Fig. 2 | Nature Communications

Fig. 2

From: CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors

Fig. 2

Characteristics of 8R70 CAR T cells. a Retroviral constructs of 8R CARs and un-mod-70CAR. b CAR transduction- efficiency 12 days after transduction. c The transduced T cell counts post-transduction in vitro. Transduced T cells were cultured in IL-2-containing medium for 25 days, and the cell counts were measured by a flow cytometer. d Representative phenotypical analysis of the CAR T-cells. The cells described in (c) were analyzed 15 days after transduction. ef CAR T-cell chemotaxis assay. The U87 cells were radiated at 6 Gy, 7 days later, the supernatant was collected. Anti-IL-8 Ab (2 μg/ml) was added to medium 15 min before the assay, cells from the bottom chamber were counted by flow cytometry 1 hr later, and medium-alone served as control. Chemotaxis index (CI) in (e) was defined by the ratio of cells that migrated to the chemoattractants over control. Absolute number of migrated cells in (f) was also used to evaluate chemotaxis [means ± SEM; n = 3; one-way ANOVA was used among the different amounts of rhIL-8 or doses of IR (black asterisks), two-way ANOVA was used for the different treatment groups (blue asterisks), and Mann–Whitney U test was used for the difference between with and without IL-8 blockade in (f) (red asterisks)]. g Tumor recognition of the CAR T cells. Luciferase-based killing assay was performed. U87-luciferase cells (U87.Luc, 2 × 104) were cocultured with CAR T cells as indicated effector-to-target ratio (E:T) (means ± SD; n = 3; unpaired t test). h CAR T cells decrease IL-8 in vitro. The transduced T cells (1 × 105/well) were cultured in T-cell media without IL-2 overnight, and rhIL-8 (5000 ng/ml) was added. The supernatant was collected for IL-8 concentration 24 h later (means ± SEM; n = 3; the Mann–Whitney U test). i CAR T-cell proliferation in the presence of IL-8. CellTrace-Violet-labeled T cells were stimulated on day 0 with anti-CD3/CD28 Dynabeads (bead: cell = 1:1) in the presence of rhIL-8, the proliferation was measured on day 4 (means ± SD; n = 3; the Mann–Whitney U test). **p < 0.01, ***p < 0.001

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