Phenotypic Analysis of PBMC Subsets

The anti-human mAbs used were as follows: PE-CD274, PE-EGFR, PE-CD3, PE-CD226 (DNAM-1), PerCP-Cy5.5-NKG2D, BV421-NKp30, BV510-granzyme B, PE-Cy5-CD107a, and PE-Smad2 (pS465/pS467)/Smad3 (pS423/pS425) (BD Biosciences, San Jose, CA); APC-CD56 (BioLegend, San Diego, CA); PE-Cy7-perforin (eBioscience, San Diego, CA), and PE-TGFβ receptor II (R&D Systems). Samples were acquired on a FACSCalibur flow cytometer or FACSVerse (Becton Dickinson, Franklin Lakes, NJ), and analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). Isotype control staining was < 5% for all samples analyzed. For human PBMC subset analysis, PBMCs from three healthy donors were obtained from the NIH Clinical Center Blood Bank (NCT00001846), as previously described (26 (link)). Frozen PBMCs were thawed and seeded at 5×10⁶ per well, and treated with IL-15SA/IL-15RA at 0, 1, 5, 25, and 50 ng/ml for 72h. Cells were harvested and assessed for the frequency of immune cell subsets by multi-parametric flow cytometry as previously described (27 (link)).