Extended Data Fig. 2: Zip-sorted dual-CAR T cells demonstrate CAR-dose-dependent upregulation of ROS and inhibitory receptor expression.
From: Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors
a, Flow cytometry analysis of BM185-ffluc-Thy1.1-Neo cell lines expressing combinations of CD19 and CD20. b, Schematic depicting BM185 syngeneic mouse model of antigen-loss escape in CAR T cell immunotherapy for acute lymphoblastic leukaemia. c–e. Sublethally irradiated BALB/c mice were injected with 1:1 mixture of BM185-CD19/BM185-CD20 at 1×105 BM185/mouse (50x increased dose vs. Figure 3g) and treated with Zip-sorted BALB/c CAR T cells on day 2. c, Leukaemia BLI (ffluc) from two combined experiments. Log-transformed BLI values were compared using a Vardi test to compare AUC values with FDR correction for multiple comparisons. d, Day 10 BLI images from representative experiment. e, Survival, compared via log-rank test. f, PD-1 expression on resting Zip-sorted BALB/c CAR T cells. g, Linear regression analysis of unstimulated T cell PD-1 expression vs. number of signalling-competent CARs expressed, n = 3 donors. h, Immunophenotype analysis of unstimulated Zip-sorted BALB/c CAR T cells; mean ± SEM from n = 3 donors. i, Total cellular ROS (CM-H2DCFDA) analysis of resting Zip-sorted CAR T cells. j, Z-score normalized mean CM-H2DCFDA MFI ± SEM results of n = 3 biological replicates. k, Mitochondrial superoxide (Mitosox Red) analysis of Zip-sorted CAR T cells. l, Z-score normalized mean Mitosox Red MFI ± SEM results of n = 3 biological replicates. Statistical differences for ROS and mitochondrial superoxide were compared using one-way ANOVA, with Tukey’s test.
