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

Abstract

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.

Keywords: NK cells; NKG7; cancer; extracellular vesicles; granzyme B.

Figure 1

Fractionation of subpopulations of NK-EVs via DG-UC or SEC. (A) Workflow for isolation of NK-EVs from 48 hrs conditioned medium of IL-15 stimulated NK-92 cells. Cleared conditioned medium was ultracentrifuged, and followed by either DG-UC or SEC as described in Materials and methods. (B) Particle concentration of DG-UC fractions 1-10 measured by NTA. Data are presented as mean ±SEM of 3 independent experiments. (C) Protein concentration of DG-UC fractions 1-10 measured by micro BCA. Data are presented as mean ±SEM of 3 independent experiments. (D) Particle size of DG-UC fractions 5-7 measured by NTA. Data are presented as mean ±SEM of 3 independent experiments. (E) Western blot analysis using 20 µg of EV isolates and bulk EVs of NK-92 cells. Representative of three independent experiments. (F) TEM of DG-UC subpopulation 5-7 (scale bar 200 nm) and (G) TEM of SEC subpopulation 3-5 (scale bar 500 nm).

Figure 2

Separation of a subpopulation of NK-EVs that induce apoptosis of HCT-116 tumor spheroids. (A) Live monitoring of apoptosis induction via IncuCyte by 20 µg NK-EVs from either DG-UC or SEC fractions measured every third hour for 48 hrs. Apoptosis was detected with Caspase 3/7 green detection reagent. Data represents 3 independent experiments ±SEM (B) Representative images of HCT-116 tumor spheroids 24 hrs after application of either DG-UC fractions 5-7 or SEC fractions 3-5 imaged by IncuCyte. PBS (in same volume as EVs) or medium alone (untreated) served as negative controls. (C) Comparison of apoptosis exerted by 20 µg NK-EVs from DG-UC or SEC fractions after 24 hrs. Data represents 3 independent experiments ±SEM. (D) Comparison of apoptosis mediated by 20 µg for either bulk EVs or DG-UC fractions 5-7 or SEC fractions 3-5 after 24 hrs as measured by IncuCyte. Data are presented as mean ±SEM of three separate experiments.

Figure 3

Comparative proteomic profiling of NK-EV subpopulations generated by DG-UC or SEC. (A) PCA analysis and (B) Venn diagram depicting differences in protein profiles between DG-UC fractions 5-7. (C) PCA analysis and (D) Venn diagram depicting differences in protein profiles between SEC fractions 3-5. (E) Heatmaps of EV markers in DG-UC fractions 5-7 or SEC fractions 3-5 represented by log2 mean intensity values of three biological replicates. (F) FunRich biological pathway analysis of proteins identified DG-UC fractions 5-7 or SEC fractions 3-5, showing percentage enrichment of proteins.

Figure 4

Identification of organelle specific signatures within DG-UC subpopulations. (A) FunRich cellular component analysis of proteins identified DG-UC fractions 5-7 or SEC fractions 3-5, showing percentage enrichment of proteins. (B) Heatmaps representing 3 three biological replicates of DG-UC fractions 5-7, represented by log2 mean intensity values of mitochondria, endoplasmic reticulum (ER), autophagosome (AP), and lysosome. Markers were identified through matching against the UniProt subcellular localization database. (C) Heatmaps representing three biological replicates of DG-UC fractions 5-7 or SEC fractions 3-5, represented by log2 mean intensity values of cytolytic granule markers identified through matching against the UniProt subcellular localization database. (D) Representative western blot analysis of sequential immunoprecipitations of DG-UC fractions 5-7 with controls IgG mAb, followed by antibodies against CD81, CD63 and NKG7. EVs from NK-92 cells were used as positive control. FT, flow-through; remaining sample after indicated pulldowns.

Figure 5

Comparison of fractionated subpopulations of primary NK-cell derived EVs via DG-UC. (A) Particle concentration of DG-UC fractions 1-10 measured by NTA. Data representing one biological experiment. (B) Particle size of DG-UC fractions 5-7 measured by NTA. Data representing one biological experiment. (C) Western blot analysis using 20 µg of EV isolates and whole cell lysate (wcl) of primary NK cells. Representative of three independent experiments. (D) TEM of DG-UC subpopulation 5-7. Scale bar 200 nm. (E) Venn diagram and (F) PCA analysis depicting differences in identified protein profiles between DG-UC fractions 5-7 by mass spectrometry. (G) FunRich biological pathway analysis of proteins identified DG-UC fractions 5-7 by mass spectrometry. (H) Heatmaps representing 3 three biological replicates of DG-UC fractions 5-7, represented by log2 mean intensity values of EV markers, (I) cytolytic granules markers and (J) NK cell receptors.