Figure 3. CARTs recognize cognate human antigen in mice.

hHer2 antigen was expressed in mouse hepatocytes following piggyBac gene transfer. Mice were then injected with 2.5 × 10⁶ anti-hHer2 CART (either HA-CAR or LA-CAR), and livers were harvested 1 week later for analysis. Control groups included mice that had hepatic Her2 expression but untransduced T cells and mice that received Her2 CART but lacked hepatic Her2 expression due to empty transposon vector transfections. (A) Her2 CARTs were detected in mouse livers (n = 4–8) by performing real-time PCR assays for CAR DNA. 2^–ΔCt values for CARTs were calculated using PCR assays that amplify the CAR intracellular signaling domain, 4-1BBz-CD3z, and were normalized to mouse PTGER2 genomic DNA content. (B) Expression of human IFN-γ mRNA from T cells was measured in murine livers (n = 4–8) using real-time PCR and normalized to mouse HPRT expression. Kruskal-Wallis test with Dunn’s multiple comparisons tests was used for statistical analysis of real-time PCR data. (C) Systemic cytokine release by T cells was detected in mouse serum (n = 4–8) by Luminex assay. A 2-way ANOVA with Tukey’s multiple comparison test was performed, and comparisons are shown between all groups and the untransduced (UTD) group (*) or between the HA-CAR and LA-CAR groups (⁺). Statistical significance is denoted as *P < 0.5, **P < 0.01,⁺⁺⁺P < 0.001, and ****^/++++P < 0.0001.