Figure 1. Cell surface expression of chimeric antigen receptor (CAR) and CD20 after transfection.
(a) Schematic representation of the CAR for CD19 and the Sleeping Beauty (SB) transposon system used in this study. PGK, phosphoglycerate kinase. mCMV, human cytomegalovirus (CMV) minimal core promoter element, (b) Expression of CAR and CD20 in 293T cells. 293T cells (8 × 105 per well in 12-well plates) were transfected with 2 μg of the SB transposon (19BBζ/CD20) without the SB10, using a standard calcium phosphate precipitation method. Twenty-four to forty-eight hours after transfection, the cells were stained with CAR [goat anti-mouse immunoglobulin G F(ab′)2] and CD20 (Rituxan) antibodies and analyzed by flow cytometry. Untransfected 293T cells were used as mock control. (c) Expression of CAR and CD20 in PBL from two donors (PBL1 and PBL2). (d) CAR and CD20 expression in umbilical cord blood (UCB) T cells. Note: neither mock treated PBL cells nor mock-treated UCB cells were sorted, and they were used for staining control only. A background CAR staining was observed in mock UCB cells. FITC, fluorescein isothiocyanate; PE, phycoerythrin.