Phenotype and antimyeloma function of SLAMF7-CAR T cells in vitro. (A) Staining with anti–epidermal growth factor receptor antibody to detect the EGFRt transduction marker on CD8⁺ and CD4⁺ SLAMF7-CAR T cells (red). Untransduced T cells were used as controls (blue, left histograms). Staining with SLAMF7 protein to detect the SLAMF7-CAR on SLAMF7-CAR T cells. CD19-CAR T cells (control) and untransduced T cells (mock) were used as references (right histograms). Expression of SLAMF7 on SLAMF7-CAR and CD19-CAR (control) T cells (dot plots). (B) Expansion of CD4⁺ and CD8⁺ T cells after anti-CD3/anti-CD28 bead stimulation and lentiviral transduction with SLAMF7-CAR and CD19-CAR as control (upper bar diagram). The left y-axis shows the absolute number of T cells, and the right y-axis shows the percentage of CAR-transduced (ie, EGFRt⁺) T cells obtained from 0.5 × 10⁶ input T cells after 9 days of culture. The subsequent antigen-dependent expansion of SLAMF7-CAR and CD19-CAR (control) T cells after stimulation with irradiated feeder cells. The diagram shows the absolute number of T cells obtained from 1 × 10⁶ input (EGFRt-enriched) T cells within 9 days of culture (lower bar diagram). The same SLAMF7⁺CD19⁺EBV-LCL feeder cells were used to stimulate SLAMF7-CAR and CD19-CAR (control) T cells. (C) Cytotoxic/cytolytic activity of CD4⁺ and CD8⁺ SLAMF7-CAR T cells within 4 and 20 hours of coculture, respectively, with myeloma cell lines. SLAMF7^– native K562 cells were used as a negative control. K562 cells that had been transduced with SLAMF7 were used as a positive control (K562/SLAMF7). (D) Secretion of IFN-γ and IL-2 analyzed by enzyme-linked immunosorbent assay in supernatants obtained after a 20-hour coculture of effector and target cells. Assays were performed with 50 000 effector T cells and 10 000 target cells in 96-well round bottom plates with a volume of 200 μl. CD19-CAR–transduced CD4⁺ and CD8⁺ T cells were used as controls, did not produce cytokines above background, and are therefore not shown in the diagram. (E) Proliferation assessed by carboxyfluorescein diacetate succinimidyl ester dilution after a 72-hour coculture of effector and target cells. Assays were performed with 50 000 effector T cells and 10 000 irradiated target cells in 96-well plates with a volume of 200 μl without the addition of exogenous cytokines. Data in panels A-E are representative of the results obtained in independent experiments with CAR T cells prepared from 4 healthy donors. ns, not significant.