Design and expression of GIFT15 fusokine. (A) Schematic representation of the GIFT15 aa sequence. (B) The predicted structural model of GIFT15. GMCSF is shown in green ribbon; intercytokine bridge, in gray ribbon; and IL-15, in cyan ribbon. The IL-15 residues experimentally identified to interact with IL-15Rα, IL-2Rβ, and IL-2Rγ are shown by yellow, purple, and red balls, respectively. (C) Denaturing immunoblot using conditioned media (CM) from genetically-modified B16F0 expressing the green fluorescent protein (GFP) or GIFT15 probed with polyclonal goat anti–IL-15 or anti-GMCSF antibodies. rIL-15 and rGMCSF were used as positive controls. (D) Biologic activity of GIFT15. To test the bioactivity of GIFT15, proliferation assays were performed by MTT incorporation using CTLL-2 and JAWSII cell line concentrations (CTLL-2, P > .05 between GIFT15 and IL-15; JAWSII, P > .05 between GIFT15 and GMCSF). Results are shown as mean of triplicates ± SEM of 1 representative experiment of 3.