Figure 1.
Constitutive expression of bioactive interleukin-12 (IL-12) inhibited the proliferation of transduced peripheral blood lymphocytes (PBLs) and was associated with increased apoptosis. (a) Human PBLs were cotransduced with MART-1 T cell receptor (TCR) and MSGV1.hIL12 vector or control truncated low-affinity nerve growth factor receptor (tLNGFR) vector. Expression of MART-1 and IL-12 were measured by fluorescence-activated cell sorter (FACS) using mouse antihuman Vβ12-phycoerythrin (PE) and IL-12-fluorescein isothiocyanate (FITC) antibody on day 6 after transduction. (b) The double-transduced PBLs were cocultured with melanoma lines: mel526 (HLA-A2+/MART-1+), mel624 (HLA-A2+/MART-1+), mel888 (HLA-A2−/MART-1+), mel938 (HLA-A2−/MART-1+). Concentration of interferon (IFN)-γ in the culture was measured using IFN-γ enzyme-linked immunosorbent assay (ELISA) (shown are the mean values of triplicate determinations, ±SD). (c) PBLs from three donors were transduced with tLNGFR or MSGV1.hIL12 to evaluate in vitro cell expansion. Viable cells were enumerated every 3–5 days by Trypan blue exclusion (n = 3; *P < 0.05; **P < 0.001). (d) Left panel: IL-12-induced apoptosis in PBLs was partially blocked by anti-IFN-γ or anti-IL12Rβ2 antibodies. On day 2 after transduction, the transduced cells were treated with 50 µg/ml of indicated antibodies. Three days later, the cells were stained with Annexin V-phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD) and analyzed by FACS. For each plot, the upper right sextant contains necrotic cells, the middle right contains late apoptotic cells, and the lower right contains early apoptotic cells. (d) Right panel: quantification of the percentage of Annexin V positive staining cells in each treatment condition (n = 3, shown are the mean values of triplicate determinations, ±SD, *P < 0.05 compared to mIgG).