Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins. 2022

Miriam Aarsund, and Tuula Anneli Nyman, and Maria Ekman Stensland, and Yunjie Wu, and Marit Inngjerdingen
Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.

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.

UI MeSH Term Description Entries
D007694 Killer Cells, Natural Bone marrow-derived lymphocytes that possess cytotoxic properties, classically directed against transformed and virus-infected cells. Unlike T CELLS; and B CELLS; NK CELLS are not antigen specific. The cytotoxicity of natural killer cells is determined by the collective signaling of an array of inhibitory and stimulatory CELL SURFACE RECEPTORS. A subset of T-LYMPHOCYTES referred to as NATURAL KILLER T CELLS shares some of the properties of this cell type. NK Cells,Natural Killer Cells,Cell, NK,Cell, Natural Killer,Cells, NK,Cells, Natural Killer,Killer Cell, Natural,NK Cell,Natural Killer Cell
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000067128 Extracellular Vesicles Membrane limited structures derived from cell membranes and cytoplasmic material, and released into EXTRACELLULAR SPACE. They circulate through the EXTRACELLULAR FLUID and through the peripheral blood in the MICROVASCULATURE where cells, much larger, cannot, thereby affecting a variety of intercellular communication processes. Apoptotic Bodies,Exovesicles,Apoptotic Body,Bodies, Apoptotic,Body, Apoptotic,Exovesicle,Extracellular Vesicle,Vesicle, Extracellular,Vesicles, Extracellular
D015415 Biomarkers Measurable and quantifiable biological parameters (e.g., specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances) which serve as indices for health- and physiology-related assessments, such as disease risk, psychiatric disorders, ENVIRONMENTAL EXPOSURE and its effects, disease diagnosis; METABOLIC PROCESSES; SUBSTANCE ABUSE; PREGNANCY; cell line development; EPIDEMIOLOGIC STUDIES; etc. Biochemical Markers,Biological Markers,Biomarker,Clinical Markers,Immunologic Markers,Laboratory Markers,Markers, Biochemical,Markers, Biological,Markers, Clinical,Markers, Immunologic,Markers, Laboratory,Markers, Serum,Markers, Surrogate,Markers, Viral,Serum Markers,Surrogate Markers,Viral Markers,Biochemical Marker,Biologic Marker,Biologic Markers,Clinical Marker,Immune Marker,Immune Markers,Immunologic Marker,Laboratory Marker,Marker, Biochemical,Marker, Biological,Marker, Clinical,Marker, Immunologic,Marker, Laboratory,Marker, Serum,Marker, Surrogate,Serum Marker,Surrogate End Point,Surrogate End Points,Surrogate Endpoint,Surrogate Endpoints,Surrogate Marker,Viral Marker,Biological Marker,End Point, Surrogate,End Points, Surrogate,Endpoint, Surrogate,Endpoints, Surrogate,Marker, Biologic,Marker, Immune,Marker, Viral,Markers, Biologic,Markers, Immune
D053804 Granzymes A family of serine endopeptidases found in the SECRETORY GRANULES of LEUKOCYTES such as CYTOTOXIC T-LYMPHOCYTES and NATURAL KILLER CELLS. When secreted into the intercellular space granzymes act to eliminate transformed and virus-infected host cells. Cytotoxic Serine Protease B,Cytotoxic T-Lymphocyte Associated 1 Protein,Cytotoxic T-Lymphocyte Proteinase 1,Fragmentin 2,Granzyme,Granzyme A,Granzyme B,Granzyme C,Granzyme K,Granzyme M,Granzyme-3,Granzyme-Like Protein III,Hanukah Factor,NK-Tryptase-2,Natural Killer Cell Granule Tryptase-2,Cytotoxic T Lymphocyte Associated 1 Protein,Cytotoxic T Lymphocyte Proteinase 1,Granzyme 3,Granzyme Like Protein III,NK Tryptase 2,Natural Killer Cell Granule Tryptase 2
D059470 Tetraspanins A large superfamily of cell surface membrane proteins characterized by their four transmembrane domains. They play a role in a variety of processes such as cellular adhesion and motility. They may be involved in the organization of cell surface MEMBRANE MICRODOMAINS that regulate the activation of LEUKOCYTES. Tetraspanin

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