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. 2008 Apr;123(4):491-9.
doi: 10.1111/j.1365-2567.2007.02714.x. Epub 2007 Oct 19.

Different types of in vitro generated human monocyte-derived dendritic cells release exosomes with distinct phenotypes

Sara M Johansson  1 , Charlotte Admyre, Annika Scheynius, Susanne Gabrielsson
Affiliations

Different types of in vitro generated human monocyte-derived dendritic cells release exosomes with distinct phenotypes

Sara M Johansson et al. Immunology. 2008 Apr.

Abstract

Human in vitro generated dendritic cells and the exosomes they release are potential tools for the modulation of immune responses. Here, we characterized differently generated monocyte-derived dendritic cells (MDDCs) and their exosomes. Culturing of peripheral CD14+ cells from the same individuals with either interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (conventional MDDCs) or alternatively with IL-4 and IL-3 generated immature MDDCs in 7 days. Fluorescence-activated cell sorting (FACS) analysis showed that the IL-4/IL-3-generated MDDCs had significantly lower percentages of CD1a+, CD40+ and CD80+ cells and a higher percentage of CD86+ cells as compared with conventional MDDCs. In addition, IL-4/IL-3-generated MDDCs had significantly higher densities of major histocompatibility complex (MHC) class I [human leucocyte antigen (HLA)-ABC], MHC class II (HLA-DR), CD11c and the tetraspanin CD81 as compared with conventional MDDCs. In a comparison of their ability to stimulate CD8+ T cells, we found that the IL-4/IL-3 MDDCs were slightly more efficient than the conventional MDDCs at inducing interferon (IFN)-gamma release in response to viral peptides. Exosome morphology was confirmed by electron microscopy and exosome phenotypes were analysed by flow cytometry and western blot. In comparison to exosomes from conventional MDDCs, exosomes from IL-4/IL-3-generated MDDCs showed significantly stronger signals for HLA-ABC, HLA-DR, CD11c, CD63 and CD81. Thus, phenotypically the exosomes largely reflected their MDDCs of origin. When exosomes were loaded with viral peptides, both types of exosomes induced IFN-gamma release from CD8+ T cells. Our findings might have significance for the development of DC- and exosome-based therapies.

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Figures

Figure 1
Figure 1
Phenotypes of interleukin (IL)-4/IL-3 monocyte-derived dendritic cells (MDDCs) differ from those of conventional MDDCs. Conventional and IL-4/IL-3-generated MDDCs were stained at day 7 with a panel of fluorescence-conjugated monoclonal antibodies (mAbs) against cell surface molecules. Histograms show fluorescence intensities with overlay above those of isotype-matched controls (grey); one representative experiment from 23–30 carried out on samples from healthy blood donors is shown.
Figure 2
Figure 2
CD8+ T-cell stimulatory capacities are similar for conventional and interleukin (IL)-4/IL-3-generated monocyte-derived dendritic cells (MDDCs). Purified autologous CD8+ T cells were stimulated with conventional MDDCs (GMDC) and IL-4/IL-3 MDDCs (IL3DC) preloaded with a viral cytomegalovirus, Epstein–Barr virus and influenza virus (CEF) peptide mix. Medium alone was the negative control (Ctrl) while CEF alone served as a positive control. (a) CD8+ T cells stimulated for 48 hr with MDDCs loaded with viral CEF peptides in an interferon (IFN)-γ-specific enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). Values are presented as number of spot-forming cells (SFCs) per 2 × 105 cells (means of triplicate experiments for five individuals). (b) CD8+ T cells stimulated as above for 4 days followed by 18 hr of [3H]thymidine incorporation for the proliferation assay. Results are presented in counts per minute (c.p.m.) for four individuals assayed in triplicate. Wilcoxon's matched pairs test was applied and P-values are presented; ns, not significant.
Figure 3
Figure 3
Similar morphology of exosomes from interleukin (IL)-4/granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4/IL-3-generated monocyte-derived dendritic cells (MDDCs). Electron microscopy pictures of exosomes from (a) IL-4/GM-CSF and (b) IL-4/IL-3 MDDC culture supernatants are shown. Cell densities were adjusted to be equal for both culture conditions and supernatants were collected after 24 hr. Exosomes were directly captured by adding anti-human leucocyte antigen (HLA) class II antibody-coated magnetic beads. Exosomes on the surface of a bead are indicated by arrows. Scale bars, 100 nm.
Figure 4
Figure 4
Exosomes from monocyte-derived dendritic cells (MDDCs) generated with interleukin (IL)-4/granulocyte–macrophage colony-stimulating factor (GM-CSF) or IL-4/IL-3 show differential expression of surface molecules. Exosomes from MDDC culture supernatants of IL-4/GM-CSF (open bars)- or IL-4/IL-3 (dotted bars)-generated MDDCs were captured on anti-human leucocyte antigen (HLA) class II beads, labelled with fluorescent antibodies and analysed by flow cytometry. Gates were set on single beads and 5 × 103 events were collected for each sample. Box-and-whisker plots illustrate the median and range, and the box includes the 25–75 percentiles of the mean fluorescence intensity (MFI) values. n, number of healthy blood donors. P-values are indicated; ns, not significant.
Figure 5
Figure 5
Exosomes from interleukin (IL)-4/IL-3 monocyte-derived dendritic cells (MDDCs) have a higher relative enrichment of human leucocyte antigen (HLA)-DR than conventional MDDC-derived exosomes. (a) Equal total protein amounts (3 µg/well) from ultracentrifuged conventional MDDC exosomes (left lane) or IL-4/IL-3 MDDC exosomes (right lane) were loaded onto 8–16% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, blotted and incubated with anti-actin and anti-HLA-DR monoclonal antibodies and detected using horseradish peroxidase and chemiluminescence. (b) Relative HLA-DR expression in ultracentrifuged conventional MDDC exosomes (open bar) or IL-4/IL-3 MDDC exosomes (black bar). Developed films were scanned and the band intensities (intensity/mm2) were measured using quantity one software. The local lane background was subtracted. Values represent mean percentage ± standard deviation (SD) of conventional exosome intensity (n = 4).
Figure 6
Figure 6
Both interleukin (IL)-4/granulocyte–macrophage colony-stimulating factor (GM-CSF)- and IL-4/IL-3-generated monocyte-derived dendritic cell (MDDC) exosomes are able to stimulate autologous CD8+ T cells in a virus peptide-specific manner. Equal amounts of exosomes from IL-4/GM-CSF (GM)- and IL-4/IL-3 (IL3)-generated MDDCs plus cytomegalovirus, Epstein–Barr virus and influenza virus (CEF) peptides were used to stimulate autologous CD8+ T cells. Unblocked T cells plus CEF peptides are displayed to demonstrate individual variation in antiviral responses. W6/32 monoclonal antibody (mAB) was used to block major histocompatibility complex (MHC) class I on the T cells. (a) Exosomes were loaded by acid elution to pH 5·2 followed by neutralization in excess CEF peptides. Unbound CEF was filtered away and as a filtration control (FCtrl) the top fraction of a filter run with only CEF peptides was included. (b) Where blocking with W6/32 mAb was efficient and exosome supply limited, exosomes were directly added together with CEF to the CD8+ T cells. Free CEF alone of the same concentration was used as a control (Ctrl). Data are presented as number of spot-forming CD8+ T cells (SFCs)/2 × 105 cells, with the mean of triplicate experiments for three (a) and two (b) individuals indicated by different symbols.
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