In this thesis, we developed and characterized novel methods to isolate of extracellular vesicles (EV) from biological fluids or tissue culture medium, analysed the molecular composition of EV from seminal plasma (spEV), and investigated effects of isolated spEV on immune cell functions.
In Chapter 2, we illustrate the many shortcomings of commonly used single step EV isolation techniques, and describe a novel three step protocol for the isolation of EV with very high purity and yield from blood or cell culture media. This protocol involves precipitation of EV by polyethyleneglycol (PEG), followed by iohexol density gradient centrifugation, and finally size exclusion chromatography (SEC). This protocol can be used for EV based biomarker discovery, or to investigate biological functions of EV on target cells.
In Chapter 3, we isolated and characterized the protein content of spEV isolated from seminal plasma of vasectomized men. With this source, contributions from the testis and epididymis were excluded. Seminal plasma contains ~0.4 mg/ml EV, which is much higher than EV concentrations in any other type of body fluid. We profiled two distinct spEV subtypes with average diameters of 50 nm and 100 nm. To separate EV with such small size differences, we used a method based on velocity gradient centrifugation. Analysis of the protein compositions of these two subtypes of spEV by quantitative Liquid Chromatography- Mass Spectrometry (LC-MS/MS) revealed 1558 proteins, ~45% of which was detected only in the 100 nm EV, 1% only in the 50 nm EV, and 54% in both 100 nm and 50 nm EV. Gene ontology enrichment analysis indicates that both classes of spEV originated in majority from the prostate, but with distinct biogenetic pathways. Moreover, we identified 9 proteins with known prostate specific expression and alternate expression levels in prostate cancer tissue. The latter data have potential for the discovery of EV associated prostate cancer biomarkers in blood. Both spEV subtypes were also found to contain proteins with potential immune regulatory functions.
In Chapters 4 and 5, we investigated in vitro the effects of isolated spEV on immune cell functions. In Chapter 4, we report that spEV were predominantly acquired by monocytes when incubated with peripheral blood mononuclear cells. Culturing of isolated CD14+ monocytes in the presence of GM-CSF and IL-4 normally drives their differentiation into CD14-CD1a+ and CD14-CD1a- populations of monocyte-derived dendritic cells (moDC). We found that spEV interfered with the differentiation of CD1a+ moDC, suggesting interference with cell-mediated immunity. Also, T cells endocytosed spEV, albeit much less as compared to monocytes. T cells that were activated in the presence of spEV were strongly inhibited in their capacity to produce the cytokines IFN-γ and TNF. Moreover, spEV reduced the surface expression of CD25 and proliferation of activated CD4+ and CD8+ T cells. spEV strongly stimulated differentiation of activated CD4+ T cells into regulatory Treg cells. Plasmacytoid dendritic cells (pDC) are essential to link innate immunity with adaptive immune responses to virus infection, including in cervical mucosa, and initiation of fetomaternal immune tolerance. In Chapter 5, spEV were tested in vitro for their effects on the pDC activation.
At physiological concentrations, spEV interfered with the activation of TLR7 challenged pDC, but not TLR9 stimulated pDC, as measured by IFN-α secretion and CD40 expression. Consistent with endosomal TLR7 signalling, spEV were also efficiently acquired and endocytosed by pDC. Proteolysis of surface exposed proteins or blocking of CD38 with antibodies interfered with the inhibitory effect of spEV on pDC activation. Collectively, the data in Chapters 4 and 5 support the idea that spEV may drive immune tolerance for allogeneic paternal antigens within the female reproductive tract. As a side effect, however, by inhibiting immune cell functions, spEV may also promote virus transmission and prostate cancer.