Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens

Abstract

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Figure 1

Visualization of individual objects by combining LSM and advanced digital image restoration. Cells were cultured in a collagen matrix to immobilize the cells and released vesicles after LPS stimulation for 24 hours. All images represent the same confocal data set taken from one cell. A: Three-dimensional reconstruction from top; B: volume rendering from the side; C–E: surface rendering from the cell (green), segmented into individual objects (cell body yellow, external objects turquoise).

Figure 2

Visualization and quantification of released exovesicles. A: LSM images of iDC compared with LPS-maturated DCs after 2 (a, d), 6 (b, e), and 24 (c, f) hours. iDCs (a–c) and LPS-DCs (d–f), in collagen matrix, showing the release of exovesicles (turquoise vesicles) at different time points. B: Total amount of exovesicles released by DCs, calculated by the IMARIS software at the different time points, ie, 2, 6, and 24 hours. Data are expressed as mean ± SD of two experiments with scanning of 8 to 10 cells each by LSM. The asterisk represents a statistically significant difference (P < 0.01) between LPS-treated and control groups. C: Size frequency of exovesicles, calculated by the Surpass module software in IMARIS, of DCs and LPS-stimulated DCs at 6 and 24 hours in the co-culture conditions. Bars are means ± SD of nine data sets.

Figure 3

TEM of human DCs processed using alginate matrix. A: LPS-DCs cultivated for 6 hours in alginate matrix showing cell bodies and ruffling of the plasma membrane. In our TEM analysis, we confirm the absence of extensive apoptosis or apoptotic bodies. Insets A′ and A″ show exovesicle (arrows) close to the cell. Inset A′ shows details of exovesicle budding (arrowhead). B: Characterization of LPS-DC-derived exovesicles. Exovesicles from 10 × 10⁶ LPS-DCs obtained after filtration and ultracentrifugation were resuspended in alginate. Different shapes of exovesicles are seen with a bilayer lipid membrane and suspended in the matrix.

Figure 4

Co-localization analysis of the incorporation of red-labeled exovesicles into green-labeled cells. Three-dimensional data of DiO-labeled iDCs (green) co-cultured with DiI-labeled LPS-treated DCs (red) in collagen matrix were taken with LSM and co-localization analysis was performed. A: Co-cultures of control cells (red and green). No red signal is seen in green cells. B: Exovesicles released from LPS-stimulated DCs fuse with the plasma membrane of iDCs, as shown by the incorporation of red material (yellow indicates co-localization of green and red, arrows). Little intracellular red material is seen (arrowhead). Images represent xy- and xz-projections; yellow arrowheads mark the position of projections. Insets represent three-dimensional reconstructions from the same data sets. C: Quantification of co-localized voxels related to the volume (μm³) in the plasma membrane of iDCs in the co-culture of nonstimulated DCs (iDC DiO − iDC DiI) or in the co-culture with LPS-stimulated DCs (iDC DiO − LPS-DC DiI). Data are expressed as mean ± SD of three experiments with LSM scanning of 10 cells each. The asterisk represents a statistically significant difference (P < 0.02) between LPS-treated and control groups.

Figure 5

Release of exovesicles from iDCs in the vicinity of mDCs. A: LSM image of DiO-labeled iDCs (green) co-cultured with DiI-labeled LPS-stimulated DCs (red) in collagen matrix for 24 hours. In A′, incorporated red material can be observed (arrows). A: Three-dimensional reconstruction; A′: xy- and xz-projections from the same data set; yellow arrowheads mark the position of projections. B: Diagram showing total amount of external objects released by iDCs in the co-culture of nonstimulated DCs (left panels) or in the co-culture with LPS-stimulated DCs (right panels). Data are expressed as mean ± SD of two experiments with LSM scanning of 8 to 10 cells each. The asterisk represents a statistically significant difference (P < 0.001) between LPS-treated and control groups.

Figure 6

Analysis of antigen-presenting function of DCs by exovesicles. MLR was used to assess the stimulatory function of exovesicles. DCs (DC1) were co-cultured with syngeneic lymphocytes (T1) at a constant concentration with or without exovesicles isolated from allogeneic pretreated DCs (DC2 and DC2-LPS) or syngeneic pretreated DCs (DC1 and DC1-LPS) as controls, as described in Materials and Methods. DC1/T1 cell ratio was 1:10. The co-cultures were incubated for 6 days and proliferation was measured by the incorporation of tritiated thymidine. One of five independent experiments, done in triplicate, is shown. Results are expressed as means ± SD. One asterisk represents a statistically significant difference (P < 0.01) compared with T1 + DC1 + Exo (DC2) control conditions, and two asterisks represents a statistically significant difference (P < 0.05) compared with T1 + DC1 + Exo (DC1-LPS) control condition.