Fig. 5
From: 4-1BB-encoding CAR causes cell death via sequestration of the ubiquitin-modifying enzyme A20
4-1BB endodomain binds A20 within the cell membrane causing A20 functional deficiency in CAR-T cells. A mRNA expression of A20 measured by qRT-PCR in NT, 19.CD28ζ, and 19.BBζ CAR-T cells collected at day 7–10 of culture. Data are represented as fold change in expression normalized to the housekeeping gene 18S, and to the expression in NT cells; NT indicates control non-transduced T cells; n = 5, **p < 0.01, one-way ANOVA. B Representative western blots showing the expression of A20 and its phosphorylated form at Ser381 in NT, 19.CD28ζ, and 19.BBζ CAR-T cells collected on day 7 of culture. C Quantification of A20 (left panel) and its phosphorylated form at Ser381 (right panel) in CAR-T cells illustrated in (B); n = 11, 15, and 15 for NT, 19.CD28ζ and 19.BBζ CAR-T cells (left panel); n = 5 (right panel), *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. D Representative confocal microscopy imaging showing the distribution of A20 in 19.CD28ζ, 19.BBζ, and BBζ ΔC10 CAR-T cells. Blue staining indicates DAPI. Shown are representative cells of a single field; magnification 63×; scale bar 5 µm. Isotype was used as a control. E Representative region including the extracellular, membrane, and intracellular areas and the horizontal change of fluorescence intensity of CAR and A20 within the region in 19.BBζ CAR-T cells. F Statistics of Pearson correlation coefficients indicating the colocalization of CAR and A20 molecules in 19.CD28ζ, 19.BBζ, and BBζ ΔC10 CAR-T cells; n = 8, 17, and 8 for 19.CD28ζ, 19.BBζ, and BBζ ΔC10 CAR-T cells; ***p < 0.001, one-way ANOVA. G Representative western blots showing A20 detection in the whole cell lysate (WCL), cytosol, and plasma membrane of NT, 19.CD28ζ, and 19.BBζ CAR-T cells collected at days 7–10 of culture. H Representative immunoprecipitation blots of the CD19-specific CAR illustrating the binding of A20 to the 19.BBζ CAR and loss of binding in BBζ ΔC10 CAR-T cells
