Fig. 4

A20 overexpression decreases cell death and improves in vitro functionality of 4-1BB costimulated CAR-T cells. A Representative immunoprecipitation blots of CAR-T cells illustrating the binding of A20 to the 19.BBζ CAR in T cells; NT indicates control non-transduced T cells. B Representative western blot showing the expression of p-IKKα/β in 19.BBζ CAR-T cells overexpressing A20. The first lane represents NT. C Representative flow cytometry histograms showing p-p65 in CAR-T cells with A20 overexpression. Isotype was used as a control, samples legends shown in D. D Quantification of the mean fluorescence intensity (MFI) showing the level of p-p65 in 19.CD28ζ and 19.BBζ CAR-T cells after A20 overexpression; n = 5, **p < 0.01, ***p < 0.001, one-way ANOVA. E Representative microscopic imaging showing the formation of cell aggregates of 19.CD28ζ and 19.BBζ CAR-T cells after A20 overexpression. CAR-T cells were analyzed at day 7–10 during the in vitro expansion; magnification 20×; scale bar 100 µm. F Quantification of Annexin-V⁺ cells described in (E) at day 7–10 of culture; n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. G Cell counts of the CAR-T cells described in (E). Cells were counted with flow cytometry with counting beads; n = 4, *p < 0.05 at day 10, two-way ANOVA. H Representative western blot testing the expression of p-RIPK1 and p-RIPK3 in 19.BBζ CAR-T cells overexpressing A20. The first line represents NT. I Quantification of the p-MLKL MFI in CAR-T cells described in (E); n = 3, *p < 0.05, **p < 0.01, one-way ANOVA. J Representative flow cytometry plots of the experiments in which CAR-T cells were cocultured with Daudi cells for five days (E:T ratio of 1:5). At day five cells were collected to measure Daudi cells (CD20⁺) and T cells (CD3⁺) by flow cytometry. K, L Summary of the coculture experiments described in (J); n = 7, *p < 0.05, paired t-test