Figure 6.

Evaluation of a human iaIL-12 construct in vitro. (A) Schematic representation of retroviral inserts for expression of human IL-12 constructs. (B, C) Human T cells were transduced to express in the construct indicated above each histogram. Two days after transduction, the T cells were activated by PMA/ionomycin overnight. (B) IL-12 cell-surface expression was detected by flow cytometry. Gating is on live lymphocytes. The frequency of IL-12⁺ cells and the geometric mean fluorescence intensity of IL-12 for each group is indicated on each histogram. (C) Transduced T cells (1×10⁶) were cultured in 1 mL of media overnight, and the supernatant IL-12 concentration was determined by an ELISA. (D) Human T cells were cotransduced to express the E7 TCR, and the IL-12 constructs are indicated on y-axis. Five days after transduction, cells were cocultured overnight with tumor cell line targets. SS4050 and CaSki are HLA-A*02:01⁺/E7⁺. 624 is HLA-A*02:01⁺/E7⁻. The concentration of cytokines in the supernatants was determined by an ELISA. Error bars represent the standard error of the mean of two technical replicates. Statistical comparisons were made to the iGFP condition using a two-way ANOVA multiple-comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. caIL-12, constitutive anchored interleukin-12; E7TCR, T cell receptor targeting human papilloma virus-16 E7; HLA, human leucocyte antigen; iaIL-12, inducible anchored interleukin-12; iGFP, inducible green fluorescent protein; IL-12, interleukin-12; LTR, long-terminal repeat; PMA, phorbol 12-myristate 13-acetate; UT, untransduced cells.