CAFs inhibit BCMA-CART cells through direct and indirect interactions. (A) BCMA-CART-cell proliferation assay with BM-CAFs and anti–TGF-β was performed in transwell or non-transwell plates. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled BCMA-CART cells were cocultured with lethally irradiated MM1.S for 5 days with BM-CAFs and anti–TGF-β. Absolute numbers of BCMA-CART cells were analyzed by using flow cytometry. BCMA-CART cells were defined as CD3-APC-H7–positive and CD45-BV421–positive cells (mean and standard error of the mean [SEM]; ***P < .001, one-way analysis of variance; n = 3, two replicates). (B-C) BCMA-CART cells were cocultured with irradiated MM1.S and BM-CAFs for 3 days, and supernatants were analyzed via singleplex or multiplex for cytokines/chemokines (mean and SEM; *P < .05, **P < .005, ***P < .0005, ****P < .0001, two-tailed Mann-Whitney U test; n = 2, two replicates). (D) BCMA-CART cells were cocultured with OPM-2 in the presence or absence of BM-CAFs. At 24 hours, cytotoxicity was assessed by luminescence relative to controls (*P < .05, ****P < .0001, two-way analysis of variance; n = 3, two replicates). (E) The evaluation of inhibitory receptors on BCMA-CART cells when they were cocultured with BCMA⁺SLAMF7⁺OPM-2 for 5 days in the presence or absence of BM-CAFs (mean and SEM; **P < .005, ****P < .0001, unpaired, two-sided t test; n = 2, two replicates). (F) The evaluation of inhibitory ligands on BM-CAFs when they were cocultured with BCMA-CART cells and irradiated MM1.S for 5 days; BM-CAFs were defined as FSP-1⁺CD38^– fractions. (Mean and SEM; ****P < .0001, unpaired, two-sided t test; n = 3, two replicates). E:T, effector-to-target ratio; n.s., not significant.