Targeting BM-CAFs and malignant cells overcome BM-CAF–induced CART-cell dysfunction. (A) CD45^– CD38^– FSP-1⁺ cells expressed SLAMF7 and FAP but not BCMA; n = 3. (B) SLAMF7 and FAP were not expressed in CD45^– cells from healthy donor BM (*P < .05, unpaired, two-sided t test); n = 4. (C) FAP-CART cells were cocultured with luciferase⁺FAP⁺WI-38. At 24 hours, cytotoxicity was assessed by luminescence relative to controls. SLAMF7-CART cells were coincubated with luciferase⁺ MM1.S JeKo-1 or Jurkat (negative control) cell lines (mean and SEM; **P = .01, ****P < .0001, two-way analysis of variance; n = 3, two replicates). (D) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled FAP- or SLAMF7-CART cells were cocultured with irradiated WI-38 (for FAP-CART cells), MM1.S (for SLAMF7-CART cells), K562 (negative control), or MM patient–derived BM-CAFs for 5 days. Medium and phorbol 12-myristate-13-acetate/ionomycin (PI) were used as negative and positive controls, respectively (n = 3, two replicates). (E) To assess degranulation and cytokine production, FAP or SLAMF7-CART cells were cocultured with WI-38 (for FAP-CART cells) or MM1.S (for SLAMF7-CART cells) for 4 hours. For negative controls, JeKo-1 (for FAP-CART cells) or Jurkat (for SLAMF7-CART cells) cells were used (mean and SEM; *P < .05, **P < .005, ***P < .001, ****P < .0001, one-way analysis of variance; n = 3, two replicates). FITC, fluorescein isothiocyanate; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; n.s., not significant; PE, phycoerythrin.