Extended Data Fig. 9: Differential signaling strength induced by individual point mutations shapes CAR T cell phenotypes in a co-stimulatory dependent manner.

a. CD69 expression levels in CD4+ (left) and CD8+ (right) 1928z as compared to E81K-modified 1928z determined by flow cytometry without further antigen stimulation (n = 10 biologically independent donors, mean ± SD; two-sided paired Student’s t-test; samples for mock 1928z partially overlap with data presented in Fig. 5a). b. Co-expression of exhaustion markers LAG3, PD-1, and TIM3 on CD4+ (left) and CD8+ (right) E81K-edited or control 1928z CAR T cells after three rounds of antigen stimulations as determined by flow cytometry (n = 5 biologically independent donors; mean + SD; two-sided paired Student’s t-test). c. Antigen-dependent cell growth of mock 1928z and E81K-edited 1928z CAR T cells (both with TRBC KO) during n = 3 weekly stimulations with Nalm6 cells at an effector to target (E:T) ratio of 2:1 (n = 3 biologically independent donors; mean± SD; two-sided paired Student’s t-test). d. Cytotoxic activity of mock 1928z compared to E81K-edited 1928z CAR T cells against Nalm6 tumor cells using an 18 h bioluminescence assay at indicated effector to target ratio (n = 5 biologically independent donors, mean ± SD, two-sided paired Student’s t-test). e. CD69 expression levels in CD4+ (left) and CD8+ (right) B7H3-28z as compared to E81K-modified B7H3-28z determined by flow cytometry without further antigen stimulation (n = 3 biologically independent donors, mean ± SD; two-sided paired Student’s t-test). f. Co-expression of exhaustion markers LAG3, PD-1, and TIM3 on CD4+ (left) and CD8+ (right) E81K-edited or mock B7H3-28z CAR T cells after one round of antigen stimulation as determined by flow cytometry (n = 4 biologically independent donors; mean + SD; two-sided Wilcoxon matched-pairs signed rank test). g. Flow cytometry-based analysis of phosphorylated (p)-AKT T308 in mock 1928z and L32P-edited 1928z CAR T cells (“L32P”) after 1 h of stimulation with Nalm6 at an effector to target (E:T) ratio of 1:1. Data are shown in triplicates from n = 3 biologically independent donors; two-sided unpaired Student’s t-test. h. CD69 expression levels in mock 19BBz as compared to L32P-modified 19BBz CAR T cells 72 h after stimulation with Nalm6 cells as determined by flow cytometry (n = 3 biologically independent donors, mean ± SD; two-sided paired Student’s t-test). i. Antigen-dependent cell growth of mock 19BBz and L32P-edited 19BBz CAR T cells after stimulation with Nalm6 cells at an effector to target (E:T) ratio of 2:1 (n = 3 biologically independent donors, mean ± SD; two-sided paired Student’s t-test). j. Cytotoxic activity of mock 19BBz compared to L32P-edited 19BBz CAR T cells against luciferase-expressing Nalm6 cells at an E:T ratio of 1:1 (n = 3 biologically independent donors, mean ± SD; two-sided paired Student’s t-test). k. Cytotoxic activity of mock 1928z compared to L32P-edited 1928z CAR T cells using an 18 h bioluminescence assay with luciferase-expressing Nalm6 cells as targets for indicated effector to target ratios (n = 4 biologically independent donors, mean ± SD; two-sided paired Student’s t-test). l.-n Granzyme B production in CD4+ and CD8+ (l.) and effector cytokine production in CD4+ (m.) and CD8+ (n.) L32P-edited and mock 1928z CAR T cells 4 h after stimulation with Nalm6 cells as measured by flow cytometry (n = 4 biologically independent donors; two-sided paired Student’s t-test). Center line shows the median, box represents 25th and 75th percentiles and whiskers minimum to maximum values.