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

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.