Dendritic cell-derived exosomes express a Streptococcus pneumoniae capsular polysaccharide type 14 cross-reactive antigen that induces protective immunoglobulin responses against pneumococcal infection in mice

Abstract

Exosomes activate T cells in vivo, but whether exosomes are able to induce humoral immune responses is still unknown. We found that dendritic cells, but not other immune cells, constitutively release an exosome-associated glycoconjugate that is cross-reactive with the capsular polysaccharide of Streptococcus pneumoniae type 14 (Cps14-CRA). Cps14-CRA was localized to the cholesterol-enriched microdomains or rafts of the exosomes and was mapped to the beta1-->6 branched N-acetyl-lactosamine derivatives of the Cps14-CRA. Injection of CFA-primed naive mice with purified dendritic cell exosomes induced immunoglobulin (Ig) anti-Cps14 responses composed predominantly of IgM, IgG3, and IgG1. These responses were associated with protection against a lethal challenge with live S. pneumoniae type 14, but not with type 3 bacteria, and was correlated with the titer of elicited IgM and IgG3 anti-Cps14. These data show, for the first time, that exosomes can induce a humoral immune response to an associated unprocessed, autologous antigen. Although anti-Cps14 Ig responses are specifically demonstrated, these could reflect a broader mechanism that modulates both natural immunity and autoimmunity to other glycotopes.

FIG. 1.

BMDC constitutively release CD9-containing microvesicles. (A) BMDC supernatants collected at different times during the culture period were filtered through 0.22-μm filters and treated with 0.2% saponin or 1% Triton X-100 for 30 min at room temperature. Microvesical CD9 content was estimated by sandwich-capture ELISA using the same anti-CD9 MAb for capture and detection. Results are expressed as μg/ml of protein exosomes using a preparation of purified BMDC-derived exosomes as a standard. Data represent the arithmetic means ± SEM of three wells tested separately. (B) CD9 in BMDC supernatants was captured using anti-CD9 (“anti-CD9 captured”) or anti-CD81 MAbs (“anti-CD81 captured”). In both cases, captured microvesicles were detected with biotinylated anti-CD9 MAb. (C) Culture supernatants filtered through 0.22-μm filters were concentrated 10-fold by ultrafiltration through 100K MWCO units, and the eluted solution was 10-fold concentrated in 5K MWCO units. Both concentrated samples were diluted to the initial volume to obtain a direct comparison with the nonconcentrated culture supernatant. As a reference, crude supernatant (unfiltered through 0.22-μm filters) was included in the assay. Data displayed in each graph are from three separate experiments. OD 405 nm, optical density at 405 nm.

FIG. 2.

An IgG1 anti-Cps14 MAb captures CD9-containing microvesicles. ELISA plates coated with 5 μg/ml of 44.1 IgG1 MAb specific for the Cps14 of Pn14 were incubated with BMDC supernatants collected at different times during the culture period, and the captured CD9 was detected with 1 μg/ml of biotinylated anti-CD9 MAb. (A) BMDC supernatants were tested directly (crude supernatant), after filtration through 0.22-μm filters, or once concentrated 10-fold in 100K MWCO units by ultrafiltration. (B) In a separate experiment, both the 10-fold 100K MWCO concentrated supernatant and the filtrate from this ultrafiltration were 10-fold reconcentrated in 5K MWCO ultrafiltration units. Wells coated with an IgG1 MAb of unrelated specificity were used as controls. In the same experiment, the concentrated supernatants were treated with 1% Triton X-100 (C) or 0.2% saponin (D) for 30 min at room temperature. OD 405 nm, optical density at 405 nm.

FIG. 3.

Detergent solubility of the Cps14-CRA. Supernatants collected at 48 h of BMDC culture were 10-fold concentrated in 100K MWCO filters units and treated with increasing concentrations of saponin or MbCD. Microvesicles were captured in ELISA plates coated with 5 μg/ml of anti-Cps14 IgG1 MAb 44.1 (“Cps14-CRA”) or with anti-CD9 MAb (“CD9”). Microvesicles captured with either MAb were detected with 1 μg/ml of anti-CD9 MAb. Data shown represent the arithmetic means ± SEM of three independent experiments run in duplicate.

FIG. 4.

Detection of Cps14-CRA in supernatants of different cultured cell types. Cell culture supernatants filtered through 0.22-μm filters and concentrated 120-fold in 100K MWCO ultrafiltration units were diluted to pretitered normalized dilutions containing amounts of CD9 microvesicles similar to those of the undiluted supernatants of BMDC cultures at day 7. These dilutions were 12-fold concentrated for the supernatant of the B-cell hybridoma expressing CD9, 6-fold concentrated for the supernatants of BM cells at day 2 of culture in GM-CSF, 2-fold concentrated for the BMM cell line supernatant (BMM cell line), 1.2-fold concentrated for the BM-derived macrophages supernatants at day 7 of the primary culture (BMM, d7), and 1-fold concentrated for the respective BMDC at day 7 of culture. Control culture media and the supernatant from splenocyte cultures were used at the maximal concentration (12-fold concentrated) due to their absent or very low content of CD9-containing microvesicles. Supernatant samples at those dilutions were incubated in ELISA plates coated with anti-CD9 MAb (“anti-CD9 captured”) and at a 10-fold-higher concentration in plates coated with the anti-Cps14 MAb 44.1 (“44.1 captured”). The captured microvesicles were detected in both ELISAs by incubation with biotinylated anti-CD9 MAb. All supernatants were collected after 48 h of cell culture, except for the supernatant of the B-cell hybridoma (28.6.20) that was collected after 4 days of culture. Bars represent the arithmetic means ± SEM of the optical density at 405 nm (OD₄₀₅) obtained in the triplicate samples in one of three independent experiments.

FIG. 5.

Specificity of the interaction between MAbs specific for the Cps14 of Pn14 and microvesicles derived from BMDC in culture. Purified IgG1 (44.1) or IgM (17.1 and 23.1) MAbs specific for Cps14 and two control IgM MAbs, one specific for the Cps of Neisseria meningitidis group A (“anti-CpsA,” 8F11.1) and the other specific for dextran (“antidextran,” clone MOPC-104E), were captured in wells precoated with anti-IgG1 or anti-IgM antibodies, and then incubated with 40 ng/ml of biotinylated Cps14 (A), 10-fold concentrated BMDC supernatant (B), or 10-fold concentrated BMDC supernatants supplemented with purified Cps14 (C). Captured biotinylated Cps14 was directly detected (A), and the CD9-containing microvesicles with anti-CD9 were biotinylated (B and C). Data in panels A and B show the arithmetic means of results for triplicate samples ± SEM of the optical density at 450 nm (OD₄₅₀) obtained in one of three independent representative experiments. Data in panel C show the percentages of inhibition obtained in those three experiments.

FIG. 6.

Phenotype of BMDC-derived microvesicles. BMDC culture supernatants collected at day 7 of the primary culture (A) or once washed and cultured for 48 h in the absence (media) or in presence of 20 ng/ml of LPS (LPS) (B) were concentrated 10-fold by ultrafiltration in 100K MWCO filter units. Microvesicles were captured in 44.1-coated ELISA plates and detected with MAbs specific for the cell markers indicated on the figure. The plates were developed when CD9 expression reached an optical density at 405 nm (OD₄₀₅) of 2.0 to normalize the content of microvesicles in the different samples. The data shown are the arithmetic means ± SEM of three separate experiments (A) or the duplicates of one experiment (B).

FIG. 7.

Cps14-specific Ig isotype responses induced by exosomes. Kinetics of the IgM and IgG anti-Cps14 response (A) or Cps14-specific IgG subclasses (B) at day 43 induced in mice immunized i.p. with an emulsion of CFA and PBS at day 0 and 24 days later (indicated by arrows) injected intravenously with 25 μg of BMDC exosomes (“exosomes”) or an equal volume of the material collected after the ultracentrifugation of fresh culture media following an identical protocol as for the purification of exosomes (“media”). The data shown are the geometric means ± SEM.

FIG. 8.

Protection conferred by the injection of BMDC-derived exosomes. (A) BALB/c mice immunized as indicated in the legend to Fig. 7 were challenged i.p. with 9,000 CFU or 12,000 CFU of strain WU-2 of Pn3 or 8 × 10⁷ CFU or 2.5 × 10⁸ CFU of Pn14 20 days after being injected with exosomes or control media. The number of mice per group injected with exosomes or “media” is indicated in each graph. Data shown are from three independent experiments. (B) Geometric means ± SEM of IgM and IgG3 anti-Cps14 titers in the sera of mice at day 0 (preimmune), at day 14 (post-CFA), and at day 43 (postexosomes), 1 day before the mice received the challenge with 25 × 10⁷ CFU of Pn14. These mice are the same shown in the lethality studies for this bacterial dose (A). IgM and IgG3 titers of the survivors and animals that died after the bacterial challenge were considered separately to determine the potential association between protection and anti-Cps14 Ig responses induced by BMDC-derived exosomes.