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. 2024 Jan 28;13(3):246. doi: 10.3390/cells13030246

Figure 1.

Figure 1

Generation of NK-92 cells expressing an NKG2D-based CAR and IL-15 superagonist RD-IL15. (A) Lentiviral transfer plasmids encoding the NKG2D-based CAR NKAR under the control of the spleen focus-forming virus promoter (SFFV). The NKAR encompasses an immunoglobulin heavy-chain signal peptide (SP), the extracellular domain of NKG2D (amino acid residues 82-216), a flexible (G4S)2 linker (L), a Myc-tag (M), a CD8α hinge region, and transmembrane and intracellular domains of CD3ζ. For co-expression of the IL-15 superagonist RD-IL15, a sequence consisting of a second signal peptide (SP), IL-15Rα sushi domain (amino acid residues 31-107), a peptide linker (L) and affinity-optimized IL-15N72D was fused in frame to the NKAR sequence via a porcine teschovirus self-cleaving peptide (P2A). NKAR and RD-IL15 sequences are followed by an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) cDNA. (B) The expression of EGFP (left), as well as NKG2D and the NKG2D-based CAR (right) by sorted NKAR-NK-92 and NKAR_RD-IL15-NK-92 cells was analyzed by flow cytometry, as indicated. Parental NK-92 cells were included for comparison. Unstained NK-92 cells served as a control.