Design, expression, and IL-6–neutralizing capacity of a bicistronic construct encoding mbaIL6 and anti-CD19 CAR. (A) Schema of the plasmid encoding both receptors (“DUAL”). (B) mbaIL6 expression in peripheral blood T cells transduced with either GFP alone (“Control”), GFP plus anti-CD19 CAR, mbaIL6, or both; cells were labeled with biotin-conjugated goat anti-human F(ab′)2 antibody and streptavidin-APC, and CD19-myc followed by PE-conjugated anti-myc. Expression of each receptor in relation to GFP is shown in supplemental Figure 4. (C) Aggregate data of mbaIL6 and CAR expression from 6 transductions with T cells from 6 donors. Mean (±SD) is shown. (D) Cell marker profile of CAR or DUAL-transduced peripheral blood T cells. Mean (±SD; n = 3) percent T cells expressing each marker is shown. (E) Proportion of naive (CD45RA⁺ CCR7⁺), effector (T_E, CD45RA⁺ CCR7^‒), central memory (T_CM, CD45RA^‒ CCR7⁺), and effector memory (T_EM, CD45RA^‒ CCR7^‒) phenotypes among T cells transduced with the various constructs (mean of 3 experiments). (F) T lymphocytes transduced as in panels B and C were cultured with 1 ng/mL IL-6. After 2 hours, IL-6 levels in the supernatant were measured by using ELISA. Symbols indicate results of 2 independent experiments.