Fig. 3

4-1BB causes hyperactive NF-κB signaling and 4-1BB-mediated cell death requires TRAF. A Venn plot illustrating the number of KEGG pathways enriched in three RNAseq or microarray datasets comparing 4-1BB to CD28-costimulated CAR-T cells (upper panel) and the detail of three shared KEGG pathways (lower panel, NF-κB pathway marked as red). B Representative western blot showing the phosphorylation of IKKα/β (p-IKKα/β, Ser^176/180) and p65 (p-p65, Ser⁵³⁶) in 19.CD28ζ and 19.BBζ CAR-T cells. NT indicates control non-transduced T cells. C Quantification of p-IKKα/β normalized to CD3ζ-CAR in NT, 19.CD28ζ and 19.BBζ CAR-T cells in the western blots shown in (B); n = 6; **p < 0.01, one-way ANOVA. D Quantification of the p-p65 expression as mean fluorescence intensity (MFI) in 19.CD28ζ and 19.BBζ CAR-T cells; n = 4, ***p < 0.001, t-test. E Quantification of the NF-κB luciferase activity in NT, 19.CD28ζ and 19.BBζ CAR-T cells. CAR-T cells were transduced with an NF-κB luciferase reporter and collected after 72 h to measure the luciferase activity. Luciferase activity was normalized by genomic luciferase level; n = 3, *p < 0.05, one-way ANOVA. F Clustering heatmap of proteomics analysis obtained by CoIP-LC-MS/MS showing the detection of peptides in the CAR pull-down from either Jurkat cells or T cells expressing either the 19.CD28ζ CAR or the 19.BBζ CAR. NT indicates control of non-transduced Jurkat cells and non-transduced T cells. G Representative microscopic imaging showing the formation of cell aggregates of CAR-T cells expressing either the 19.CD28ζ or the 19.BBζ CARs and CAR-T cells expressing the mutated form of the 4-1BB endodomain (BBζ mut1, BBζ mut2, and BBζ ΔC10). NT indicates control non-transduced T cells; magnification 20×; scale bar 100 µm. H Quantification of Annexin-V⁺ cells of the CAR-T cells described in (G) at day 7–10 of culture; n = 5, **p < 0.01, ***p < 0.001, one-way ANOVA. I Cell counts of the CAR-T cells described in (G). Cell numbers were counted by flow cytometry with counting beads; n = 4, *p < 0.05, **p < 0.01 at day 10, two-way ANOVA