Fig. 4.

IL-2 withdrawal results in enrichment of NK-92 cells co-expressing IL-15 and a chimeric antigen receptor. a Schematic representation of the lentiviral transfer vector pS-31.28.z-I-IL15-W that encodes under the control of the Spleen Focus Forming Virus promoter (SFFV) the chimeric antigen receptor 31.28.z (CAR) that consists of an immunoglobulin heavy chain signal peptide (SP), the EpCAM-specific scFv(MOC31) antibody fragment (scFv), a Myc-tag (M), the CD8 α hinge region (CD8α), transmembrane and intracellular domains of CD28 (CD28), and the intracellular domain of CD3 ζ chain (CD3ζ), followed by an internal ribosome entry site (IRES) and cDNA encoding human IL-15. b After transduction with S-31.28.z-I-IL15-W vector particles, IL-15-expressing cells were selected by IL-2 withdrawal for 14 days. Then, proliferation of the selected cells in growth medium containing or lacking IL-2 was analyzed in MTT metabolization assays by determination of the absorbance at 595 nm as a measure for the relative number of viable cells. Parental NK-92 cells were included for comparison. Mean values ± SEM are shown (n = 3); ***p < 0.0001. NK-92/31.28.z-IL-15 cells with high CAR surface expression were isolated from the selected pool by flow cytometric cell sorting with Myc-tag-specific antibody 9E10. c The presence of IL-15 protein in sorted NK-92/31.28.z-IL-15 cells was investigated by intracellular cytokine staining with anti-IL-15 antibody and flow cytometry (open area). Parental NK-92 cells served as a control (gray area). d Homogeneous CAR surface expression in NK-92/31.28.z-IL-15 cells was confirmed by flow cytometry with 9E10 antibody (open area). Parental NK-92 cells were included for comparison (filled area)