eGSHs differentially regulate CAR expression and CAR T-cell function in vitro. (A) Experimental schema for weekly antigenic stimulation of purified CAR⁺ T cells starting 7 days after transduction. Flow cytometry for CAR expression on day 0, 7, and 14 was performed before plating onto CD19⁺ aAPCs. (B) CAR expression profile of CAR⁺ T cells over 2 weeks of antigenic stimulation. (C) Proliferation in response to weekly antigenic stimulation over 2 weeks for cells shown in Figure 2B. Data are shown as cumulative fold change in T-cell numbers, mean, and range of 2 technical replicates. (D) rAAV6 cassette design incorporating chromatin insulator element C1. (E) CAR expression profile of all CAR⁺ T cells over 2 weeks of antigenic stimulation. Data shown are a representative example of 2 technical replicates. See supplemental Figure 3A-B for quantification of data. (F) Cytotoxicity assay for all CARs shown in panel E, at day 0. Data are shown as mean ± SD of 3 technical replicates. (G) Proliferation of GSH-CAR⁺ cells shown in panel E over 2 weeks in culture. Data are shown as mean and range from 2 technical replicates. Data from E-G are a representative example from 1 T-cell donor. All experiments have been performed for each construct with at least 2 T-cell donors. aAPCs, artificial antigen presenting cells; UT, untransduced cells used as controls.