Exosomes : nano-vesicles in immune regulation

Abstract

Nano-vesicles (30-100 nm) with an endosome-derived limiting membrane are called exosomes. These are released from the cell when the endosome fuses with the outer cell membrane. Exosomes from antigen presenting cells (APC) carry MHC class I and class II as well as integrins, tetraspanins and co-stimulatory molecules. They can either stimulate T cell responses or induce tolerance. Exosomes are presently being evaluated as therapeutic tools but still little is known about their biological roles. The overall aim of this thesis was to study exosomes as immune-regulators in vitro and to investigate how exosomes found in vivo may exert immune regulatory effects. To achieve this, new methods were developed.

It is debated whether exosomes can stimulate T cells directly or if APC are needed. Exosomes produced by human monocyte-derived dendritic cells (MDDC) were analyzed for their CD8+ T cell stimulatory capacity. This was explored in a virus peptide specific, highly sensitive enzyme-linked immunospot (ELISPOT)-assay measuring IFN-gamma or TNF-alpha release. The stimulation occurred without APCs and was dependent on exosome-dose and MHC class I. Exosomes from lipopolysaccharide (LPS)-matured MDDC were more efficient stimulators than exosomes from immature MDDC. Thus, the method proved to be a sensitive way to measure exosome T cell stimulatory capacity.

Further, MDDCs generated in two different ways and their released exosomes were compared. Exosomes from IL-4/IL-3 generated MDDCs showed significantly higher levels of HLA-ABC, HLA-DR, CD11c, CD63 and CD81 than exosomes from conventional MDDCs. Both kinds of exosomes could stimulate IFN-gamma release from CD8+ T cells using the virus peptide-specific ELISPOT, however, no difference in stimulatory capacity was detected. On the cellular level, the IL-4/IL-3 generated MDDC showed a slightly more efficient T cell stimulatory capacity as compared to conventionally generated MDDC. Thus, exosomes inherited some features from their parent cells, but not all, and both types were able to stimulate virus specific T cells and could potentially be used in therapy.

Next we investigated whether human breast milk contains exosomes. By immuno-capture and flow cytometry, electron microscopy, sucrose density gradient centrifugation, Western blot, and mass-spectrometry, we identified exosomes. Interestingly, these exosomes inhibited anti-CD3 induced cytokine production, which correlated with an induction of CD4+CD25+Foxp3+ regulatory T cells. For the first time the presence of exosomes in human breast milk has been shown and they have a potential to influence immune responses.

To assess if exosomes from different sources have preferences for specific cell types in peripheral blood mononuclear cells (PBMC), we compared the association patterns of exosomes isolated from MDDC, B cells and human breast milk. Flow cytometry and confocal laser scanning microscopy showed that both MDDC- and milk-exosomes preferred monocytes, which mainly ingested the exosomes, whereas B cell exosomes targeted CD19+ B cells and remained surface associated. Multispectral imaging flow cytometry supported our findings and made possible the objective analysis of a high number of cells in each sample. This novel technique can be useful for the development of different exosomes for therapies and for exploring biological roles of exosomes.

In conclusion, the work presented in this thesis has increased our understanding of the presence of human exosomes in vivo, and addressed how different exosomes may exert distinct immunomodulatory effects.