sBCMA binds BCMA CAR T cells and inhibits cytokine production. (A) sBCMA concentration in plasma from MM and control patient BM measured by ELISA. Samples were stratified by the percentage of CD138⁺ cells of live BMMCs as measured by flow cytometry for MM patient samples. (B) Surface staining of CD4⁺ BCMA⁻ and CD19 CAR T cells with APC-conjugated BCMA/Fc and anti-EGFR Ab. (C) Staining with APC-conjugated BCMA/Fc of CD4⁺ BCMA CAR T cells after 30 minutes preincubation with exogenous recombinant BCMA. (D) Intracellular IFN-γ (left), IL-2 (middle), or TNF (right panel) staining of CD4⁺ T cells after 5 hours of stimulation with 8226 or U266 (BCMA CAR T) or K562-CD19 (CD19 CAR T) in the presence and absence of exogenous recombinant BCMA at an E:T ratio of 4:1. (E) Cytolytic activity of CD4⁺ BCMA CAR T cells against K562/BCMA (left), U266 (middle), and 8226 (right) at varying concentrations of recombinant BCMA analyzed by a 4-hour ⁵¹Cr-release assay at an E:T ratio of 30:1 (squares) and 10:1 (circles). Data in panels B and C are representative of 2 independent experiments with T cells from different donors; data in panels D and E show summarized data with T cells from 2 different donors. Error bars represent mean plus SEM. *P < .05; as determined by 1-way ANOVA with the Tukey posttest. Data for CD8⁺ T cells are shown in supplemental Figure 1A.