Expression of mbaIL6 does not affect anti-CD19 CAR function. (A) IFN-γ production in T cells cocultured with CD19⁺ OP-1 ALL cells for 6 hours at 1:1, measured by flow cytometry after labeling with anti-human IFN-γ–PE. Symbols are triplicate measurements with T cells from 3 donors. CAR expression was 73% to 85% for CAR alone and 71% to 80% for CAR plus mbaIL6 (“DUAL”). (B) CD107a expression in T cells cocultured with OP-1 for 4 hours at 1:1, measured by flow cytometry after labeling with anti-human CD107a-PE. (C) T-cell cytotoxicity against OP-1 after 4 hours at 1:1. (D) T cells were cultured for 30 minutes with 5 ng/mL IL-6 before coculture with Daudi-luciferase cells at 2:1 or 1:1. Bars show mean (±SD) cytotoxicity in triplicate measurements. (E) T-cell proliferation in cultures with or without OP-1 for 21 days at 1:1, with 120 IU/mL IL-2. Irradiated OP-1 cells were added on days 0, 7, and 14. Symbols are percentage of cells recovered after culture compared with the number of cells at the beginning of the culture and are the mean (±SD) of triplicate measurements with cells from 3 donors. (F) PD1, TIM3, and LAG3 expression in T cells cocultured with irradiated OP-1 for 7 days at 1:1 with 10 IU/mL IL-2. Data were plotted with Python 3 using Matplotlib package (https://matplotlib.org/). The profile of unstimulated cells is shown in supplemental Figure 5.