Violet tracer

One to three days after transduction, selection and expansion, inducible CD38-CAR T cells were incubated with Luc-GFP-transduced human malignant cell lines or violet tracer (Thermo Fisher) labeled primary BM-MNC for 24 hours. The luciferase signal produced by surviving malignant cell lines was determined after 24 hours with a GloMax® 96 Microplate Luminometer (Promega) within 15 minutes after the addition of 125 μg/mL beetle luciferin (Promega). % lysis cells = (1 − (BLI signal in treated wells / BLI signal in untreated wells)) × 100%. To analyze surviving primary BM-MNCs Flow-Count™ Fluorospheres (Beckman 7547053) were added, cells were harvested and stained for different CD markers (see above). Viable cells were then quantitatively analyzed through Flow-Count-equalized measurements. Percentage cell lysis was calculated as % lysis cells = (1 − (absolute number of viable target cells in treated wells / absolute number of viable target cells in untreated wells)) × 100%.

Violet tracer (Thermo Fisher, Waltham, MA, USA) labeled target cells were pre-incubated with serial dilutions of antibodies for 15 min in 96-well U bottom plates, and then cultured for 24 h without additional effector cells or with effector cells at a fixed (10:1) T-cell to target (E:T) cell ratio. Effector cells were allogeneic or autologous effector PBMCs or, from specified samples, CD3⁺ lymph node-residing T-cells, isolated using magnetic-activated-cell sorting (MACS) following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Viable target cells were identified with multicolor flow cytometry. Data were analyzed using FACS DIVA software. Cytotoxicity was calculated only if >500 viable target cells were counted in untreated wells and with the following formula: $\mathrm{\%}\mathrm{Cytotoxicity}=\left(100-\mathrm{\%}\mathrm{viability}\right),$ $\mathrm{\%}\mathrm{Viability}=100*\left(\frac{\#7\mathrm{AAD}\_\mathrm{negative}\mathrm{events}\mathrm{of}\mathrm{test}\mathrm{sample}}{\#7\mathrm{AAD}\_\mathrm{negative}\mathrm{events}\mathrm{of}\mathrm{control}\mathrm{sample}}\right).$
Further details are described in online supplementary material and methods.
The concentration of granzyme B released in cell-free supernatants of cytotoxicity assays was measured by a sandwich ELISA kit (Mabtech, Nacka Strand, Sweden) following manufacturer’s protocol.