Human NK cells were isolated from fresh or frozen peripheral blood of healthy volunteer donors (NIH Clinical Center Blood Bank (NCT00001846)) using a negative selection Human NK Cell Isolation Kit (Miltenyi Biotech, Auburn, CA) following the manufacturer’s protocol, resulting in >80% purity (CD3-/CD56+). Each experiment and experimental repeat utilized distinct healthy donors. NK cells were treated with 50 ng/ml of IL-15SA/IL-15RA (IL-15 N72D superagonist/IL-15RαSu-Fc; ALT-803, Altor Bioscience, Miramar, FL) and/or 2 ng/ml of TGF-β1 (R&D Systems, Minneapolis, MN), and/or 1 μg/ml of the TGFβ receptor I kinase inhibitor SD208 (Tocris Bioscience, Bristol, UK) for experiments. The concentration of IL-15SA/IL-15RA treatment was determined by previous reports (20 (link), 25 (link)). The concentration of TGF-β1 treatment was determined by the TGF-β1 level in plasma of cancer patients in previous studies (4 (link), 6 (link)). For select experiments, NK cells were isolated from frozen peripheral blood obtained from prostate cancer patients.
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Isolation and Stimulation of Human NK Cells
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Corresponding Organization :
Other organizations : National Cancer Institute, National Institutes of Health, Center for Cancer Research, Altor BioScience (United States)
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Variable analysis
independent variables
- Treatment with 50 ng/ml of IL-15SA/IL-15RA (IL-15 N72D superagonist/IL-15RαSu-Fc; ALT-803, Altor Bioscience, Miramar, FL)
- Treatment with 2 ng/ml of TGF-β1 (R&D Systems, Minneapolis, MN)
- Treatment with 1 μg/ml of the TGFβ receptor I kinase inhibitor SD208 (Tocris Bioscience, Bristol, UK)
- NK cell response to the treatments
- Peripheral blood samples from healthy volunteer donors (NIH Clinical Center Blood Bank (NCT00001846))
- Isolation of NK cells (>80% purity, CD3-/CD56+) using a negative selection Human NK Cell Isolation Kit (Miltenyi Biotech, Auburn, CA)
- Positive control: NK cells treated with IL-15SA/IL-15RA
- Negative control: NK cells without any treatment
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