Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could clearly separate two EV populations, namely SEVs (average diameter <200 nm) and LEVs (diameter range between 200 and 1000 nm). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD009074.

Extracellular vesicles mediate intercellular communication: Transfer of functionally active microRNAs by microvesicles into phagocytes.

Cell activation and apoptosis lead to the formation of extracellular vesicles (EVs) such as exosomes or microvesicles (MVs). EVs have been shown to modulate immune responses; recently, MVs were described to carry microRNA (miRNA) and a role for MVs in the pathogenesis of autoimmune diseases has been discussed. Here we systematically characterized MVs and exosomes according to their release stimuli. The miRNA content of viable or apoptotic human T lymphocytes and the corresponding MVs was analyzed. miRNA, protein and surface marker expression, as well as cytokine release by human monocytes was measured after EV engulfment. Finally, miRNA expression in T lymphocytes and MVs of healthy individuals was compared with those of systemic lupus erythematosus (SLE) patients. We demonstrate that, depending on the stimuli, distinct subtypes of EVs are released, differing in size and carrying a specific RNA profile. We observed an accumulation of distinct miRNAs in MVs after induction of apoptosis and the transfer of functional miRNA by MVs into human monocytes. MVs released from apoptotic cells provoke less of an inflammatory response than those released from viable cells. MiR-155*, miR-34b and miR-34a levels in T lymphocytes and corresponding MVs were deregulated in SLE when compared to healthy individuals.