Fig. 1: MCL-1 expression limits MM cell killing by anti-BCMA CAR T cells.

A Graphical representation of experimental findings. B Representative histograms showing MCL-1 protein expression measured by intracellular flow cytometry in alive H929 or L363 MM cells after 24 h co-culture with anti-BCMA CAR T cells using the indicated effector to target cell ratio’s (E:T). The dotted line indicates the median fluorescence intensity of MCL-1 in untreated (0:1) L363 or H929 cells. The gray histograms shows isotype control staining (iso). C H929 and L363 cells were co-cultured for 24 h with either anti-BCMA CAR T (H929, n = 5; L363, n = 4) or CD19 CAR T (n = 3) cells at the E:T ratios specified. Graphs show the percentage of viable H929 or L363 with MCL-1 expression above the median expression in untreated cells. Dots represent separate experiments with SEM. Statistical testing was performed using one-way ANOVA, followed by multiple comparison testing. D Representative gating strategy of L363 MM cells co-cultured with CellTrace Violet (CTV)-labeled anti-BCMA CAR T cells and stained with nucleic acid dye TO-PRO-3, and measured by flow cytometry after 24 h of culture. Co-cultures were simultaneously incubated with 100 nM MCL-1 inhibitor S63845 (lower panels) or without (upper panels). Indicated percentages of viable cells are calculated within CTV-negative MM cells. E Quantified specific apoptosis of H929 or L363 cells as detailed for (C) and by using the gating strategy shown in (D). Percentages were calculated based on absolute cell numbers using counting beads. Specific apoptosis was determined by measuring the altered percentage of TO-PRO-3^- (live) cells compared with untreated cells and was defined as follows: ([% cell death in treated cells−% cell death in control]/% viable cells control) × 100. For H929 a concentration of 10 nM MCL-1i and for L363 a concentration of 100 nM MCL-1i was used. Dots show averages of separate experiments with H929 (n = 3) or L363 (n = 6) with SEM. Statistical testing was performed using one-way ANOVA, followed by multiple comparison testing. F Specific apoptosis (calculated as in (E)) induced by anti-BCMA CAR T cells in primary MM cells pre-treated with MCL-1i (blue circles) or without pre-treatment (white circles). Bone marrow mononuclear cells (BMNCs) were cultured for 48 h in the presence or absence of MCL-1i (S63845, 100 nM). After pre-treatment, the number of viable MM cells (CD38 + CD138+) on each condition was quantified by FACS following the gating strategy shown in Supplementary Fig. 1c. MCL-1i was washed away and BMNCs cells were subsequently co-cultured with anti-BCMA CAR T cells for 24 h at a 1:5 E:T ratio, calculated based on the number of alive MM cells present on each condition. Data are from 3 independent experiments. Each circle represents a patient sample. Statistical analysis was performed using a two-tailed paired t test. Source data are provided as a Source Data file.