Extended Data Fig. 5: E81K-modified 19BBz CARs exhibit sustained effector function.

a., b. Absolute CD4+ counts of mock and E81K-edited 19BBz CAR T cells in spleen (a.) and bone marrow (b.) of mice 16 days after CAR T cell infusion (n = 5 mice, mean; two-sided unpaired Student’s t-test). c., d. Flow cytometric analysis of CD57 + CD8+ (c.) and CD57 + CD4+ (d.) E81K- edited or mock 19BBz CAR T cells in blood, spleen and bone marrow of mice 16 days after CAR T cell infusion (n = 5 mice, mean; two-sided unpaired Student’s t-test). e. Percentage of effector memory cells in unstimulated CD4+ and CD8+ mock 19BBz and E81K-edited 19BBz CAR T cells generated from tumor patients (both with TRBC KO) as determined by flow cytometry (n = 8 biologically independent tumor patients, mean ± SD; two-sided paired Student’s t-test). f.-k. CAR T cells were stimulated two (f.,i-k.), or three (g.,h.) times with Nalm6 before undergoing further assays. f. Cytotoxic activity of mock 19BBz compared to E81K-edited 19BBz CAR T cells (both with TRBC KO) after two stimulations with Nalm6 cells using an 18 h bioluminescence assay with luciferase-expressing Nalm6 cells as targets. E:T, effector to target ratio (n = 8 biologically independent donors, mean ± SD; two-sided Wilcoxon matched-pairs signed rank test). g., h. Cytotoxic activity of mock CD22-specific BBz CAR T cells compared to E81K-edited 22BBz CAR T cells against luciferase-expressing Nalm6 tumor cells (g.) and of mock 19BBz CAR T cells compared to E81K-modified 19BBz CAR T cells against luciferase-expressing Daudi tumor cells (h.) using an 18 h bioluminescence assay. E:T, effector to target ratio (n = 3 technical replicates, reproduced in two biologically independent donors, mean). i., j. Flow cytometric analyses of effector cytokine and Granzyme B production of CD8+ (i.) and CD4+ (j.) E81K-edited compared to mock 19BBz CAR T cells (both with TRBC KO) after co-culture with Nalm6 cells for four hours (n = 6 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. k. GSEA of differentially expressed genes between E81K-edited and control 19BBz CAR T cells (both with TRBC KO) from one healthy donor after two Nalm6 tumor cell stimulations based on bulk RNA sequencing. Dot size represents adjusted P-value, permutation test. NES, normalized enrichment score.