Figure 3.

VSV23 Produces Active IL-23. A) vIL23 production by VSV23 infected BHK21 cells. BHK21 cells were infected with VSV23, VSVST, or VSVXN2 at MOI = 0.1 and incubated overnight at 37 °C. Virally infected supernatant was harvested and subjected to UV inactivation to inactivate virus. Supernatant from uninfected BHK21 cells was used as a negative control. Samples were subjected to the Quantikine Mouse IL-12/IL-23 p40 (non-allele-specific) Immunoassay ELISA kit (R&D Systems Minneapolis, MN). Supernatant from VSV23 infected cells contained 750 pg/ml of the p40 subunit. The experiment indicates that there are no detectable levels of p40 secreted by cells infected with VSVST or VSVXN2. BHK21 cells do not produce IL-23 and as expected control samples did not produce detectable levels of the cytokine component. B) IFN-γ is induced by IL-23: Lanes represent amplified mRNA specific for IFN-γ from CD4⁺ cells that were treated as follows –1: rIL-12 for 6 hours, 2: rIL-23 for 6 hours, 3: rIL-12 for 12 hours, 4: rIL-23 for 12 hours, 5: rIL-12 for 18 hours, 6: rIL-23 for 18 hours,7: vIL-12 for 6 hours, 8: vIL-23 6 hours, 9: vIL-12 for 12 hours, 10: vIL-23 for 12 hours, 11: vIL-12 for 18 hours, 12: vIL-23 for 18 hours, 13: untreated CD4⁺ T cells. Upper band represents IFN-γ signal at 400 bp, lower band is GAPDH loading control at 200 bp. Each reaction was run in triplicate, and the experiment was repeated twice. Image is typical of all results.

Abbreviations: IFN, interferon; mRNA, messenger ribonucleic acid.