(a, b) The 28-z, BB-z and 28-ΔIL2RB-z (YXXQ) CAR-transduced CD8+ T cells were stimulated with NALM-6, and their gene expression profiles were compared by microarray analysis at 4, 24 and 72 hours after stimulation (n=4 different donor samples). The data shown are unsupervised hierarchical clustering (a) and principal component analysis (b) of differentially expressed genes (raw P values<0.01 with repeated measures one-way ANOVA). (c, d) Gene set enrichment analysis (GSEA) on the expression profiles of the 28-ΔIL2RB-z (YXXQ) CAR-T cells compared with the 28-z or BB-z CAR-T cells using genes induced by the IL-21 treatment (c) or STAT3 target genes (d) as gene sets. Nominal p values and false discovery rates (FDR) to adjust for multiple hypothesis testing are presented. (e) Relative expression of the genes associated with cytolytic activity was analyzed by qPCR in the CAR-T cells stimulated for 24 hours. Expression levels were normalized to the UBC expression (n=4; log2-transformed values were compared with repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F=24.52 for GZMA, F=43.68 for GZMB, F=32.5 for GZMH, F=43.43 for GZMK, F=32.54 for PRF1; degree of freedom=11). Horizontal lines denote the mean values. (f, g) The CAR-transduced T cells were repeatedly stimulated with NALM-6 at an effector to target (E:T) ratio of 1:3 (f). The cytotoxic activity against NALM-6 on days 0, 3, and 6 was evaluated by flow cytometry. The data shown are four technical replicates for individual CAR-T cells with mean ± s.d. (ordinary one-way ANOVA with Tukey’s multiple comparisons test for each round of stimulation; F=0.41 for Day 0, F=44.94 for Day 3, F=46.75 for Day 6; degree of freedom=19). Similar results were obtained in a repeat experiment. * P<0.001. ns, not significant.