Figure 6

In vitro activation of T4⁺ human T cells. Human T cells were engineered to coexpress the IL-4-responsive 4αβ chimeric cytokine receptor together with the T1E28z CAR (combination called “T4”). After expansion in IL-4, comparison was made with control T cells that were untransduced (Untrans) or that express T1NA (truncated endodomain). (A) To define targeting specificity for ErbB receptors/dimers, 1 × 10⁶ of the indicated T-cell populations were cocultivated with an equal number of 32D cells that express the specified ErbB receptor(s). Supernatants were harvested at 72 h and analyzed for interferon (IFN)-γ (mean ± SD; ***P < 0.001; **P < 0.01; *P < 0.05). (B) 1 × 10⁶ T cells were cocultivated with confluent monolayers of the indicated head and neck tumor cells cultured in 24-well (1.9-cm²) plates. Supernatants were harvested at 72 h and analyzed for IFN-γ (mean ± SD of three independent experiments; ***P < 0.001; LUC, luciferase). (C) T cells (number indicated above each well) were cocultivated for 72 h with the indicated head and neck tumor cell monolayers, cultured in 24-well plates. Nonadherent cells were then removed and, after fixation, residual tumor monolayers were stained using crystal violet (representative of three experiments). (D) T cells were established in triplicate cultures as described in (C). Cytokine analysis was performed on supernatants harvested after 24 h (IL-2, IL-4) and 72 h (IFN-γ). (E) T-cells (1 × 10⁶ cells) were established in triplicate cultures with the indicated monolayers, in the presence or absence of IL-4 (30 ng/mL). Supernatants were analyzed after 72 h for IFN-γ.