Figure 2.
hnCD16-iNK cells demonstrate improved in vitro ADCC against multiple tumor types. (A) hnCD16-iNK cells and unmodified iNK cells produce CD107α and IFN-γ in response to Raji cells with or without anti-CD20 antibody, in response to SKOV-3 cells with or without anti-HER2, and in response to Cal27 cells with or without anti-EGFR. hnCD16-iNK cells or unmodified iNK cells were left unstimulated or were stimulated with a 1:1 ratio of target cells with or without antibody and stained for CD107a and IFN-γ 4 hours later. (B) Quantification of CD107a (left panel) and IFN-γ (right panel) expression by cells in panel A. An increase in CD107a+ or IFNγ+ positive cells in the antibody group was normalized to the without-antibody group (fold increase: antibody/without antibody). Studies were repeated independently 3 times, and data are mean ± standard deviation. (C) Quantification of flow cytometric analysis of TNF-α and IFN-γ production and CD107a surface expression after a 4-hour incubation with culture media only (unstimulated) or with the indicated stimuli. Heat maps quantify the frequency of NK cells that are positive for IFN-γ, TNF-α, or CD107a and are scaled from 0% (black) to 30% (yellow), with background expression subtracted such that unstimulated = 0. (D) ADCC against Raji cells was analyzed using a caspase-3/7 green flow cytometry assay. Raji cells were incubated with NK cells, with or without anti-CD20 antibody, for 4 hours. (E) ADCC against Raji cells was analyzed over a 24-hour period using an IncuCyte real-time imaging system. Anti-CD20 was titrated from 0.001 μg/mL to 20 μg/mL. (F-G) Long-term (66-hour) ADCC assays using the IncuCyte real-time imaging system. ADCC against the lung cancer cell line A549 with and without anti-EGFR mAb (F) and against the ovarian cancer cell line SKOV-3 with and without anti-HER2 mAb (G). Data in panels F-G are presented as the normalized frequency of target cells remaining, where target cells without NK effectors = 100%. Data in panels D-G were repeated independently in 3 separate experiments. ***P < .001, 2-tailed Student t test.

hnCD16-iNK cells demonstrate improved in vitro ADCC against multiple tumor types. (A) hnCD16-iNK cells and unmodified iNK cells produce CD107α and IFN-γ in response to Raji cells with or without anti-CD20 antibody, in response to SKOV-3 cells with or without anti-HER2, and in response to Cal27 cells with or without anti-EGFR. hnCD16-iNK cells or unmodified iNK cells were left unstimulated or were stimulated with a 1:1 ratio of target cells with or without antibody and stained for CD107a and IFN-γ 4 hours later. (B) Quantification of CD107a (left panel) and IFN-γ (right panel) expression by cells in panel A. An increase in CD107a+ or IFNγ+ positive cells in the antibody group was normalized to the without-antibody group (fold increase: antibody/without antibody). Studies were repeated independently 3 times, and data are mean ± standard deviation. (C) Quantification of flow cytometric analysis of TNF-α and IFN-γ production and CD107a surface expression after a 4-hour incubation with culture media only (unstimulated) or with the indicated stimuli. Heat maps quantify the frequency of NK cells that are positive for IFN-γ, TNF-α, or CD107a and are scaled from 0% (black) to 30% (yellow), with background expression subtracted such that unstimulated = 0. (D) ADCC against Raji cells was analyzed using a caspase-3/7 green flow cytometry assay. Raji cells were incubated with NK cells, with or without anti-CD20 antibody, for 4 hours. (E) ADCC against Raji cells was analyzed over a 24-hour period using an IncuCyte real-time imaging system. Anti-CD20 was titrated from 0.001 μg/mL to 20 μg/mL. (F-G) Long-term (66-hour) ADCC assays using the IncuCyte real-time imaging system. ADCC against the lung cancer cell line A549 with and without anti-EGFR mAb (F) and against the ovarian cancer cell line SKOV-3 with and without anti-HER2 mAb (G). Data in panels F-G are presented as the normalized frequency of target cells remaining, where target cells without NK effectors = 100%. Data in panels D-G were repeated independently in 3 separate experiments. ***P < .001, 2-tailed Student t test.

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