CD4⁺ T cell activation promotes the differential release of distinct populations of nanosized vesicles

Abstract

Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the plasma membrane. Vesicles actively released by CD4+ T cells have immune-modulatory characteristics. Using our recently developed high-resolution flow cytometry-based method for the analysis of individual nanosized vesicles, we here investigated how T cell receptor (TCR)-triggering and co-stimulatory signals influence the quantity and characteristics of nanosized vesicles released by CD4+ T cells. We found that the number of released nanosized vesicles within the buoyant density range characteristic for exosomes (1.10–1.19 g/ml) was increased by TCR-triggering and that additional co-stimulatory signals had a potentiating effect on vesicle release. However, the increase in the number of released vesicles varied substantially between density fractions within the 1.10–1.19 g/ml range and was highest for the vesicle populations in 1.14 and 1.17 g/ml fractions. Heterogeneity was also observed within the individual density fractions. Based on lipid bilayer fluorescent labelling intensity and light scattering, 3 distinct vesicle subpopulations were identified. One vesicle subpopulation increased significantly more upon T cell activation than the other subpopulations, and this was dependent on high levels of co-stimulation. These data show that T cells release a heterogeneous population of nanosized vesicles and indicate that T cells differentially regulate the release of distinct vesicle subpopulations depending on their activation status.

The role of microRNA in nutritional control

“…(ii) In a Dicer-independent mechanism, miRNA is cleaved by Ago2 to form a mature miRNA. targeting of vesicles released by dendritic and T cells [6,7]. Analysis of these vesicles requires dedicated nanotechnology, such as the newly developed high-resolution flow cytometric technique for high-throughput multiparameter analysis of individual nano-sized vesicles [8].…”

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

“…More importantly, the dot plots in Figure 3D clearly illustrate a change in light scatter-based heterogeneity in EVs when measured at too high vesicle concentrations. Besides light scatter profile of EVs, we previously demonstrated high-resolution flow cytometric identification of different EV populations based on fluorescent antibody labeling (3,4,10). We here show that co-staining of CFSE-labeled mast cell-derived vesicles …”

“…The release of EVs and their content, i.e., proteins, lipids and RNAs, is tightly regulated and varies not only between different cell types but also depends on the physiological state of the producing cell (2)(3)(4). Consequently, EV release is very dynamic and the EV population is very heterogeneous.…”

“…3). This method has previously also facilitated the identification of activation status-dependent changes in EV subsets released by dendritic cells and T cells (3,4,10). However, to gain more insight in the composition and function of specific EV subsets and to identify EV-based biomarkers, accurate methods are needed for EV-subset isolation.…”

Post-translational add-ons mark the path in exosomal protein sorting

“…The existence of heterogeneous MVBs populations becomes apparent when different components of endosomal trafficking or the ESCRT pathway are silenced or overexpressed, and when exosomal secretion is disturbed and not all exosomal markers are affected to the same extent [10,15]. This enables distinguishing between global markers, e.g.…”