Extended Data Fig. 2: Characterization of the tricistronic CAR T-cell PET-reporter system.

Gene map of PET and reporter genes for CAR T cells containing the sodium Iodide symporter (hNIS), PSMA targeting scFv (P28z), and a BLI reporter system (exCLuc) to produce the CAR-T-cell hNIS.P28z.exCLuc. b, Flow plot of PC3 cells engineered lacking PSMA. c, PSMA-positive engineered cells. d, Flow plot of CAR T cells expressing the anti-PSMA-scFv. e, In vitro activation of the BLI reporter in tricistronic CAR T cells shows functional incorporation of the genes. f, Addition of ¹²⁴I to wild-type or tricistronic CAR T cells shows an increase with only tricistronic CAR T cells, with mild blocking with sodium perchlorate, confirming the specific uptake of ¹²⁴I through hNIS. g, Confocal imaging of wild-type and hNIS tricistronic CAR-T cells for nuclear staining, hNIS and WGA (wheat germ agglutinin). Scale bar, 5 µm. h, In vitro cytotoxicity of PSMA targeting CAR-T cells reduces cell population in PSMA-positive co-cultures, whereas PSMA-null cells were unaffected through 48 hours by the co culture with CAR-T cells. I, In vivo delivery of CAR T cells targeting PSMA-positive tumours led to reduced tumour growth in PSMA-positive tumours, whereas PSMA-negative tumours continued to grow. In e, f, h, I, the lines denote the mean with error bars representing the s.e.m. b-d, Representative flow-cytometry contour quadrant plot to show cell-intensity characteristics. The gating scheme can be found in Supplementary Fig. 11. e, Box plot representing min and max values, with the middle line as the mean. In f, h, n = 3 technical replicates per condition. In i, n = 5 mice per arm. In e,h, the P values are significant, using a multiple-comparison unpaired t-test and assuming the same s.d. in the population. %IA/CC represents percent injected activity per cubic centimetre.