Exosomes from Bone Marrow Dendritic Cells Pulsed with Diphtheria Toxoid Preferentially Induce Type 1 Antigen-Specific IgG Responses in Naive Recipients in the Absence of Free Antigen

Mice

BALB/c mice were obtained from The Jackson Laboratory and used at 8–10 wk of age for the experiments. Mice were maintained in a pathogen-free environment at Uniformed Services University of the Health Sciences. The experiments in this study were conducted according to the principles set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Production and culture of BMDC

DC were cultured from bone marrow of BALB/c mice in the presence of 10 ng/ml GM-CSF as described previously (15), using culture medium supplemented with 6% FCS that was ultracentrifuged two times at 100,000 × g for 18 h at 10°C for depletion of bovine exosomes and protein aggregates.

Preparation of exosomes

BMDC were cultured at 1 × 10⁶ cells/ml in the presence or in the absence of 30 μg/ml detoxified DT provided by Dr. J. Mond (Biosynexus, Gaithersburg, MD). After 24 h of culture, both DT-pulsed and unpulsed BMDC were stimulated with 20 ng/ml LPS to induce DC maturation or maintained in the absence of LPS, followed by addition of another 30 μg/ml DT to the DT-pulsed BMDC cultures. After another 24 h of culture, BMDC culture supernatants were collected and cell debris was removed by centrifugation at 300 × g for 10 min at 4°C. Cleared culture supernatants were filtered through 0.22-μm cellulose acetate filters (GE Osmonics). Exosomes are 60–90 nm in diameter and filter freely through 0.22-μm filters. We did not observe significant losses of tetraspan-containing microvesicles after filtration. Filtered supernatants were centrifuged at 10,000 × g for 30 min, and the exosomes collected from the supernatant at 100,000 × g for 1 h at 4°C, washed with cold PBS, and then resuspended in PBS at a 1/500 volume. Sterility was preserved throughout the process. Purified exosomes contained less than one viable bacteria per 25 μg of protein. Protein content was determined by the bicinconinic acid method (Pierce).

Exosomes purified from culture supernatants of BMDC pulsed with DT are referred to as “DT-exosomes,” and those from BMDC cultured in the absence of DT as “control-exosomes.” Preparations obtained from LPS-stimulated BMDC cultures are referred to as “mature control- or DT-exosomes,” and those from cultures lacking LPS as “immature control- or DT-exosomes.” A similar preparation obtained from culture medium is referred to as “Media” and was used to control for the potential contaminant carryover during the purification. This preparation contained no detectable protein (<6 μg/ml).

Coupling of exosomes to latex beads and immunofluorescence analysis

A total of 10⁶ aldehyde/sulfate polystyrene latex beads (Interfacial Dynamics), 3.9 μm in diameter, was coated with 1 μg of exosomes for 16 h at 20°C, washed, and incubated for 1 h with 1% BSA. For FACS analysis, 50,000 latex beads coated with exosomes were incubated with 0.2 μg of fluorochrome-conjugated mAbs specific for several DC markers or isotype-matched negative control mAbs. Samples were analyzed in triplicate. All mAbs used were obtained from BD Pharmingen, except for anti-CD9 mAb (clone Nyn.H3), which was obtained from Emfret. Latex beads incubated with the Media preparations and blocked with BSA were used as a control for nonspecific binding of mAb to beads. Only single beads were gated for analysis. Acquired data were analyzed using the WinList3D program. The mean fluorescence intensity of the phenotypic markers was used to compare their relative expression in mature and immature exosomes.

Quantitation of bovine contaminants in the preparations of exosomes

Bovine haptoglobin (Hp) was quantified essentially as previously reported for human Hp (16) using both a direct adsorption ELISA and a capture ELISA with affinity-purified rabbit anti-bovine Hp (Immunology Consultants Laboratory). Hp purified from fresh bovine acute-phase serum (Life Diagnostics) was used to generate a standard curve. The limit of detection was 0.5 ng/ml (22 ng/ml aggregated Hp) on direct ELISA and 0.75 ng/ml (250 ng/ml aggregated Hp) in capture ELISA.

BSA content was determined by direct ELISA in wells coated with exosomes, and blocked with 1.5% OVA. BSA was detected with mouse IgG2a anti-BSA species-specific mAb BSA-33 (Sigma-Aldrich). The limit of detection was 0.7 ng/ml BSA.

Bovine exosomes were detected by a sandwich-capture ELISA using two mAbs specific for bovine tetraspans CD9 (capture) and CD63 (detection). Therefore, the ELISA detects CD9-CD63 complexes, but not free CD9 or CD63. Wells were coated with 1 μg/ml purified mouse IgG2 anti-bovine CD9 mAb (Abcam; clone IV50), blocked with 2% BSA in PBS, and incubated overnight at 4°C with exosomes. Captured CD9-containing vesicles were detected with a mouse IgG1 anti-bovine CD63 mAb (clone CC25; Serotec). Bovine FCS (not ultracentrifuged) used as a reference, typically gives an OD₄₀₅ of 3.0. Guinea pig sera and wells not incubated with anti-CD63 mAb were used as negative controls. More than 93% of the detectable CD9-CD63 vesicles were pelleted by a single ultracentrifugation of the FCS.

Bovine microvesicles were detected by a sandwich-capture ELISA using two mAbs specific for bovine tetraspans CD9 and CD63. Therefore, the ELISA detects CD9-CD63 complexes likely assembled in microvesicles, but not free CD9 or CD63. Wells were coated with 1 μg/ml purified mouse IgG2 anti-bovine CD9 mAb (clone IV50; Abcam), blocked with 2% BSA in PBS, and incubated overnight at 4°C with exosomes. Captured CD9-containing vesicles were detected with a mouse IgG1 anti-bovine CD63 mAb (clone CC25; Serotec). Bovine FCS (not ultracentrifuged) used as a reference, typically gives an OD₄₀₅ of 3.0. Guinea pig sera and wells not incubated with anti-CD63 mAb were used as negative controls. Approximately 93% of the detectable CD9-CD63 was pelleted by a single ultracentrifugation of the FCS at 100,000 × g for 18 h.

Quantitation of murine cytokines

IL-6 and IL-12 concentrations in the purified exosomes were measured using a quantitative sandwich ELISA previously described in detail (15).

Quantitation of DT

The content of intact DT in the purified exosomes was determined by competitive inhibition ELISA. Briefly, wells coated with 5 μg/ml DT, and blocked with 2% BSA, were incubated with serial 2-fold dilutions of purified DT standard or DT-exosomes diluted in a 1/200,000 dilution of a high-titered mouse anti-DT sera. Control-exosomes were used as negative controls. The Ig binding was detected with a goat anti-mouse IgG conjugated to AP. The limit of detection of the assay was 5 ng/ml DT. DT was also quantified in exosomes following treatment for 1 h at room temperature with 1% Triton X-100 (TX-100). Both samples and standards were treated with detergent during incubations.

Mouse immunizations

Groups of 20 female BALB/c mice at 8–10 wk of age were immunized i.p. with 250 μl of an emulsion of CFA and PBS or CFA and a solution of 25 μg of DT in PBS at day 0. Twenty-four days later, five mice of each group received one i.v. injection of 25 μg of mature or immature DT-exosomes or 25 μg of mature or immature control-exosomes in 200 μl of PBS. Additional groups of mice immunized with CFA or DT plus CFA emulsion at day 0, received at this time (day 24) one i.v. injection with an equal volume of media, or with 25 pg of LPS and 0.02 μg of intact DT in PBS, two times the upper limit of DT that could be detected in a 25-μg dose of exosomes. All mice were bled at day 0 to obtain preimmune sera, and at days 14, 23, 38, and 44 for measurement of anti-DT Ig isotype titers by ELISA.

Measurement of serum titers of Ig anti-DT isotypes

DT-coated and 2% BSA-blocked ELISA plates were incubated with serial 3-fold serum dilutions overnight at 4°C. Replicate plates were then incubated for 1 h at 37°C with polyclonal goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM, or IgG (γ-chain) conjugated to alkaline phosphatase (Southern Biotechnology Associates). Titers were expressed as the dilution of sera giving an absorbance at 405 nm equal to 1.0, following the development of the enzymatic reaction for 1 h, using p-nitrophenyl-pyrophosphate as substrate. Preimmune sera were used as negative controls.

Statistics

Data were expressed as arithmetic mean ± SEM of the individual titer. Levels of significance of the differences between the groups were determined by the Student’s t test. Values of p < 0.05 were considered statistically significant.

Purified exosomes from BMDC express high levels of Ag-presenting and costimulatory molecules

Our major goal was to determine the ability of BMDC-derived exosomes to induce Ag-specific humoral immunity in vivo, following preincubation of BMDC cultures in vitro with a soluble protein. DT, a highly immunogenic protein widely used as a carrier in conjugate vaccines, was selected as our model protein (17, 18). BMDC were cultured in the presence or in the absence of DT for 48 h, and the exosomes were then purified from the culture supernatant. This early time point in the culture was selected to reduce the risk of contamination with apoptotic microvesicles, because BMDC undergo some spontaneous apoptosis following the first 48 h of culture (19). Exosomes from these immature BMDC showed high levels of tetraspans (CD9 and CD81), predominant in exosomes, CD11b and one of its ligands (CD54), but very low levels of CD11c (Fig. 1⇓). These exosomes also expressed relatively high levels of MHC-II, MHC-I, CD1d, and CD86, with CD80 expressed at lower levels (Fig. 1⇓). Because exosomes from immature BMDC have been reported to have anti-inflammatory (9) and even T cell-immunosuppressive properties (8), we wanted to compare these immature exosomes with those obtained from BMDC stimulated during the last 24 h of culture with 20 ng/ml LPS. This stimulation induced BMDC maturation as evidenced by up-regulation of cell surface MHC-II and CD86 (data not shown). Exosomes secreted from LPS-matured BMDC, showed an identical expression level of tetraspans relative to immature BMDC, consistent with the attachment to the beads of equal numbers of exosomes, but demonstrated an increased expression of MHC-II, CD86, CD80, and especially of CD54 (Fig. 1⇓). CD54 has been shown to be critical for priming of naive T cells (4). The increased expression of theses markers was similar in two independent experiments run in triplicate (p < 0.01). The phenotype of the control-exosomes obtained from BMDC cultured in the absence of DT, was identical with that shown for DT-exosomes in Fig. 1⇓, suggesting the pulse with DT do not affect the phenotype of the secreted exosomes. Collectively, these data suggest that exosomes will have immunostimulatory properties that may be enhanced by BMDC maturation.

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FIGURE 1.

Phenotype of exosomes. Latex beads coated with exosomes purified from the culture supernatant of BALB/c (MHC-II^d) BMDC pulsed with DT in the absence (immature) or presence of 20 ng/ml LPS after 24 h of culture (LPS-matured) were analyzed by FACS. Latex beads coated with Media were used as controls. The histograms obtained for one of the negative isotype controls (rat IgG2b) is shown. The phenotype of the control-exosomes obtained from BMDC cultured in the absence of DT was identical with that shown for DT-exosomes.

Purified exosomes are essentially free of bovine contaminants

Next, we determined the level of medium-derived protein contaminants within the purified exosomes, because these contaminants could produce artifacts following mouse immunization. Three bovine proteins: tetraspan (CD9/CD63) complexes, Hp, and BSA, were used as tracers. Tetraspans are highly expressed in cell membranes and were thus used as markers of contamination for bovine vesicles. Bovine tetraspans were readily detectable in de-complemented FCS, but following the first and second ultracentrifugation of FCS used for BMDC culture, >93% and essentially 100% of the detectable tetraspans were removed, respectively. Furthermore, bovine tetraspans were undetectable in BMDC-derived exosomes (Table I⇓), suggesting that all the purified exosomes were murine.

Hp present at low concentrations in FCS (1.6–2.6 ng/ml), is readily polymerized by heating, generating very high m.w. aggregates that could be copurified with exosomes (12). Thus, Hp is a useful protein marker to follow both purity and copurification of aggregated proteins. Aggregated Hp was completely removed following FCS ultracentrifugation (<0.3 ng/ml) and was undetectable in the purified exosomes (<0.005% of total protein; Table I⇑), suggesting that aggregated bovine proteins are not a significant contaminant of our preparations of purified exosomes.

Ultracentrifugation removed <14% of total BSA, which is a major component of FCS (>600 μg/ml culture medium). In this regard, BSA was detected in the purified exosomes used in the experiments illustrated in Figs. 1–3⇑⇓⇓ (Table I⇑), although it was <0.065% of the total protein. A similar content of BSA was detected (0.42 ± 0.05 ng/μg of protein; n = 32) in other preparations of exosomes. The tendency of BSA to copurify with exosomes has been observed (12). Collectively, these data suggest that, although some major protein component of FCS may be copurified in very low but quantifiable amounts, we can consider our BMDC-derived exosomes as being essentially free of bovine contaminants.

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FIGURE 2.

DT-specific Ig isotype responses induced by exosomes derived from DT-pulsed BMDC. BALB/c mice (five mice per group) were immunized i.p. with an emulsion of CFA (A) in PBS (CFA) or (B) containing 25 μg of DT (CFA+DT), and 24 days later, mice received an i.v. injection (indicated by arrows) of 25 μg of exosomes purified from the culture supernatant of BMDC loaded with DT and stimulated with LPS (mature DT-exosomes) or not (immature DT-exosomes), or from BMDC not exposed to DT but stimulated with LPS (mature control-exosomes). Additional control groups were injected with 25 pg of LPS plus 20 ng of intact DT. Data are expressed as the geometric mean ± SEM. Statistical significance between the Ig titers induced by DT-exosomes and control-exosomes is indicated by an asterisk (∗) and between those induced by mature and immature DT-exosomes by a pound sign (#). The data shown are representative of two independent experiments.

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FIGURE 3.

Isotype distribution of the IgG responses induced by intact DT and exosomes derived from BMDC loaded with DT. DT-specific serum titers of IgG subclasses in the experiment illustrated in Fig. 2⇑ were determined by ELISA. A, Primary IgG subclass responses to intact DT, 14 (left panel) and 21 (center panel) days after i.p immunization with a DT plus CFA emulsion (day 0); right panel, i.p. immunization of DT-processed in exosomes 20 days after the injection of DT exosomes (day 24) in mice i.p. immunized at day 0 with an emulsion of CFA in PBS (naive mice). B, Secondary IgG subclass responses to a boosting immunization (day 24) with 25 pg of LPS plus 20 ng of intact DT (left panel), control-exosomes (center panel), or DT-exosomes (right panel) in mice primed at day 0 with a DT plus CFA emulsion. Data are presented as the geometric mean ± SEM. For mice primed with DT, only the IgG subclass responses to mature control- or DT-exosomes are shown, because titers did not differ from those obtained in mice injected with the corresponding immature exosomes.

Purified exosomes derived from BMDC pulsed with DT do not contain serologically detectable DT

Next, we wanted to determine the potential presence of serologically intact DT within the purified exosomes. Using a high-titered mouse anti-DT sera in a competitive inhibition ELISA, we determined that DT was not serologically detectable (<0.04% protein or <10 ng of DT in 25 μg of exosomes) in either intact (Table I⇑) or 1% TX-100 solubilized (data not shown) control- or DT-exosomes. The pulse of BMDC with DT did not affect the yield or purity of the exosomes. LPS-matured BMDC consistently yielded lower amounts of exosomes than immature BMDC (Table I⇑). Collectively, the purified preparations of immature exosomes (n = 6) yielded for control exosomes 0.82 ± 0.09 μg/ml supernatant, similar (p = 0.07) to that of DT-exosomes (0.76 ± 0.08 μg/ml supernatant). In contrast, the yield of mature exosomes was lower than for immature exosomes in both control exosomes (0.65 ± 0.07 μg/ml supernatant; p = 0.01) and DT-exosomes (0.59 ± 0.06 μg/ml supernatant; p = 0.008)

Exosomes from DT-pulsed BMDC induce IgG DT-specific responses in naive recipients

Immunization of naive mice with intact CFA plus DT induced DT-specific IgM and IgG responses with the delayed kinetics and isotype distribution typical of responses to T cell-dependent Ags (Fig. 2⇑B). Thus, IgG anti-DT was the prevalent isotype, peaking at 24 days postimmunization. In contrast, IgM anti-DT titers were relatively low, peaked at day 14, and gradually decreased to undetectable levels by day 30. However, a major part of the decreased detection of IgM over the time is likely due to competition with IgG anti-DT, present in sera in overwhelming amounts, at time points later than 14 days. Sera from naive mice contained no detectable IgG anti-DT (titer <1/10) and CFA alone did not induce IgG anti-DT responses, or significantly affect preimmune natural IgM anti-DT levels (Fig. 2⇑A).

We next determined the ability of exosomes, derived from either immature or mature DT-pulsed BMDC (DT-exosomes), to initiate primary Ig isotype responses in naive mice, and/or to boost secondary Ig responses in mice primed 24 days earlier with intact DT. Exosomes (25 μg of total protein) were injected i.v, without adjuvant, allowing exosomes direct access to the spleen in the absence of local inflammation. Exosomes purified from both DT-pulsed immature and mature BMDC induced primary IgM and IgG anti-DT responses, more sustained than that observed in naive mice immunized with intact DT (Fig. 2⇑A), and boosted the IgG anti-DT response in DT-immune mice (Fig. 2⇑B). However, mature DT-exosomes induced a primary IgG anti-DT response (Fig. 2⇑A) that was 10-fold higher than the IgG response induced by immature DT-exosomes (p < 0.0003), whereas the primary IgM (p > 0.11) and boosted IgG (p > 0.05) anti-DT responses were not significantly different between the two groups. In contrast, injection of Media or control-exosomes from immature (data not shown) or mature (Fig. 2⇑) BMDC had no effect on these responses. These responses were unlikely due to free intact DT, because serologically detectable DT in the dose injected was <0.01 μg. Furthermore, we measured <1 pg of LPS per microgram of exosome protein and the injection of 25 pg of LPS and 0.02 μg of intact DT had no effect on these responses (Fig. 2⇑). These results suggest that exosomes are a competent stimulus to initiate primary humoral Ag-specific responses, as well as to stimulate a secondary response in primed mice. Furthermore, the maturation state of the DC releasing the exosomes has a major impact in the efficiency of those exosomes to induce primary Ab responses in naive recipients.

IgG2b and IgG2a are the major subclasses induced by DT-exosomes, in contrast to the predominant IgG1 anti-DT response induced by intact DT

We next wanted to determine the IgG subclass distribution induced by either intact DT or DT-exosomes. Immunization with intact DT induced IgG responses that were mostly IgG1 (type 2 response). Thus, serum titers of IgG1 anti-DT were 30–100 times higher than those of IgG2a or IgG2b anti-DT at any given time point postimmunization (Fig. 3⇑A). IgG3 was only a minor part of the IgG anti-DT response. Serum titers and kinetics for the IgG1 anti-DT response following immunization with intact DT, was very similar to that obtained for total IgG (Fig. 2⇑).

In striking contrast, both immature and mature DT-exosomes induced primary DT-specific IgG responses that were predominantly IgG2b and IgG2a (type 1 response), with only a relatively modest contribution by IgG1 (3- to 5-fold lower serum titers than IgG2b or IgG2a) (Fig. 3⇑A). As mentioned earlier for IgG, higher overall IgG isotype responses, with the exception of IgG3 (p = 0.056), were observed in response to mature, relative to immature, DT-exosomes. Thus, exosomes induce primary IgG responses in nonimmune recipients through a type 1-associated mechanism. This result lends strong support to the notion that exosomes are directly involved in the induction of IgG anti-DT responses, and not copurified traces of free DT.

In mice primed with intact DT, secondary immunization with mature DT-exosomes significantly boosted IgG1 anti-DT (p = 0.03–0.01), IgG2a anti-DT (p = 0.0001–0.000006), and IgG2b anti-DT (p = 0.006–0.003) responses relative to either mature or immature control-exosomes, Media, or 20 ng of intact DT plus LPS. In contrast, IgG3 titers were unaffected (p = 0.74–0.56). Immature DT-exosomes also boosted each anti-DT IgG subclass in a similar manner as the mature DT-exosomes (Fig. 3⇑B; p = 0.87–0.19). Of interest, the IgG1/IgG2a anti-DT titer ratio, which was 76 ± 12, 21 days after primary immunization with intact DT, was essentially maintained 20 days after a subsequent injection with control exosomes (IgG1/IgG2a ratio, 51 ± 14; p = 0.14). However, following secondary immunization with either immature or mature DT-exosomes, this ratio was significantly reduced (IgG1/IgG2a ratio, 10 ± 2; p = 0.0002), suggesting that exosomes could mediate a relative shift to a type 1 response in mice previously primed for a type 2 response.

Exosomes do not contain detectable IL-12 or IL-6

BMDC activation with pathogen-associated molecular patterns result in secretion of proinflammatory cytokines, particularly IL-12 and IL-6 (15, 19). We observed >30 ng/ml of both IL-12 and IL-6 in BMDC culture supernatants after 24 h of stimulation with 20 ng/ml LPS (data not shown). IL-12 is a key type 1-polarizing cytokine produced by DC (20), whereas IL-6 can act directly on B cells to induce maturation to Ab secretion (21). IL-12 and IL-6 have been implicated in DC priming for antibacterial IgG responses (15). However, we were unable to detect either IL-12 or IL-6 in the intact (Table I⇑) or 1% TX-100 solubilized exosomes (data not shown), arguing against their direct role in the exosome-mediated induction of type 1 polarizing activity and Ig induction, respectively.