Extended Data Fig. 2: E81K editing modulates PI3K signaling and enhances cytotoxic capacity and antigen sensitivity in 19BBz CAR T cells.
From: CAR-adapted PIK3CD base editing enhances T cell anti-tumor potency
a. Immunoblot of primary 19BBz CAR T cells with TRBC1/2 KO (“mock”) or additional E81K editing (“E81K”) after 30 min of stimulation with Nalm6 at an effector to target (E:T) ratio of 1:1. p-AKT T308 and S473, p-GSK3β Ser9 and p-S6 S235/236 are shown, Tubulin served as loading control. Data represent one biological donor. b. Flow cytometry-based analysis of phosphorylated (p)-AKT T308, S473 and p-S6 S235/236 in 19BBz, E81K-edited 19BBz and 1928z CAR T cells, all with additional TRBC knockout (KO) after 1 h of stimulation with Nalm6 at an effector to target (E:T) ratio of 1:1. Data are shown as technical triplicates from one representative donor (out of three biologically independent donors). c. Immunoblot of single cell clones derived from unstimulated SUP-T1 cells either with the wild-type sequence (wt) or a homozygous E81K mutation. p-AKT T308 and S473, and p-S6 S235/236 are shown, Tubulin served as loading control. Data represent one independent experiment. d. Flow cytometry-based analysis of phosphorylated (p)-AKT T308 in control or E81K-edited TRBC KO 19BBz CAR T cells generated from T cells of pre-treated tumor patients 1 h after co-culture with Nalm6 cells (E:T 1:1; n = 4 biologically independent tumor patients, mean ± SD; two-sided paired Student’s t-test). e. Flow cytometry-based analysis of phosphorylated (p)-AKT T308 in unstimulated control or E81K-edited TRBC KO 19BBz CAR T cells generated from pre-treated tumor patients (n = 4 biologically independent tumor patients, mean ± SD; two-sided paired Student’s t-test). f. Flow cytometry-based analysis of phosphorylated (p)-ERK1/2 Y204 in TRBC KO 19BBz, E81K-edited TRBC KO 19BBz and TRBC KO 1928z CAR T cells 1 h after stimulation with Nalm6 cells. Data are shown in triplicates from one representative donor out of n = 3 biologically independent donors. g. Cytotoxic activity of TRBC KO 19BBz compared to TRBC KO E81K-edited 19BBz CAR T cells using an 18 h bioluminescence assay with luciferase-expressing Nalm6 wild type cells as targets for indicated effector to target (E:T) ratios (n = 9 biologically independent donors, mean ± SD; two-sided Wilcoxon matched-pairs signed rank test). h. Heatmap of genes of interest related to proliferation (prolif.), metabolic fitness, immune suppression (suppr.) and effector function comparing unstimulated E81K-edited (“E81K”) and control 19BBz CAR T cells (both with TRBC KO) (n = 3 technical replicates per group from one healthy donor) analyzed by bulk RNA sequencing; rows normalized to mean of control group. i. Flow cytometric analysis of CD19 expression levels in CD19-low und CD19 wild type Nalm6 cells expressing luciferase. CD19 surface counts were analyzed by Quantum™ Simply Cellular® beads. j. Antigen-dependent cell growth of E81K-edited 19BBz relative to control 19BBz CAR T cells after stimulation with CD19-low Nalm62884 cells at an effector to target (E:T) ratio of 2:1 (n = 6 biologically independent donors, mean ± SD; two-sided one-sample t-test). k. Cytotoxic activity of TRBC KO 19BBz compared to TRBC KO E81K-edited 19BBz CAR T cells using an 18 h bioluminescence assay with luciferase-expressing CD19-low Nalm6 cells as targets for indicated effector to target (E:T) ratios (n = 6 biologically independent donors, mean ± SD; two-sided Wilcoxon matched-pairs signed rank test).
