Type Culture Collection) and WEHI-303.1.5 (WEHI-303) (29) were transfected with palm-GFP (WEHI-231-GFP) or palm-tdTomato (WEHI-303-tdTomato) lentiviral plasmids, generous gifts from Charles Lai, Institute of Atomic and Molecular Sciences, Taiwan (13). Briefly, these were cotransfected with pCMV-VSV-G-M5 and pCMV-δR8.91 (from Dorothee von Laer, Medical University of Innsbruck, Austria) into HEK293T cells (American Type Culture Collection). Virus particles were collected at 12 h and 36 h posttransfection and added to 12-well plates containing WEHI-231 or WEHI-303 cells, which were then centrifuged at 2000 rpm (750 × g) for 1 h at 21°C. Cells were cultured for 48 h before enrichment by FACS. Cells were resorted regularly with the Beckman Coulter MoFlo Astrios EQ (Medical Laboratory Services, Memorial University) to maintain >90% fluorescently labeled cells (Supplemental Fig. 1A).
Primary bone marrow B cell isolation
Femurs were removed from euthanized 6–8-wk-old male or female CD24KO mice and bone marrow was flushed out with Quin saline (25 mM NaHEPES, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 1 g/l glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM 2-ME, pH 7.2), using a 21-gauge needle. Cells were pooled from two mice and single-cell suspensions produced using a 100-µm nylon mesh. The EasySep Mouse B cell isolation kit (19854; StemCell Technologies) was used to enrich bone marrow isolates following the manufacturer’s protocol. Primary bone marrow B cell purity was confirmed by flow cytometry to be >80% B cells using anti-mouse CD19-Per-CP-Cy-5.5 (45-0193-82; eBioscience), IgM-APC/Cy7 (406515; BioLegend), CD45R (B220)-PE-Cy7 (25-0452-82; eBioscience) and CD24-PE (12-0242-82; eBioscience) Abs. All analysis by flow cytometry was performed on the FACSAria (BD Biosciences; Cold-Ocean Deep-Sea Research Facility, Memorial University).
WEHI-231-GFP donor cells (5 × 105 cells/ml in 500 µl) were treated with stimulating Abs as described below.
For stimulation of CD24, 10 µg/ml functional grade primary monoclonal M1/69 rat anti-mouse CD24 Ab (16-0242-85; eBioscience) or 10 µg/ml matching primary isotype Ab (16-4031-85; eBioscience) were preincubated with 5 µg/ml goat anti-rat secondary Ab (112-005-003; Jackson ImmunoResearch) at a 2:1 ratio to ensure efficient cross-linking of primary Ab and that no excess secondary Ab was present. We have confirmed that isotype preincubated with secondary Ab does not bind to cells (24). After stimulation for 15 min at 37°C in complete medium, donor cells were centrifuged at 400 × g for 5 min to remove Ab-containing medium and then resuspended in complete medium followed by coculture with recipient cells, WEHI-303-tdTomato (5 × 105 cells in 500 μl), giving a final concentration of 106 cells/ml in 1 ml, at 37°C in a humidified incubator containing 5% CO2 for a total of 24 h. The cocultured cells were analyzed by flow cytometry. Cleared supernatant (cSN) was collected from WEHI-231-GFP cells, stimulated as above for 1 or 2 h, after centrifugation at 500 × g for 5 min, then 2000 × g for 5 min. cSN was added to recipient cells as indicated.
When primary bone marrow B cells were used as recipients, they were prestained with proliferation dye eFluor 670 (65-0840; eBioscience) following the manufacturer’s protocol and then cocultured with WEHI-231-GFP cells as indicated. eFluor 670 positivity was determined by flow cytometry (Supplemental Fig. 2G).
To assess anti-CD24–induced apoptosis in primary bone marrow B cells, WEHI-231-GFP and primary bone marrow B cells were plated at a density of 5 × 105 cells each in 500 µL complete medium. The cultured cells were mixed 1:1, giving a final concentration of 106 cells/ml in 1 ml, and were treated with Ab as indicated for 24 h.
To stimulate IgM, WEHI-231-GFP cells were treated with 10 µg/ml anti-mouse IgM (115-005-020; Jackson ImmunoResearch) or left untreated for 1 or 2 h, then centrifuged at 400 × g for 5 min to remove Ab-containing medium and then resuspended in complete medium followed by coculture with recipient cells CD24KO B cells (5 × 105 cells in 500 μl), giving a final concentration of 106 cells/ml in 1 ml, at 37°C in a humidified incubator containing 5% CO2 for a total of 24 h. The cocultured cells were analyzed by flow cytometry.
To analyze anti-IgM–induced apoptosis of WEHI-303-tdTomato cells, WEHI-231-GFP and WEHI-303-tdTomato cells were plated at a density of 5 × 105 cells each in 500 µL complete medium. The cultured cells were mixed 1:1, giving a final concentration of 106 cells/ml in 1 ml, and were treated with 10 µg/ml anti-mouse IgM (115-005-020; Jackson ImmunoResearch) or left untreated for 24 h.
For stimulation of CD40, WEHI-231-GFP cells were treated with 10 µg/ml anti-mouse CD40 (16-0401-82; eBioscience) or 10 µg/ml isotype control Ab (16-4321-82; eBioscience) for 2 h, or left untreated, then centrifuged at 400 × g for 5 min to remove Ab-containing medium and then resuspended in complete medium followed by coculture with recipient cells WEHI-303-tdTomato as above.
To stimulate CD48, WEHI-231-GFP were stimulated with 10 µg/ml anti-mouse CD48 (103402; BioLegend) or 10 µg/ml isotype control Ab (400902; BioLegend) for 1 h, or left untreated, followed by centrifugation at 400 × g for 5 min to remove Ab-containing medium and then resuspended in complete medium followed by coculture with recipient cells WEHI-303-tdTomato as above.
EV isolation by size exclusion chromatography
cSN was collected from WEHI-231-GFP cells as described above. Four milliliters of supernatant, from both 1 h stimulations (2 ml) and 2 h stimulations (2 ml), were loaded onto a qEV2/70nm size exclusion chromatography (SEC) column (Izon Science, Medford, MA, USA) following the manufacturer’s instructions. Fractions 7–12 were pooled and then concentrated to ∼200 μl using Amicon ultra-15 centrifugal filter units (UFC901024; Millipore, Etobicoke, ON, Canada) at 3000 × g for 50 min. The concentrated fraction was diluted in complete medium and incubated with WEHI-303-tdTomato cells for 24 h at 37°C in a CO2 incubator.
Nanoparticle tracking analysis
Isotype- and CD24-stimulated conditioned medium (CM) were diluted 5-fold in sterile 0.1-µm filtered 1× PBS if from cells cultured in EV-free medium and left undiluted if from SEC. EV-free medium was prepared according to the previously established protocol (30); briefly, complete medium with 20% FBS was centrifuged at 100,000 × g at 4°C for 16 h, passed through a 0.22-µm filter, and then mixed with serum-free RPMI 1640 in a 1:1 ratio. For each measurement, quintuplicate 1-min videos were captured at a temperature of 25°C and syringe pump speed of 25 µl/s. Postcapture, videos were analyzed by the Nanosight NS300 software version 3.4 Build 3.4.003.
EV isolation by Vn96 peptide-based affinity isolation
One milliliter of CM was incubated with 30 µg of Vn96 peptide at 4°C, overnight on a rotator. The CM-Vn96 mix was then centrifuged at 17,000 × g for 15 min and the supernatant removed. A second milliliter of CM was added to the pellet, followed by a brief vortex to disrupt the pellet and then incubation with rotation for 3 h at 4°C. Vn96 EVs were pelleted by centrifugation at 17,000 × g for 15 min at 4°C, followed by three washes with 0.1-µm filtered 1× PBS supplemented with 1.2 mM PMSF.
Vn96 EV pellets isolated as above were dissolved in 0.1-μm filtered 1× PBS, mixed with Laemmli reducing sample buffer and 50% of the volume separated using 12% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, then blocked with 5% (w/v) skimmed milk in 0.1% TBST. Primary Abs (all from Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in 5% (w/v) skimmed milk in TBST as follows: 1:1000 HSP90 α/β (SC-13119), 1:1000 CD81 (SC-166029), 1:1000 full length GFP (SC-8334). HSP90 and CD81 were detected using HRP-conjugated goat anti-mouse IgG (SC-13119). GFP was detected using HRP-conjugated mouse anti-rabbit IgG (SC-2357). IgM was detected using goat anti-mouse IgM HRP (1:1000, SC-2064). All secondary Abs were diluted 1:1000 in 5% (w/v) skimmed milk in TBST. Western chemiluminescent HRP substrate (Immobilon ECL Ultra Western HRP Substrate) was used for detection. Western blot images were acquired using a ChemiDoc gel documentation system (Bio-Rad, Hercules, CA). Image manipulation involved adjustments to brightness and contrast only.
IgM and CD24 detection by flow cytometry
Cells were resuspended with FACS buffer (PBS 1×, pH 7.4, 10010-023; Life Technologies, containing 1% heat-inactivated FBS) and stained with 0.5 µg of IgM-PE-Cy7 (25-5890; eBioscience) or with 0.25 µg of CD24-PE (12-0242-82; eBioscience) for 30 min at 4°C. Cells were then washed with FACS buffer and analyzed by flow cytometry. See Supplemental Figs. 1 and 2 for gating strategies.
Phospho-ERK detection by flow cytometry
After 24 h of coculture, the cells were stimulated with 10 µg/ml anti-mouse IgM (115-005-020; Jackson ImmunoResearch) for 5 min at 37°C, and the reaction stopped with 0.4 ml of cold 1× PBS containing 1 mM vanadate (13721-39-6; Sigma Aldrich). The cells were centrifuged at 400 × g for 5 min and then resuspended with 100 µl of prewarmed fixation buffer (00-8222-49; eBioscience) for 35 min at room temperature (RT) followed by resuspension in 100 µl of permeabilization buffer (00-8333-56; eBioscience) and incubation with 1 µg of phospho-ERK1/2 conjugated with Alexa Fluor 647 (675503; BioLegend) for 35 min at 4°C in the dark. The reaction was stopped with 0.9 ml of permeabilization buffer before centrifugation at 400 × g for 5 min. The cell pellets were washed with FACS buffer and analyzed by flow cytometry.
Apoptosis assay by flow cytometry
Cells were resuspended in Annexin V binding buffer (10 mM HEPES buffer, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Three microliters of Annexin V-Alexa Fluor 647 (A23204; Invitrogen) or 5 µl of Annexin V-PE (640908; BioLegend) was added to 100 µl of cell suspension following the manufacturer’s instructions. Cells were then washed with FACS buffer and analyzed by flow cytometry.
WEHI-303 cells were incubated with 5 µM CFSE (ThermoFisher) for 10 min at RT and then quenched with FBS following the manufacturer’s instructions. WEHI-231 were stimulated with anti-CD24 for 15 min, washed, and then cocultured with WEHI-303 for 24 h at 37°C, as above. Cells were adhered to coverslips precoated with 1 µg/ml anti–MHC class II (MABF33; Millipore) overnight at 4°C. Cells were fixed in 4% paraformaldehyde for 10 min at RT. After washing three times with PBS, slides were blocked with 2% BSA in 1× PBS for 30 min at RT. Coverslips were incubated for 1 h at RT with Alexa Fluor 647-conjugated goat anti-mouse IgM (112-545-175; Jackson ImmunoResearch) in 2% BSA/PBS, washed in PBS and then mounted using Prolong Diamond (ThermoFisher) mounting medium. Cells were imaged with spinning disk confocal microscopy using a Quorum Technologies system based on a Zeiss Axiovert 200 M microscope with a 100× NA 1.45 oil objective and a QuantEM 512SC Photometrics camera for image acquisition. Z-stacks were acquired in 0.5-µm increments. Images were assembled using ImageJ after export of 16-bit TIFF using Slidebook v6.0.4 software (3i; Denver, CO).
All statistical analysis was performed in GraphPad (version 8.4.3) with specific tests indicated in each figure legend.
EV trafficking of lipid and membrane proteins between murine B lymphoma cells in response to CD24 stimulation
The WEHI-231 murine B cell lymphoma cell line is used extensively as a model for B cell responses to receptor engagement. This cell line has surface receptor characteristics of immature B cells that undergo growth arrest and apoptosis in response to BCR cross-linking (31). To analyze the transfer of lipid and protein owing to the action of EVs, we took advantage of the WEHI-231 variant, WEHI-303, which lacks membrane IgM (Supplemental Fig. 1B) (29).
To test the hypothesis that lipids and proteins can be transferred between murine B cells in response to CD24 stimulation, we used a coculture model to capture the maximum amount of exchange in this dynamic system. After Ab-mediated stimulation of CD24 on WEHI-231-GFP cells, excess Ab was washed out to ensure that the recipient WEHI-303-tdTomato cells were not exposed to stimulating Ab. Equal numbers of anti-CD24–stimulated or control Ab-stimulated (isotype) WEHI-231-GFP cells were then cocultured with recipient WEHI-303-tdTomato cells (Fig. 1A). We found a statistically significant increase in GFP+tdTomato+ cells in response to CD24 engagement showing exchange of lipid between cells (Fig. 1B, 1C). These data could represent WEHI-303-tdTomato cells that have taken up GFP from WEHI-231-GFP cells or vice versa. To determine the direction of the transfer more precisely, we made the assumption that tdTomato+ cells with lower levels of GFP are most likely WEHI-303-tdTomato cells that have incorporated GFP+ EVs and GFP+ cells with lower levels of tdTomato are most likely WEHI-231-GFP that have incorporated tdTomato EVs. Analysis of low, mid, or high levels of GFP in tdTomato+ cells revealed a statistically significant increase of GFP events in the low and mid range but not in the high range (Fig. 1D and Supplemental Fig. 1D). These data suggest that the increase in double-positive events is most likely due to incorporation of EVs into tdTomato cells and not a cellular fusion event, which would have high levels of both fluorophores. We also observed an increase in GFP+ cells that were tdTomato+, with statistically significant increases at the low and mid tdTomato levels with the largest increase in the mid range (Fig. 1E, 1F and Supplemental Fig. 1C–E). Thus, there is a bidirectional EV exchange between cells that is increased when CD24 is stimulated on the donor cells. It is not clear if the transfer of tdTomato+ from the WEHI-303-tdTomato cells to the WEHI-231-GFP donor cells reflects an increase in the uptake of EVs by the WEHI-231-GFP cells or an increase in the release of EVs by WEHI-303-tdTomato cells in response to the acquisition of CD24 that has been clustered by anti-CD24 Abs.
To determine if membrane proteins are also transferred in response to CD24 simulation we analyzed the acquisition of IgM by WEHI-303-tdTomato cells. After coculture with anti-CD24–treated WEHI-231-GFP cells, we found a significant and substantial increase in the number of cells expressing cell surface IgM as well as an increase in the level of IgM on WEHI-303-tdTomato cells, albeit at a lower level than WEHI-231 cells, Fig. 1F, 1G and Supplemental Fig. 1F). Neither the percentage positive nor the level of IgM was changed in WEHI-231-GFP after stimulation (Fig. 1G and Supplemental Fig. 1G, 1H), indicating that the transfer of IgM to recipient cells did not substantially deplete IgM from the surface of the donor cells.
We then used confocal microscopy to visualize IgM transfer to WEHI-303 cells that had been labeled with CFSE because tdTomato fluorescence is destroyed upon fixation. We found that WEHI-303 cells displayed patches of IgM on their cell surface and that the size of these patches appeared to be greater when the donor WEHI-231-GFP cells had been cocultured with anti-CD24 as opposed to an isotype-matched control Ab (Fig. 1H).
To further support our hypothesis that EVs mediate the transfer of lipids and proteins from donor to recipient cells, we used low speed centrifugation to generate a cell-free cSN from WEHI-231-GFP cells that had been stimulated with anti-CD24 Abs, and then incubated the recipient cells with this cSN. Because of their small size, EVs would remain in the cSN, as much greater centrifugal forces are required to pellet them. When donor cSN isolated from an equal number of cells was added (1:1 ratio), there was no difference in the number of recipient cells gaining GFP between CD24 and control stimulation (Fig. 2A, 2B). However, when we used cSN at a 4:1 ratio we found a clear and statistically significant increase in the number of GFP+ tdTomato+ cells when the WEHI-303-tdTomato cells were cultured with the cSN of anti-CD24–treated WEHI-231-GFP cells, compared with when the WEHI-303 cells were exposed to cSN from isotype control-treated donor cells (Fig. 2A, 2B). Interestingly, with both a 1:1 and a 4:1 ratio, we found a significant increase in the percentage of recipient cells that acquired IgM after being cultured with cSN from CD24-stimulated donor cells (Fig. 2C). However, the amount of IgM acquired was substantially greater when a 4:1 cSN ratio was used, as indicated by the significantly higher level of anti-IgM fluorescence associated with the cSN-treated WEHI-303-tdTomato cells (Fig. 2D, 2E). Thus, there is a dose-dependent increase in the transfer of IgM.
To further characterize the cSN, we analyzed the particles released following isotype or CD24 stimulation from WEHI-231-GFP cells cultured in EV-free medium, using nanoparticle tracking analysis (Supplemental Fig. 3A). The mean EV size increased with 1 h of CD24 stimulation, but not 24 h, (Supplemental Fig. 3B left) whereas the concentration of released EVs was increased at 24 h but not 1 h (Supplemental Fig. 3B right). These data are generally consistent with our previous work and showed EVs in the 100–200-nm size range (25). Next, we determined if both GFP and IgM are present in the EVs. EVs were isolated and characterized by Western blot using the Vn96 peptide-based purification, which precipitates EVs based on interaction of the Vn96 peptide with heat-shock proteins, which we previously used for this purpose (25, 32). We found that both GFP and IgM coprecipitated with the EV surface protein markers CD81 and HSP90 showing the GFP and IgM are packaged in EVs (Supplemental Fig. 3C). We also found GFP and IgM present in EVs isolated using SEC (data not shown). To determine if EVs were able to transfer IgM, we then isolated EVs from cSN using SEC followed by incubation with recipient cells for 24 h. The EVs isolated by SEC were slightly larger than the bulk EV population with no significant difference between isotype and CD24 stimulation (204 ± 13 nm versus 181 ± 15 nm, respectively). Similar to our results with cSN, stimulation of donor cells with CD24 resulted in a significant increase in GFP and IgM on recipient cells (Fig. 2F, 2G). Thus, these results provide further evidence that GFP and IgM are transferred between B cells by EVs. Surprisingly, we did not observe transfer of IgM or CD24 when donor and recipient cells were cocultured but separated by a filter with 0.4-µm pores in a Transwell system (data not shown). This suggests that the exchange is likely dependent on close contact between cells.
Overall, we conclude that donor cells secrete EVs carrying lipids and membrane proteins that can be taken up by recipient cells. Consistent with our past work (24, 25), and given that IgM is a transmembrane cell surface receptor that we detect with an Ab directed against its extracellular domain, the simplest explanation for these data are that CD24 promotes the release of EVs from the plasma membrane that contain lipids and proteins, which are then incorporated into the plasma membrane of the recipient cells.
CD24 engagement on donor cells induces transfer of lipids and CD24 to recipient cells
We next asked if primary bone marrow B cells from CD24KO mice could also function as recipient cells. To do this, we labeled primary bone marrow cells with eFluor 670, which binds to cellular proteins in the cytoplasm, to track these as recipient cells. We found a statistically significantly increase in GFP+eFluor 670+ cells in response to CD24 engagement on the donor cells, showing transfer of the lipid-associated palm-GFP from donor to recipient cells (Fig. 3A). Analysis of low and mid levels of GFP in eFluor 670+ cells revealed statistically significant increases in GFP+ events in bone marrow B cells (Fig. 3B). Over 95% of the cells that took up GFP were B cells based on expression of CD19 or B220 (Fig. 3A, 3B). Conversely, there was also a CD24-mediated increase in GFP+ cells that acquired eFluor 670 fluorescence, with a statistically significant increase in the mid range. Thus, this shows that cytoplasm was transferred from the eFluor 670+ CD24KO bone marrow B cells to the anti-CD24–stimulated WEHI-231-GFP cells (Fig. 3C). Thus, similar to when WEHI-303 are recipient cells, there is a bidirectional exchange between cells that is increased when CD24 is stimulated on the donor cell, either by increasing uptake by the donor cell or by causing recipient cells to release more EVs in response to the transferred receptor.
We next investigated CD24 transfer and found a significant increase in surface CD24 expression on the CD24KO primary B cells after they were cocultured with CD24+ WEHI-231-GFP cells. Although this transfer of CD24 occurred in the absence of stimulating the WEHI-231-GFP cells, we found enhanced transfer of CD24 to the bone marrow B cells after stimulation of CD24 on the donor cells (Fig. 3D). In addition, we found that CD24 was transferred during the 4°C centrifugation step as indicated by the ∼5% positive cells in the nonincubated control. (Fig. 3D). Similar to the transfer of GFP, the vast majority of recipient cells (>89%) are primary B cells, indicating that contaminating cells are responsible for only a small percentage of uptake. Thus, CD24 is readily transferred to primary B cells and this transfer is increased when the donor cells are stimulated via CD24.
CD24 engagement on donor cells induces transfer of signaling-competent IgM and CD24 to recipient cells
We next asked if transferred receptors are functional in the recipient cells and can induce cellular responses. Ab-mediated engagement of the BCR induces phosphorylation of ERK1/2 within minutes and we used this as a readout of BCR signaling (33). We confirmed that phospho-ERK1/2 is detected using intracellular flow cytometry upon IgM stimulation of WEHI-231-GFP but not the IgM-negative WEHI-303-tdTomato cells (Supplemental Fig. 2E). Interestingly, we observed an increase in phospho-ERK cells in response to anti-IgM stimulation in WEHI-303-tdTomato cells that had been cocultured with either isotype- or anti-CD24–stimulated donor cells (Fig. 4A). To more carefully analyze the response of WEHI-303-tdTomato cells that were capable of responding to IgM stimulation, we analyzed only those WEHI-303-tdTomato cells that were GFP+, an indicator of cargo transfer from the donor WEHI-231-GFP cells. We found that anti-IgM treatment stimulated ERK phosphorylation in GFP+ WEHI-303-tdTomato cells that had been cocultured with unstimulated WEHI-231-GFP donor cells and a larger percentage of the WEHI-303-tdTomato cells exhibited increases in phospho-ERK if the donor WEHI-231-GFP cells had been stimulated through CD24 (Fig. 4B). Therefore, the transferred IgM present on the surface of WEHI-303-tdTomato cells is functional, with a greater fraction of the cells acquiring IgM when donor cells were stimulated with CD24.
We next tested if engagement of CD24 on the donor cell can induce transfer of functional CD24 to recipient cells. Previously, we and others have shown that CD24 engagement can induce apoptosis in WEHI-231 and primary bone marrow B cells (22, 24). When CD24KO B cells that had been cocultured with anti-CD24–stimulated WEHI-231-GFP cells were exposed to anti-CD24 Abs, we observed that the recipient cells (>95% B cells) that had acquired CD24 underwent apoptosis (Fig. 4C). Focusing on GFP+ CD24KO B cells (i.e., evidence of cargo transfer), we found an even more pronounced increase in CD24-induced apoptosis (Fig. 4D). Therefore, CD24 that is transferred to CD24-negative primary B cells in response to CD24 stimulation of the donor cells retains its functionality.
IgM stimulation of donor cells induces transfer of lipids and CD24 to recipient cells
Next, we set out to determine whether IgM stimulation of donor B cells can also induce transfer of lipids and proteins. To do this, we used WEHI-231-GFP cells as the donors and CD24KO primary cells as recipients, as in (Fig 3. We found a statistically significantly increase in GFP+eFluor 670+ recipient CD24KO cells (>95% B cells) when the donor WEHI-231 GFP cells had been stimulated with anti-IgM for 1–2 h, compared with when the recipient cells were cocultured with untreated donor cells (Fig. 5A, 5B). We also observed the donor cells had an increase in eFluor 670 fluorescence, showing that anti-IgM stimulation of the donor cells increased the transfer of cytoplasm from the recipient cells (Fig. 5C). To determine if surface proteins are transferred in response to IgM stimulation, we analyzed the acquisition of CD24 by CD24KO primary B cells. Although untreated or control isotype-treated donor cells were able to transfer CD24 to recipient cells (>90% B cells), stimulating the donor cells with anti-IgM for 1–2 h induced a statistically significant higher level of CD24 transfer to the CD24KO primary B cells (Fig. 5D). Thus, IgM stimulation of the donor cells increased the percentage of recipient cells that gained CD24.
IgM stimulation of donor cells induces transfer of signaling-competent IgM to recipient cells
We then determined whether IgM stimulation of donor cells could induce the transfer of signaling-competent IgM to recipient cells. Donor WEHI-231-GFP were mixed with WEHI-303-tdTomato recipient cells and stimulated with anti-IgM. In this case the stimulating Ab was not removed because the IgM-negative WEHI-303 cells do not undergo apoptosis in response to anti-IgM. As above, we found a statistically significantly increase in GFP+tdTomato+ WEHI-303 cells after coculture with anti-IgM–stimulated WEHI-231-GFP cells (Fig. 6A) with a statistically significant increase of low and mid range GFP+ cells, which are likely WEHI-303 cells that acquired GFP from the GFPhi WEHI-231-GFP cells (Fig. 6B). Similar to CD24 stimulation of donor cells, anti-IgM stimulation of the WEHI-231-GFP donor cells induced transfer of lipid from recipient cells to donor cells (Fig. 6C).
Next, we analyzed IgM-mediated apoptosis, which allows us to detect transfer of functional BCRs to the WEHI-303 cells. We were not able to analyze IgM transfer to recipient cells in response to anti-IgM stimulation of donor cells because the stimulating anti-IgM Ab blocks all available epitopes for detecting transferred IgM on the surface of recipient cells (data not shown). We found that anti-IgM–induced apoptosis of WEHI-303-tdTomato cells was significantly increased after they had been cocultured with WEHI-231-GFP cells (Fig. 6D). Not all of the WEHI-303-tdTomato cells that had acquired GFP from the donor cells became sensitive to anti-IgM–induced apoptosis, perhaps because they did not also acquire IgM (Fig. 6E). Thus, similar to CD24 signaling, BCR signaling is also able to induce the transfer of functional membrane proteins to recipient cells. Moreover, the transferred BCR is able to activate signaling pathways that initiate apoptosis, demonstrating that the transferred receptor can interact with host cellular machinery.
In this study, we have demonstrated that stimulation of two receptors on B cells, which differ in structure, can both cause the release of EVs that can alter recipient cell responses by transferring functional membrane proteins. Transfer of membrane proteins was accompanied by a concurrent transfer of lipids to further support that the mechanism of transfer was via EVs. Furthermore, because cSN and isolated EVs were able to transfer lipids and proteins, cell-cell contact was not necessary for this process. To the best of our knowledge, this is the first time that transfer of functional receptors by EVs has been shown to occur in B cells and the first time that stimulation of one receptor resulting in transfer of a second receptor has been shown in any cell type.
In these experiments, the number of cells that acquired lipids was consistently lower than the number that acquired either CD24 or IgM. This is most likely due to differences in the threshold of detection for the fluorophores combined with the limited packaging ability of palm-GFP into EVs. The interior lumen of EVs that are released in response to CD24 stimulation is estimated to be 20–90 nm3 (34). GFP is a 28 kDa cylindrical protein that is 4.2 nm long by 2.4 nm wide (∼19 nm3) (35). Therefore, we can estimate that there could be one to four GFP molecules inside one EV. In contrast, there could potentially be 10-fold more CD24 and IgM molecules on the surface of individual EVs (36), each with the potential to be bound by more than one detection Ab. Thus, the apparent difference in the uptake by recipient cells is most likely due to a limitation of this technique rather than a true difference in uptake of the lipids versus membrane proteins by recipient cells and the amount of lipid transferred is likely underestimated.
Another limitation of this study is that we are using Ab-mediated engagement of CD24 to induce signaling. These conditions may not properly mimic endogenous activation of CD24 in vivo. The ligand for CD24 on bone marrow B cells has not been identified. Identification of the relevant activating ligand would allow us to confirm that ligand-induced clustering of CD24 also stimulates EV release.
Interestingly, even though our data clearly show that EVs are responsible for transfer of lipids and IgM, we did not observe transfer when donor and recipient cells were separated by a membrane (data not shown). Thus, close contact between cells is necessary for the EV-mediated transfer between cell populations. Our previous data showed that CD24 is exchanged between identical cellular populations (24), suggesting that autocrine uptake by the same cell type is likely contributing to the total amount of exchange in the coculture. Thus, we are likely underestimating the extent of exchange mediated by EVs in our coculture system as autocrine mechanisms likely account for a significant fraction of EV uptake.
Using Western blot analysis, we were able to clearly visualize GFP and IgM present in isolated EVs, as validated by HSP90 and CD81 expression. However, we were unable to detect CD24 in a similar manner despite using multiple Abs and conditions (data not shown). However, in our previous work we were clearly able to show by flow cytometry that CD24 was present on subcellular size objects in the culture supernatant that also expressed phosphatidylserine (PS) as detected by Annexin V (24). Stimulation of CD24 increased the abundance of the CD24+Annexin V+ particles (24). In addition, we previously detected CD24 by flow cytometry using bead-based isolation of EVs (25). We suspect that the lack of detection of CD24 in isolated EVs by Western blot may be due to the reduced sensitivity of this technique because of the low levels of protein that are present in EVs. Thus, although we have not conclusively shown that CD24 is also transferred via EVs in this study, the evidence thus far suggests that this is a likely mechanism for the transfer of CD24.
IgM is a transmembrane component of the BCR, which induces apoptosis in immature B cells (37–39). CD24 is a GPI-anchored protein that induces apoptosis in vitro and during B cell development (18, 21–24). CD24 is constitutively located in lipid rafts and the BCR translocates to lipid rafts after stimulation (40, 41). Both can activate similar signaling pathways including those leading to apoptosis. For example, both activate the ERK signaling pathway and both activate the caspase-3 pathway (24, 40 42). It is not known how CD24 or IgM regulates EV release, nor is it clear what specific properties of these receptors induce release of EVs. We found that stimulation of CD40, a lipid raft-resident TNF receptor family member, that is a proproliferative costimulatory receptor for B cells (43, 44) did not induce a statistically significant amount of lipid transfer, potentially owing to limitations in detecting GFP as discussed above, and only a minor amount of IgM transfer (Supplemental Fig. 4A–C). Stimulation of CD48, a GPI-anchored lipid raft-resident, proadhesion and proproliferative costimulatory receptor of CD40 (45–47) was able to induce a small but statistically significant transfer of lipids but not IgM (Supplemental Fig. 4D–F). It is unclear if the small degree of transfer in either case is biologically meaningful. Together, these observations suggest that specific receptors can induce robust receptor-mediated EV release but that others do not. More work is needed to define the precise pathway(s) that regulate both EV release and EV uptake.
The data from this study and our previous studies suggest that the EVs induced by CD24 stimulation are MVs budded off the plasma membrane and not exosomes derived from multivesicular bodies (24, 25). A number of stimuli have been shown to induce MV release from platelets and endothelial cells, including destabilization of the membrane by complement proteins, shear stress, and proapoptotic stimuli (48). Increased [Ca2+]i can also cause lateral redistribution of membrane components to promote membrane curvature (Ref. 49 and C. Allolio and D. Harries, manuscript posted on bioRxiv, DOI: 10.1101/2020.04.29.068221). This physical alteration in membrane curvature, as well as protein crowding on the surface of cellular membranes, can directly contribute to MV formation (50). However, more work is needed to determine how MV release is regulated in response to stimulation of CD24 or IgM.
EV uptake can occur via EV internalization by phagocytosis or micropinocytosis (51), as well as theoretically by direct fusion between EVs and the plasma membrane. The lipid raftlike membrane composition of EVs is also known to contribute to fusion with recipient cell membranes (52). In addition, PS and P-selectin on the exterior of cells are necessary for fusion of tissue factor–expressing MVs with platelets, and PS is necessary for fusion of EVs with glioma cells (53–55). Interestingly, PS on EVs is increased upon stimulation of CD24, suggesting that PS binding may be a mechanism by which the EVs that are released in response to CD24 stimulation bind to target cells (24). In this study, we observed the maintenance of the transmembrane orientation of both CD24 and the BCR by flow cytometry, the appearance of patches of BCR by confocal microscopy, and the transfer of cytosol from primary B cells to WEHI-231 cells, as assessed by the transfer of eFluor 670. Recycling of the BCR to the plasma membrane from the cytosol would result in an inverted orientation of the BCR and CD24. Thus, the maintenance of the orientation of these receptors suggests that there is some fusion of the EVs with the plasma membrane. However, the precise mechanism responsible for EV uptake, and whether these EVs are targeted to recipient cells via specific receptors or PS, is not yet known.
During B cell development, the expression of the BCR is limited to one rearranged H chain and one L chain allele via allelic exclusion. This ensures that B cells can only be activated in response to one, or a closely related set of, antigenic determinants. The data that we present in this study suggest that B cells could acquire additional BCRs, with differing antigenic specificities, owing to the action of EVs in response to stimulation of either CD24 or the BCR. Furthermore, the newly acquired BCRs retain functionality in the recipient cells. Our data show that between 5 and 20% of cells in the bulk culture can acquire new receptors, suggesting that a minority of cells in the population would acquire new receptors in this manner. We speculate that this may be a mechanism to maintain cellular homeostasis. In this model, an increase in CD24+ cells, stimulation of CD24 by an unknown ligand, and/or stimulation of IgM, depending on the cellular compartment, would result in a concurrent increase in the release of EVs bearing CD24 and/or IgM. Uptake of these EVs by neighboring cells would induce cell death in the presence of ligand or Ag. In the bone marrow, an increase in CD24+ pre–B cells could result in increased apoptosis in CD24– B cells, an effect that is consistent with the leaky block in B cell development observed in CD24-transgenic mice (23). Overall, the paracrine effect of EVs could result in the appropriate reduction of cell number needed to maintain homeostasis. However, the presence of a second BCR with a different antigenic recognition could also result in the nonspecific activation of B cells in the presence of costimulation. Future work is needed to determine the contribution of EVs to B cell development and activation.
Interestingly, the transfer of associated receptors in response to stimulation of a particular receptor (i.e., transfer of IgM by CD24 and vice versa) suggests that cells could transfer receptors with different functions than the activated receptor. The consequence of transferring different receptors would depend on the presence of the appropriate ligand for the transferred receptor. Nevertheless, this creates substantial potential for cross-activation of cells owing to their acquisition of new functionalities.
Overall, our data demonstrate that both the BCR and CD24 can enable the transfer of functional receptors, via EVs in the case of BCR. This transfer allows recipient cells to become susceptible to novel antigenic or ligand stimulation. The effects of this acquisition are likely localized in time and space during B cell development or activation in vivo. However, the impact of EV-mediated receptor transfer in vivo remains to be determined.
We thank Nicole C. Smith at the Cold-Ocean Deep-Sea Research Facility, Memorial University of Newfoundland, and Stephanie Tucker and Chris Corkum at the Medical Laboratories, Memorial University of Newfoundland, for expert assistance with flow cytometry. We thank the staff from Animal Care Services, Memorial University of Newfoundland, for excellent assistance. The visual abstract was created in Biorender.com.
This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant 2017-04630 to S.L.C.), a Vietnam International Education Development scholarship (to H.-D.P.), the Natural Sciences and Engineering Research Council of Canada (Undergraduate Student Research Award to R.H.S.), and the Canadian Institutes for Health Research (Grant PJT-152946 to M.R.G.). M.N.L. is a trainee in the Cancer Research Training Program of the Beatrice Hunter Cancer Research Institute, with funds provided by GIVETOLIVE Cancer Studentship.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.