Fig 2: Identification by unbiased proteomic analysis of groups of proteins likely released in the same EV subtypes by Jurkat cells Overlay of proteomic profiles of CD63 and CD81 (left) versus CD81 and ITGB4 (right) showing the relative abundance distribution across the 3 × 3 subfractions obtained from untreated Jurkat cells. Although all three proteins are strongly enriched in F3 fractions, the profiles of CD63 and CD81 are reproducibly different, whereas profiles of CD81 and ITGB4 are extremely similar.Neighbourhood Network plot of CD3G as a single query (replicate tolerance = 50, **network members, cut-off for replicates = 2, 25% distance percentile for edges). Nodes: red = query, orange = close neighbour in all three replicates, grey = close neighbour in two out of three replicates. Edges: percentile within the local distance distribution (thicker edge and darker shade = smaller distance, i.e. closer neighbour); see Materials and Methods for details.Multiple query Neighbourhood Network plot for CD63, CD81 and CD3G. The top 30 close neighbours of each query were jointly used for the network layout (B ranking network members, 50% distance percentile of edges). Nodes: red = query, light red = close neighbours in 2 or 3 replicates, blue = neighbours validated by Immunoisolation, Immuno-EM or MacsPlex Exo in figure 3. Edges: percentile within the local distance distribution (thicker edge and darker shade = smaller distance, i.e. closer neighbour); see Materials and Methods for details. The three networks are remotely connected, but clearly separate.

Fig 3: Unbiased proteomic profiling analysis of EV subtypes released by Jurkat cells AProteomic profiles of different proteins, showing the relative abundance distribution across the 3 × 3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1-F2-F3A, F1-F2-F3B and F1-F2-F3C).BAbundance profiles of over 3,000 proteins in EVs recovered after 10K (F1), 30K (F2) and 100K (F3) centrifugations (see Appendix Fig S1), from Jurkat cells were subjected to principal component analysis (PCA). Each scatter point represents one protein; proximity indicates similar profiles and hence similar distributions across EVs. The PCA scores plot was annotated with known marker proteins of intracellular organelles, and with markers identified previously (Kowal et al, 2016) as specific for CD9- or CD63- or CD81-bearing sEVs in human dendritic cells (Appendix Table S1), as indicated in the legend. Non-marker proteins are shown as grey dots. EVs of different subcellular origin are clearly separated by the profiling analysis. PC1 and PC2 account for 44.5 and 19.1% of the variability in the data, respectively.CViolin plots showing enrichment of the protein markers of intracellular organelles across the F1-F2-F3 subfractions as compared to expression in the total cell proteome. Solid horizontal lines indicate medians, and dashed line indicates quartiles (n = 3). Mitochondria and ER markers are de-enriched, and plasma membrane markers are enriched in EV fractions, with progressively pronounced effects from F1 to F3.D, ENeighbourhood Network plots (top 25% quantile edges) for single queries CD63 (E, **network members, cut-off for replicates = 3) and CD81 (D, *network members, cut-off for replicates = 2), show several ESCRT components in the CD63 network, and ARRDC1 and integrins ITGA4/B1 in the CD81 network. A multi-query network for these two proteins and CD3G, shown in Fig EV1, shows that all three networks are separated, indicating presence of the markers in different EV subtypes. Nodes: red = query, orange = close neighbour in all three replicates, grey = close neighbour in two out of three replicates. Edges: percentile within the local distance distribution (thicker edge and darker shade = smaller distance, i.e. closer neighbour); see Materials and Methods for details.

Fig 4: Biochemical analysis of the composition of EVs released by primary CD4+ T cellsEVs were purified by SEC from supernatant of activated CD4+T cells. EVs were subjected to immunoisolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, CD81, ADAM10 and CD3G. A representative image and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD + FT) in samples obtained from four independent donors are shown.Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 × 109 particles. Captured EVs were detected with either APC-labelled anti-CD81, anti-CD63 or anti-CD3E. Left: Median APC fluorescence values for the different bead populations are shown as a ratio to the median APC fluorescence of control beads (log10 scale). Mean ± SD for four independent experiments is shown. Right: Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81 (mean value of 4 independent donors).