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Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins

NK cells can broadly target and kill malignant cells via release of cytolytic proteins. NK cells also release extracellular vesicles (EVs) that contain cytolytic proteins, previously shown to induce apoptosis of a variety of cancer cells in vitro and in vivo. The EVs released by NK cells are likely...

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Autores principales: Aarsund, Miriam, Nyman, Tuula Anneli, Stensland, Maria Ekman, Wu, Yunjie, Inngjerdingen, Marit
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9520454/
https://www.ncbi.nlm.nih.gov/pubmed/36189227
http://dx.doi.org/10.3389/fimmu.2022.977353
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author Aarsund, Miriam
Nyman, Tuula Anneli
Stensland, Maria Ekman
Wu, Yunjie
Inngjerdingen, Marit
author_facet Aarsund, Miriam
Nyman, Tuula Anneli
Stensland, Maria Ekman
Wu, Yunjie
Inngjerdingen, Marit
author_sort Aarsund, Miriam
collection PubMed
description NK cells can broadly target and kill malignant cells via release of cytolytic proteins. NK cells also release extracellular vesicles (EVs) that contain cytolytic proteins, previously shown to induce apoptosis of a variety of cancer cells in vitro and in vivo. The EVs released by NK cells are likely very heterogeneous, as vesicles can be released from the plasma membrane or from different intracellular compartments. In this study, we undertook a fractionation scheme to enrich for cytolytic NK-EVs. NK-EVs were harvested from culture medium from the human NK-92 cell line or primary human NK cells grown in serum-free conditions. By combining ultracentrifugation with downstream density-gradient ultracentrifugation or size-exclusion chromatography, distinct EV populations were identified. Density-gradient ultracentrifugation led to separation of three subpopulations of EVs. The different EV isolates were characterized by label-free quantitative mass spectrometry and western blotting, and we found that one subpopulation was primarily enriched for plasma membrane proteins and tetraspanins CD37, CD82, and CD151, and likely represents microvesicles. The other major subpopulation was enriched in intracellularly derived markers with high expression of the endosomal tetraspanin CD63 and markers for intracellular organelles. The intracellularly derived EVs were highly enriched in cytolytic proteins, and possessed high apoptotic activity against HCT-116 colon cancer spheroids. To further enrich for cytolytic EVs, immunoaffinity pulldowns led to the isolation of a subset of EVs containing the cytolytic granule marker NKG7 and the majority of vesicular granzyme B content. We therefore propose that EVs containing cytolytic proteins may primarily be released via cytolytic granules.
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spelling pubmed-95204542022-09-30 Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins Aarsund, Miriam Nyman, Tuula Anneli Stensland, Maria Ekman Wu, Yunjie Inngjerdingen, Marit Front Immunol Immunology NK cells can broadly target and kill malignant cells via release of cytolytic proteins. NK cells also release extracellular vesicles (EVs) that contain cytolytic proteins, previously shown to induce apoptosis of a variety of cancer cells in vitro and in vivo. The EVs released by NK cells are likely very heterogeneous, as vesicles can be released from the plasma membrane or from different intracellular compartments. In this study, we undertook a fractionation scheme to enrich for cytolytic NK-EVs. NK-EVs were harvested from culture medium from the human NK-92 cell line or primary human NK cells grown in serum-free conditions. By combining ultracentrifugation with downstream density-gradient ultracentrifugation or size-exclusion chromatography, distinct EV populations were identified. Density-gradient ultracentrifugation led to separation of three subpopulations of EVs. The different EV isolates were characterized by label-free quantitative mass spectrometry and western blotting, and we found that one subpopulation was primarily enriched for plasma membrane proteins and tetraspanins CD37, CD82, and CD151, and likely represents microvesicles. The other major subpopulation was enriched in intracellularly derived markers with high expression of the endosomal tetraspanin CD63 and markers for intracellular organelles. The intracellularly derived EVs were highly enriched in cytolytic proteins, and possessed high apoptotic activity against HCT-116 colon cancer spheroids. To further enrich for cytolytic EVs, immunoaffinity pulldowns led to the isolation of a subset of EVs containing the cytolytic granule marker NKG7 and the majority of vesicular granzyme B content. We therefore propose that EVs containing cytolytic proteins may primarily be released via cytolytic granules. Frontiers Media S.A. 2022-09-15 /pmc/articles/PMC9520454/ /pubmed/36189227 http://dx.doi.org/10.3389/fimmu.2022.977353 Text en Copyright © 2022 Aarsund, Nyman, Stensland, Wu and Inngjerdingen https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Aarsund, Miriam
Nyman, Tuula Anneli
Stensland, Maria Ekman
Wu, Yunjie
Inngjerdingen, Marit
Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins
title Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins
title_full Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins
title_fullStr Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins
title_full_unstemmed Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins
title_short Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins
title_sort isolation of a cytolytic subpopulation of extracellular vesicles derived from nk cells containing nkg7 and cytolytic proteins
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9520454/
https://www.ncbi.nlm.nih.gov/pubmed/36189227
http://dx.doi.org/10.3389/fimmu.2022.977353
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