May 25–29, 2022 | Lyon, France
King's College London, London, United Kingdom
As important mediators of intercellular communication, alterations in exosomes have been reported in various diseases, including cancer and viral infections. Here we explore the profile of exosomes from recovered COVID-19 patients and their possible impact on the T cell–mediated immune response.
Our cohort included healthy donors (pre and post vaccination) and donors recovered from mild or severe COVID-19. Cell-free plasma was used to purify exosomes with the Exosome Isolation Kit CD63 from Miltenyi Biotec. Exosome origin was established by staining exosomes for more than 30 cell-specific surface markers by flow cytometry with Miltenyi Biotec’s MACSPlex Exosome Kit, human.
We found that exosome profiles related to disease severity, indicating that different subsets of cells were contributing to the anti-viral response in mild and severe infection, while vaccination mimicked aspects of viral infection. Changes were maintained for several weeks post recovery and after vaccination. Interestingly, alterations in T cell–derived exosomes were amongst the most pronounced detected. Donor-derived exosomes influenced cytokine production, activation status, and metabolic profile of T cells in vitro. Exosomes derived from donors recovered from mild disease overall induced immune suppression, while those from donors recovered from severe disease allowed for increased T cell activation and corresponding metabolic reprogramming.
In summary, we find that exosomes in the circulation sustain distinct profiles both post vaccination and during an immune response, making them interesting candidates as biomarkers. Furthermore, we show that patient-derived exosomes influence T cell function indicating their contribution to shaping anti-viral immunity and clinical outcome.
Molly George received her degree in biochemistry at the University of Exeter, UK in 2017. She completed a master’s degree in biotechnology at Imperial College London in 2018. She then went on to work as an assistant at the Francis Crick Institute, before taking on a role as an Analytical Scientist at Oxford Biomedica. In 2020, Molly began her PhD at King’s College London where she researches T cell metabolism, with a dual focus on CAR T cells and the role of extracellular vesicles.
Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
Extracellular vesicles (EVs) carry information from the originating cells, and the EV mixture reflects cell composition and intercellular communication. Different EVs can be distinguished by surface marker profiling. We used immunomagnetic enrichment and a multiplex bead assay to analyze the composition of exosomal surface proteins by flow cytometry.
For immunomagnetic isolation, EVs were incubated with MicroBeads, separated on a magnetic column, washed, and eluted. For comparison, EVs were isolated from plasma by serial centrifugation, filtration, and ultracentrifugation.
The separated fractions were incubated with a mix of 39 distinctly labeled beads conjugated to antibodies which bind to different known exosome surface epitopes. Beads were discriminated by flow cytometry and bound EVs were detected by labeled antibodies.
Compared to isolation by ultracentrifugation, immunomagnetic enrichment resulted in a higher sensitivity of the subsequent analysis. The increasing signal intensities observed for starting volumes from 0.5 to 2 mL of plasma demonstrated effective enrichment of EVs and the possibility of obtaining semiquantitative results with the multiplex bead assay (MACSPlex Exosome Kit, human). Prominent signals for CD41b or CD42a indicated that platelet EVs were dominant in plasma. The tetraspanin markers CD9 and CD81 were present on exosomes in the isolated fraction. Furthermore, markers for lymphocytes (CD45), T cells (CD8), or epithelial cells (CD31) were detected.
We also investigated surface proteins of EVs directly from ascites of ovarian cancer patients. Besides the common exosome markers CD9, CD63, and CD81, we detected markers indicating blood cell-derived vesicles like CD4 and CD8 (for T cells) and CD14 (for monocytes). Interestingly, different positive signals for key tumor markers such as EpCAM, CD133 (PROM1), CD44, and CD49e were detected in ascites from different patients, indicating the presence of tumor-derived exosomes.
We present an easy workflow comprising immunomagnetic enrichment and a multiplex bead assay for surface marker profiling of EVs.
Stefan Wild received his degree in chemistry at the Ludwig-Maximilians-Universität Munich, Germany in 1998. He completed his PhD in biochemistry in 2001. From 2002 to 2005 he worked as research scientist for Memorec Stoffel GmbH. Since 2005 he is senior scientist R&D at Miltenyi Biotec. As a group leader, he is responsible for the development of new tools to investigate extracellular vesicles.
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