Analysis of extracellular vesicles and their application in immunotherapy

Abstract

Extracellular Vesicles (EVs) are nano-sized particles secreted by most, if not all, cell types. They are important mediators in intercellular communication and carry a broad array of biomolecules such as lipids, nucleic acids and proteins. Because of their ability to deliver functional biomolecules, EVs have been studied and utilized in both physiological and pathological settings. The EV family is composed of multiple types of vesicles, including microvesicles, deriving from the cell membrane, and exosomes which are of endosomal origin. Despite the two different biogenesis pathways of these vesicles, they share many characteristics which makes it difficult to separate them during EV isolation. Hence, the name EVs is preferred when working with vesicles.

Many different EV isolation methods exist based on distinct EV characteristics, e.g. size and density, but it still remains largely unknown if different isolation methods also enrich for certain EV subpopulations. Furthermore, it is known that to isolate a pure EV population, a combination of methods needs to be applied. However, depending on the study set up, this is not always possible. Therefore, we isolated EVs with five different methods using two different sample types, i.e. human plasma and conditioned cell culture supernatant, and performed a thorough analysis of the isolated EV fractions (study I). Although each isolation method enriched for EVs, there was a rather large heterogeneity between the samples, both for plasma and cell culture supernatant, regarding particle count, particle size, RNA amount and protein amount. Furthermore, we identified that certain methods enriched for proteins related to cell organelles such as Golgi or endoplasmic reticulum, which originally are not classified as EV-related proteins. We concluded that, depending on the sample type and volume, different isolation methods are preferred.

EVs are being applied in a therapeutic setting and especially Dendritic cell (DC) derived EVs have been used, as they can induce antigen-specific T and B cell responses. However, it is not clear whether different EV subtypes can have different immunostimulatory capacities. Therefore, we compared the immunostimulatory capacities of DC derived microvesicles and exosomes, loaded with the antigen Ovalbumin (OVA), in vivo (study II). The results showed that exosomes induce significantly more antigen-specific T cells than microvesicles and higher levels of OVA-specific IgG in the serum. This was likely due to the higher levels of OVA on exosomes, indicating that different EV subsets have different capabilities to load and transfer antigen. To further investigate the immunostimulatory capacities of DC derived EVs, allogeneic exosomes and syngeneic exosomes were compared side by side, as the usage of allogeneic exosomes would enhance the applicability of EVs in the clinic (study III). Both OVA-loaded allogeneic and syngeneic exosomes induced high levels of antigen-specific T cells. Interestingly, allogeneic exosomes induced significantly more T follicular helper cells and increased OVA specific IgG-levels, indicating that allogeneicity might serve as an adjuvant. Since DC derived exosomes are able to induce strong antigen-specific immune responses, we tested if they could sensitize a tumour model that is non-responsive to anti-PD-1 or anti-PD-L1 treatment, into a responsive tumour (study IV). Indeed, we saw that in a prophylactic B16 melanoma tumour model, exosomes and anti-PD-L1 combination improved survival as compared to exosomes or PD-L1 treatment alone. This suggests that exosomes can be used in combination treatment, in which they sensitize the tumour to checkpoint blockade.

In conclusion, this thesis provides an improved understanding of EV isolation and specifically on the purity and EV enrichment of different methods. Furthermore, this thesis describes new knowledge on DC-derived EVs, suggesting that exosomes are the preferred EV subtype for EV-based immunotherapies and that allogeneic exosomes can promote the humoral immune responses. Lastly, DC-derived EVs can sensitize non-responsive melanoma to anti-PD-1/PD-L1, showing that DC-derived EVs can be used in cancer combination therapy.