A mixed lymphocyte reaction as a functional assay for extracellular vesicles of different origins

Abstract

Background & Aim Extracellular vesicles (EVs), such as exosomes and microvesicles, are shed by all cell types and found in all body fluids. EVs transmit specific information from their cells of origin to specific target cells and are key factors in a novel form of intercellular communication. Depending on their origin, EVs can modulate immune responses and either act proinflammatory (e.g. mature DC-EVs) or anti-inflammatory (e.g. mesenchymal stem cell (MSC) and many tumor cell-derived EVs). Methods, Results & Conclusion Aiming to analyze immune-modulating properties of EVs from different sources, in vitro, we established a novel form of a mixed lymphocyte reaction (MLR) assay. Here, human peripheral blood-derived mononuclear cells (MNCs) were pooled from up to 12 different healthy donors warranting high cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed MNCs are cultured for 5 days in the absence or presence of EVs. Thereafter, cell morphologies are documented and cells are phenotypically characterized by flow cytometry. By analyzing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by therapeutically active MSC-EVs. For example we observed that in the presence of active MSC-EVs more CD14+ (monocytes) and CD56+ (natural killer cells) are recovered from the MLR than in corresponding control samples. In contrast, in the presence of active MSC-EVs contents of CD4+ and CD8+ T cells got slightly decreased. Focusing on T cells, we learned that active MSC-EVs reduced the content of CD4 and CD8 T cells expressing T cell activation markers like CD54 and CD25. Currently, we compare the immunomodulatory capabilities of EVs of different cell types. Furthermore, we proceed in optimizing the marker panel to distinguish immune cell subtypes such as the different types of CD4+ cell types (T 1, T 2, T 17 and T).