Exosomes in immune regulation and allergy

Abstract: Exosomes are nano-sized vesicles of endosomal origin which are secreted from several different cell types. Depending on their cellular source they have been suggested to have different functions, like having a role in antigen delivery and T cell activation (antigen presenting cell-derived exosomes) or a role in tolerance induction (epithelial cell-derived exosomes). In addition, exosomes have shown potential to be used in immunotherapy both against infections and cancer. This thesis aimed at elucidating the presence of exosomes in vivo, develop methods to asses their function as immune regulators and to investigate if they may have a role in inflammatory responses such as allergies. A lot of investigations have been published on exosomes derived from in vitro culture supernatants but very few studies have been performed showing the presence of exosomes in vivo. Since the lung contain many antigen presenting cells (APCs) and is a site of antigen entry we hypothesized thatbronchoalveolar lavage fluid (BALF) might contain exosomes. By flow cytometry analysis and immune electron microscopy we describe the novel finding of exosomes in BALF. These exosomes expressed the antigen presenting molecules MHC class I and II, the co-stimulatory molecule CD86 and the tetraspanin protein CD63, suggesting them to be of APC origin, and to have a role in the immune defense of the lung. Exosomes from APCs have been proposed to have a role in T cell activation. However, there are contradictory data on how this activation is achieved; i.e. if exosomes can directly activate T cells or if they exert their effect via APCs. We show that dendritic cell (DC)-derived exosomes loaded with viral peptides can activate human autologous peripheral CD8+ T cells to produce IFN-gamma and TNF-alpha without the addition of APCs by using the sensitive enzyme-linked immunospot (ELISPOT) assay. This stimulation was more efficient when using exosomes from mature DCs and was dependent of exosomal MHC class I. These data suggest that DC-derived exosomes may have a role in T cell activation during an immune response and show that the ELISPOT assay is a suitable method to use for evaluating exosome induced immune responses. Breast milk is a complex liquid with immune competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant s immune system. We further demonstrate the presence of exosomes in vivo with the finding that exosomes expressing molecules such as MHC, CD63, CD81, CD86, MUC-1 and heat shock proteins are present in human breast milk. These exosomes inhibited anti-CD3 induced cytokine production from peripheral blood mononuclear cells (PBMC). Furthermore, PBMC incubated with milk-derived exosomes showed a higher number of Foxp3+CD4+CD25+ T regulatory cells. Our results show that human breast milk contains exosomes with the capacity to influence immune responses, and this suggests that they might influence the development of the infant immune system. Since both we and others have shown that exosomes from APCs can have a function in T cell activation, we wanted to further investigate if APC-derived exosomes could have a role in activation of allergen specific T cells. We demonstrate that B cell-derived exosomes loaded with peptides from the major birch pollen allergen Bet v 1 can activate Bet v 1 specific T cells to both proliferate and produce the Th2-like cytokines IL-5 and IL-13 in a dose-dependent manner. This finding suggests that APCderived exosomes may have a role in activation of T-cells during allergic immune responses. In conclusion, the work presented in this thesis has increased our knowledge of the presence, phenotype and function of exosomes found in vivo. Furthermore, it sheds light on the function of APCderived exosomes in T cell stimulations with the implication of a possible role in allergic responses.