Fig 1: CAR gene delivery with mCD8-LV4 × 104 BALB/c splenocytes were cultivated alone or in co-culture with 4 × 103 A20 tumor cells stained with Cell Trace Violet (CTV). Cultures were treated with defined particle amounts of mCD8-LV encoding GFP or a chimeric antigen receptor (CAR) directed against mCD19. After spinfection, cells were cultivated for 2 days in the presence of rhIL7 before analysis by flow cytometry and qPCR. (A) Workflow. (B) CAR structure. The CAR used in this experiment was previously published. It employs a single-chain variable fragment (scFv) of the ID3 monoclonal antibody against mouse CD19. It is a second-generation CAR with a CD3? stimulatory and CD28? costimulatory domain. The first and third immunoreceptor tyrosine-based activation motifs (ITAMs) were ablated by mutation to decrease T cell apoptosis and increase survival in vivo.10 A myc tag (purple) between scFv and transmembrane domain (tm) facilitates detection of the CAR. (C) Similar VCNs were obtained for both vector stocks. Symbols represent data of lymphocytes from n = 5 mice. Bars represent means. Error bars represent 95% CIs. DNA was extracted from total cells, without prior sorting. (D–G) Killing of CD19+ B lymphocytes in splenocyte-only cultures. (D) Representative flow cytometry plots from one mouse showing CD19 signals of viable singlet cells after no treatment or transduction with 2 × 109 particles/well. Percent CD19 positivity and CD19+ cell counts are reported. (E) Percentage of CD19+ cells relative to untransduced control. (F) Percentage of viable cells relative to untransduced control. (G) Geometric MFI of CD8+ cells. (E–G) Symbols represent data of lymphocytes from n = 5 mice treated with mCD8-LVGFP (green squares) or mCD8-LVCAR (yellow circles). (H and I) Killing of A20 target cells in cocultures. Flow cytometry data, obtained from viable singlet cell populations are reported. (H) Representative flow cytometry plots from one mouse after no treatment or transduction with 2 × 109 particles/well. CTV+ cells are A20 tumor cells. (I) Percentage of CTV+ (A20) cells relative to untransduced control. Symbols represent data from n = 5 mice treated with mCD8-LVGFP (green squares) or mCD8-LVCAR (yellow circles). See also Figures S3 and S4.