Fig. 1

4-1BB signaling causes cell aggregation and Annexin-V expression in CAR-T cells. A Representative microscopic imaging showing the formation of cell aggregates in NT, 19.CD28ζ, and 19.BBζ CAR-T cells at day 7–10 during the in vitro expansion; magnification 20×; scale bar 100 µm. NT indicates control non-transduced T cells. B Quantification of the size of cell aggregates illustrated in (A); n = 10, ***p < 0.001, one-way ANOVA. C Quantification of Annexin-V⁺ cells of 19.CD28ζ and 19.BBζ CAR-T cells at day 7–10 of culture; n = 6, ***p < 0.001, t-test. D Cell counts of 19.CD28ζ and 19.BBζ CAR-T cells in vitro. Cell numbers were counted by flow cytometry with counting beads; n = 4, **p < 0.01, ***p < 0.001, two-way ANOVA. E Representative confocal microscopy imaging showing the distribution of the GFP-tagged 19.CD28ζ and 19.BBζ CARs on the cell surface of CAR-T cells. Blue staining indicates DAPI. Shown are representative cells of a single field; magnification 63×; scale bar 5 µm. F Schematic of the 19.CD28ζ and 19.BBζ CARs with loss-of-function mutation in the ITAMs (CD3ζ mut); F represents phenylalanine. G Representative microscopic imaging showing the formation of cell aggregates in 19.CD28ζ and 19.BBζ CAR-T cells, and 19.BB CD3ζ mut CAR-T cells at day 7–10 during the in vitro expansion; magnification 20×; scale bar 100 µm. H Quantification of the size of cell aggregates illustrated in (G); n = 10, ns represents no significance. I Quantification of Annexin-V⁺ cells of 19.CD28ζ and 19.BBζ CAR-T cells, and 19.BB CD3ζ mut CAR-T cells at day 7–10 days of culture; n = 5, *p < 0.05, ns represents no significance, one-way ANOVA. J Cell counts of 19.CD28ζ and 19.BBζ CAR-T cells, and 19.BB CD3ζ mut CAR-T cells in vitro. Cell numbers were counted by flow cytometry with counting beads; n = 4, ns represents no significance at day 10, two-way ANOVA