Journal: Cancer Immunology, Immunotherapy

Article Title: Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18

doi: 10.1007/s00262-022-03161-0

Figure Lengend Snippet: Comparison of EVs derived from IL-15 or IL-12/15/18-stimulated NK cells, NK-92 or KHYG-1 cells. a TEM image of EVs derived from IL-12/15/18-stimulated NK cells. Scale bar 200 nm. b NTA analysis of EVs derived from indicated stimulations of NK cells, NK-92 cells or KHYG-1 cells. Representative of one of three separate experiments. Particle size (c) and particle concentration (d) as measured by NTA of EVs derived from indicated stimulations of NK cells, NK-92 cells or KHYG-1 cells. Data are presented as the mean ± SEM of three separate experiments. e BCA analysis of EVs derived from indicated stimulations of NK cells, NK-92 cells or KHYG-1 cells. Data are presented as the mean ± SEM of five separate experiments. f Western blot analysis using 20 µg of indicated EV isolates and corresponding cellular lysates. Representative of three independent experiments

Article Snippet: Freshly isolated NK cells (2 × 10 6 /ml), NK-92 cells (5 × 10 5 /ml), or KHYG-1 cells (5 × 10 5 /ml) were cultured in 10 ng/ml human recombinant IL-15 alone, or in combination with 10 ng/ml human recombinant IL-12 and IL-18 (all from R&D Systems) in serum-free AIM-V medium for 48 h. Additionally, NK cells were stimulated with anti-CD16-coated pan-mouse Dynabeads (ThermoFisher Scientific) for 48 h in AIM-V medium.

Techniques: Comparison, Derivative Assay, Concentration Assay, Western Blot

Journal: Cancer Immunology, Immunotherapy

Article Title: Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18

doi: 10.1007/s00262-022-03161-0

Figure Lengend Snippet: EVs from primary NK cells and NK-92 cells broadly target tumor spheroids. a-i Measurement of apoptosis of indicated tumor spheroids with EVs from IL-15 or IL-12/15/18-stimulated primary NK cells or NK-92 cells measured as increase in the MFI signal of Caspase 3/7 green detection reagent (left panels) or spheroid size (right panels) using the Incucyte S3 platform. Spheroids were monitored every hr for 5 days. j-r Cytotoxic killing assay (left panels) or degranulation assay (right panels) of donor NK cells or NK-92 cells stimulated for 48 h with IL-15 or IL-12/15/18 toward indicated cancer cells

Article Snippet: Freshly isolated NK cells (2 × 10 6 /ml), NK-92 cells (5 × 10 5 /ml), or KHYG-1 cells (5 × 10 5 /ml) were cultured in 10 ng/ml human recombinant IL-15 alone, or in combination with 10 ng/ml human recombinant IL-12 and IL-18 (all from R&D Systems) in serum-free AIM-V medium for 48 h. Additionally, NK cells were stimulated with anti-CD16-coated pan-mouse Dynabeads (ThermoFisher Scientific) for 48 h in AIM-V medium.

Techniques: Degranulation Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18

doi: 10.1007/s00262-022-03161-0

Figure Lengend Snippet: Proteomic comparison of EVs derived from NK cells, NK-92 and KHYG-1 cultured in presence of IL-15 or IL-12/15/18. Venn diagrams depicting number of identified proteins in at least two of three replicates of EVs from (a) primary NK cells, (b) , NK-92 cells or (c) KHYG-1 cells stimulated with IL-15 or IL-12/15/18 for 48 h, and CD16 for 48 h for NK cells. d Comparative Venn diagram depicting percent similarities in the protein profile of the 7 different EV isolates. e PCA analysis of the protein profile of the seven EV isolates based on the median intensity of proteins. The variability explained by the first and second components of the PCA is indicated by arrow direction and length. f Cellular component analysis of proteins identified in EVs from NK cells, NK-92 or KHYG-1 by FunRich analysis showing percentage of identified proteins within the depicted pathways. Enrichment within depicted pathways were significant for all EV isolates ( p < 0.001). g Heatmap showing the median protein intensity values of cytolytic proteins detected in the EV isolates. h Western blot analysis of 20 µg EVs derived from NK cells, NK-92 or KHYG-1 cells stimulated by the indicated cytokines or CD16. Whole cell lysates of indicated cells as controls. The data is representative of three independent experiments

Article Snippet: Freshly isolated NK cells (2 × 10 6 /ml), NK-92 cells (5 × 10 5 /ml), or KHYG-1 cells (5 × 10 5 /ml) were cultured in 10 ng/ml human recombinant IL-15 alone, or in combination with 10 ng/ml human recombinant IL-12 and IL-18 (all from R&D Systems) in serum-free AIM-V medium for 48 h. Additionally, NK cells were stimulated with anti-CD16-coated pan-mouse Dynabeads (ThermoFisher Scientific) for 48 h in AIM-V medium.

Techniques: Comparison, Derivative Assay, Cell Culture, Western Blot