Figure 1.

Optimization of caIL-12 constructs. (A) Schematic representation of retroviral vector inserts for expression of designer murine IL-12 constructs. (B) 293cells were transfected with vectors encoding IL-12 fused with different transmembrane domains. IL-12 on surface of the cell membrane was measured by flow cytometry performed 48 hours after transfection. Gating is on live cells. (C) The IL-12 level in the supernatant of transfected 293 cells was measured by ELISA 48 hours after transfection with the construct indicated on the x-axis. (D, E) Murine T cells from C57BL/6 mice were transduced with MSGV1-based retroviruses to express the constructs indicated on the x-axis. Approximately 48 hours after transduction, 1×10⁶ genetically modified T cells were incubated in 1 mL of culture medium overnight. The levels of (D) mouse IL-12 and (E) mouse IFN-γ in the culture were measured by ELISA. caIL-12, constitutive anchored interleukin-12; csIL-12, constitutively expressed secreted interleukin-12; IFN-γ, interferon-γ; IL-12, interleukin-12;; LTR, long-terminal repeat; UT, untransduced cells.