Fig. 3: In vitro comparison of the split-design CAR approach with conventional CAR. | Nature Communications

Fig. 3: In vitro comparison of the split-design CAR approach with conventional CAR.

From: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

Fig. 3

a Schematic representations of ligand-based conventional and split-design CAR approaches. The extracellular domains of BAFF and APRIL were used as target moieties to generate conventional CAR-T cells, referred to as APRIL CAR and BAFF CAR, respectively. b, d Representative images of cell‒cell conjugates captured at 100× oil objective magnification using a laser scanning confocal microscope (Nikon, A1R). APRIL or 9E10-IgG4m (pre-incubated with Myc-APRIL) CAR-T cells were co-cultured with RPMI8226-GFP cells (b), while BAFF or 9E10-IgG4m (pre-incubated with Myc-BAFF) CAR-T cells were co-cultured with IM9-GFP cells (d). Fluorescent labels included Hoechst (blue), anti-PKC-θ (red), and GFP (green) and a merged view of all stains. Scale bar = 10 μm. c, e Statistical analysis of the mean fluorescence intensity of PKC-θ at the IS in panels b and d, respectively. In panel c, sample sizes: APRIL CAR, n = 37; 9E10-IgG4m, n = 39. In panel e, BAFF CAR, n = 34; 9E10-IgG4m, n = 44. All n values represent individual cells. P values were determined by paired two-tailed t-tests. f, g Cytotoxicity assays of conventional and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. h, i Inflammatory cytokine release assay. Conventional CAR-T cells or sCAR-T cells along with 1 nM corresponding switches were co-cultured with the specific target cells for 24 h at an E:T ratio of 1:1 in triplicate. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. j, l Schematic representations of ligand-based split-design CAR and FDA-approved CAR, referred to as BCMA CAR (j) and CD19 CAR (l), respectively. k, m Cytotoxicity assays of FDA-approved CAR-T cells and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. Data in this figure are representative of three independent experiments. Error bars represent mean ± SD. NS indicates not significant. Source data are provided in the Source Data file.

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