Functional consequences of IL-6 neutralization with mbaIL6-T cells. (A) mbaIL6 and GFP expression in peripheral blood T cells transduced with either GFP alone (“Control”) or GFP plus mbaIL6, after labeling with biotin-conjugated goat anti-human F(ab′)2 antibody and streptavidin-APC. (B) IL-6 binding to Control or mbaIL6-transduced peripheral blood T cells, labeled with IL-6 biotin and streptavidin-APC. (C) Cell marker profile of Control or mbaIL6-transduced T cells from 3 donors. Mean (±SD) of percent T cells expressing each marker is shown. (D) Control or mbaIL6-transduced T lymphocytes at the indicated concentrations were cultured for 2 hours with 1 ng/mL human IL-6; IL-6 in the supernatant was measured by using ELISA. Mean (±SD; n = 3) is shown. ***P < .001. (E) DS-1-mCherry cells were cocultured with Control or mbaIL6-transduced T cells at a 1:1 ratio, with IL-6 (0.5 ng/mL). DS-1 proliferation was quantitated by using the IncuCyte Live Imaging System; shown are mean (±SD) of red calibrated units (RCU) × μm²/well in triplicate measurements. *P = .02; **P < .01 for data at 120 hours. (F) Control or mbaIL6-transduced T lymphocytes at the indicated concentration were cultured for 2 hours with 1 ng/mL IL-6. THP-1 cells were then exposed to either 1 ng/mL IL-6 or to the supernatant of the lymphocyte cultures for 15 minutes at 37°C. Flow cytometry histograms show labeling of THP-1 cells with anti-STAT3 pY705; the graph on the right shows the decrease in pSTAT3 relative to that of THP-1 cells exposed for 15 minutes to 1 ng/mL IL-6. (G) THP-1 cells were exposed to IL-6 for 15 minutes, after 30-minute incubation with the indicated concentrations of tocilizumab. Cells were then labeled with anti-STAT3 pY705 and analyzed as in panel F.