Igg4 isotype control

Cytotoxicity was assessed with an annexin V-FITC staining kit (Miltenyi Biotech) or by ImageStream analysis. The viability dye was chosen based on the panel necessary for flow cytometric analyses. Except for DCs, all target cells were stained with CellTrace Violet (CTV, Thermo Fisher Scientific) prior to the cytotoxicity assays. For that, cells were centrifuged (5 min, RT, 450xg) and the supernatant was completely removed. Cells were resuspended in 1 mL DPBS and stained with 500 nM CTV for 20 min (37°C, protected from light). Afterwards, the reaction was stopped with medium containing 10% FBS and the cells were washed once. CD19-CAR-iNKT cells or untransduced iNKT cells were incubated for 4–24 hours with CTV+ target cells (SEM, Raji, Jurkat, K562 cells or patient samples) or DCs at determined effector to target ratios (E:T). After coincubation, the cells were stained and analyzed by flow cytometry. Where stated, 10 µg/mL nivolumab (Novartis) or its IgG4 isotype control (BioLegend), 100 ng/mL α-GalCer (Abcam, Cambridge, UK) or dimethyl sulfoxide (DMSO, Sigma-Aldrich) were added to the assays. The results are expressed as percentage of cytotoxic activity using the formula: cytotoxic activity (%)=(1−(% annexin V^negviability dye^neg target cells incubated with CD19-CAR-iNKT cells/% annexin V^negviability dye^neg target cells incubated alone))×100.

The proliferation of CD19-CAR-iNKT cells and T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) dilution. First, CD19-CAR-iNKT cells or MACS-isolated T cells were resuspended in phosphate-buffered saline (PBS, Gibco) and stained with CellTrace CFSE cell proliferation kit (BioLegend) for 5 min at room temperature. Immediately after staining, cells were washed with pure FBS and then two times in PBS supplemented with 5% FBS and finally resuspended in CAR medium. CFSE-labeled cells were tested in a mixed lymphocyte reaction (MLR) and against modified Raji cells. Where stated, 10 µg/mL nivolumab (Novartis) or its IgG4 isotype control (BioLegend) was added. Results are expressed as a percentage of proliferating cells or as mean fluorescence intensity.