HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection

Peer reviewe

Analysis of the microRNA profile and origin of exosomes in plasma of melanoma patients and healthy individuals

Exosomen sind extrazelluläre Vesikel, die von lebenden Zellen in einem Energie-abhängigen Prozess sekretiert werden. Studien haben gezeigt, dass Exosomen von Tumorzellen und Immunzellen Immunreaktionen stimulieren und unterdrücken können. Daher sollte in dieser Arbeit untersucht werden, ob sich Plasmaexosomen von Melanom-Patienten und gesunden Spendern in ihrem microRNA-Profil unterscheiden. Wir entdeckten kein microRNA-Set, das alle Melanom-Patienten als solche identifizieren konnte. Die Signale schwankten jedoch zwischen den Spendern und wir können nicht ausschließen, dass einige microRNAs bei einzelnen Patienten auf den Tumor hinweisen können. Im Durchschnitt zeigten die Plasmaexosomen der Melanom-Patienten einen 6.9-fachen Signalanstieg aller detektierten microRNAs im Vergleich zu gesunden Kontrollen. Um die Zelltypen zu identifizieren, die den Anstieg der exosomalen miRNA-Signale bewirken, haben wir uns dazu entschieden, die Proteinbeladung der Exosomen zu charakterisieren da es kaum Zelltyp-spezifische miRNAs gibt. Dazu haben wir eine multiplex bead-based platform entwickelt, die die parallele Detektion von 39 Oberfächenproteinen ermöglicht. Manche Proteine, die zur Identifikation von Zelltypen verwendet werden, werden auf die entsprechenden Exosomen übertragen und lassen so auf die Herkunftszellen der Exosomen schließen. Zum Beispiel wurde CD14 auf Monozyten-Exosomen detektiert, CD19 auf B-Zell-Exosomen und CD41b, CD42a, CD61 und CD62P auf Thrombozyten-Exosomen. Die Aktivierung von B-Zellen und die Differenzierung von dendritischen Zellen aus Monozyten (moDCs) zeigten, dass auch manche Reifungs- und Differenzierungsmarker auf Exosomen übertragen werden. Die Kombination verschiedener Fangantikörper-Partikel mit verschiedenen Detektionsantikörpern innerhalb der multiplex bead-based platform ermöglichte die Entdeckung unbekannter Exosomen-Subpopulationen wie CD19lowCD20high Exosomen in der Exosomen-Präparation von stimulierten B-Zellen. Interessanterweise fehlte der so-genannte Exosomen-Marker CD9 auf Exosomen von B-Zellen und NK-Zellen und die CD81-Signale waren auf Thrombozyten-Exosomen kaum nachweisbar. Diese Erkenntnisse wurden mittels hochauflösender Fluoreszenz-Mikroskopie (STED) validiert. CD63 wurde in allen Exosomen-Präparationen nachgewiesen und ist daher möglicherweise ein verlässlicherer Exosomen-Marker. Die Protein-Profile verschiedener Immunzell-Exosomen wurden mit denen von Plasmaexosomen verglichen und zeigten, dass sich Exosomen von Thrombozyten, moDCs, B-Zellen, T-Zellen und NK-Zellen im Blut befinden. Plasmaexosomen von Melanom-Patienten und gesunden Spendern zeigten unterschiedliche Signalintensitäten für die Thrombozyten-Marker CD42a und CD62P, was auf eine veränderte Exosomen-Sekretion oder eine geänderte Proteinbeladung der Thrombozyten-Exosomen hinweist. Das schwächere CD8-Signal auf den Melanom-Plasmaexosomen könnte auf eine verringerte Exosomen-Sekretion von zytotoxischen Zellen wie T-Zellen und NK-Zellen hinwiesen. Die Melanom-Marker MCSP, CD146 und CD49e wurden zwar auf Exosomen aus Melanom-Zellkulturen detektiert, aber nur das CD49e-Signal war auf den Plasmaexosomen von zwei von vier Melanom-Patienten im Vergleich zu gesunden Spendern erhöht. Wir schließen aus unseren Ergebnissen, dass Melanom-Exosomen nicht den Großteil der Plasmaexosomen ausmachen. Da sich die miRNA-Profile der Melanom-Patienten und der gesunden Spender stark ähneln, wurde der Signalanstieg der exosomalen miRNAs im Plasma der Melanom-patienten ebenfalls nicht durch miRNAs vom Tumor verursacht. Wir denken, dass das Melanom die Exosomen-Sekretion von Blutzellen (wie Thrombozyten und zytotoxischen Zellen) verändert. Indem die Zusammensetzung und die Funktion verschiedener Blutzell-Exosomen aufgedeckt werden, könnten wichtige Erkenntnisse gewonnen werden, um die interzelluläre Kommunikation über Exosomen während der Tumorentwicklung besser zu verstehen.Exosomes are extracellular vesicles released from living cells in an energy-dependent process. They differ from other extracellular vesicles such as apoptotic vesicles and membrane vesicles due to their endocytic origin. Exosomes from tumor cells and immune cells were shown to activate as well as inhibit immune responses. Therefore, we aimed to investigate whether plasma exosomes from melanoma patients and healthy donors differ in their microRNA (miRNA) content. However, we could not identify a subset of tumor-related microRNAs for all patients. Instead, we observed variations between donors and cannot exclude that some microRNAs might be an indicator of tumor burden in individual patients. On average, the exosomal microRNA signal intensity increased by the factor of 6.9 for melanoma plasma exosomes as compared to healthy donors. To identify the cell types that are responsible for this signal increase of exosomal microRNAs, we decided to continue with the protein characterization of plasma exosomes because there are hardly any cell type-specific miRNAs. For this purpose, we established a multiplex bead-based platform that allows the detection of 39 surface proteins in parallel. Our analysis revealed that some surface proteins that are used to identify cell populations are transferred to the respective exosomes and can be used to relate exosomes to their originating cells. For example, CD14 was detected on monocyte-derived exosomes, CD19 on exosomes from B cells and CD41b, CD42a, CD61, and CD62P on platelet exosome preparations. B cell stimulation and the generation of monocyte-derived dendritic cells (moDCs) further demonstrated that some markers for activation and differentiation are also transferred to exosomes. The combination of different capture and detection antibodies with the multiplex platform revealed unidentified exosome subpopulations such as CD19lowCD20high exosomes in exosome preparations from stimulated B cells. Interestingly, two of the so called exosome markers were not detected on every exosome sample. CD9 was missing on exosomes from B cells and NK cells, while CD81 was hardly detectable on platelet exosomes. These observations were validated by high-resolution fluorescence microscopy (STED) that was established to visualize the protein distribution on single exosomes. CD63 was present on every exosome preparation and might be more reliable as an exosome marker. The comparison of surface protein profiles from various immune-cell derived exosomes with plasma exosomes revealed the contribution of platelets, moDCs, B cells, T cells, and NK cells to the exosome pool found in plasma. On plasma exosomes from melanoma patients, we observed changed signals for the platelet markers CD42a and CD62P as compared to plasma exosomes from healthy donors, indicating an altered exosome secretion or protein loading of exosomes by platelets. A weaker CD8 signal on melanoma plasma exosomes as compared to healthy plasma exosomes might point at a diminished exosome secretion by cytotoxic cells such as T cells and NK cells. The melanoma markers MCSP, CD146, and CD49e were detected on exosomes from melanoma cell culture, but only the CD49e signal was elevated in two out of four plasma exosome samples from melanoma patients as compared to healthy donors. We conclude that melanoma exosomes are not a major component of the pool of plasma exosomes. Consistent with the similar microRNA profiles for melanoma patients and healthy controls, the increase of exosomal microRNAs in plasma is likely not caused by microRNAs being secreted from the tumor. We suggest that melanoma alters the exosome secretion of blood cells such as platelets and cytotoxic cells. Revealing the contribution and the function of different immune cell-derived exosomes in plasma might advance our understanding of cell-cell communication via exosomes during tumor progression

Melanoma affects the composition of blood cell-derived extracellular vesicles

Extracellular vesicles are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell and extracellular vesicles in melanoma patient’s plasma could be indicative for the tumor. Likewise, extracellular vesicles might influence tumor progression by regulating immune responses. We performed a broad protein characterization of extracellular vesicles from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer cells, monocytes, monocyte-derived dendritic cells and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on extracellular vesicles. Hierarchal clustering of protein intensity patterns grouped extracellular vesicles according to their originating cell type. The analysis of extracellular vesicles from stimulated B cells and monocyte-derived dendritic cells revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of extracellular vesicles from platelets, antigen presenting cells and natural cells as shown by platelet markers, costimulatory proteins, and a natural killer cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers indicating a changed vesicle secretion or protein loading of extracellular vesicles by platelets and a lower CD8 signal that might be associated with a diminished activity of natural killer cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of extracellular vesicles in melanoma plasma, but rather argue for an indirect influence of melanoma cells on the vesicle secretion or protein loading by blood cells

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions