HUMAN IL-12 P40 HOMODIMER BINDS TO THE IL-12 RECEPTOR BUT DOES NOT MEDIATE BIOLOGIC ACTIVITY

IL-12, a heterodimeric cytokine, consists of two disulfide-iinked subu nits, p40 and p35. We investigated the role of p40 in ligand binding a nd signal transduction by expressing this subunit alone in COS cells. Culture media of the transfected COS cells exhibited specific dose-dep endent binding to KIT225/K6 cells, a human T cell line that expresses IL-12R. Analysis of the culture media by SDS-PACE and Western blotting demonstrated the presence of 40-kDa monomers and 80-kDa disulfide-lin ked homodimers. The two p40 species were purified and identified by N- terminal sequencing and proteolytic peptide mapping. Characterization of the p40 proteins for binding and bioactivity showed that both the p 40 monomer and dimer inhibited I-125-labeled IL-12 binding to IL-12R, but the 80-kDa species, having a 50% inhibitory concentration (IC50) o f 20 to 70 ng/ml, was at least 20-fold more effective than the monomer . Although neither the monomer nor the dimer stimulated human PHA-blas t proliferation, the 80-kDa dimer inhibited IL-12-induced proliferatio n in a dose-dependent manner with an IC50 of 65 ng/ml. The results sug gest that the IL-12 p40 subunit contains the essential epitopes for re ceptor binding. However, a proper conformation required for high affin ity binding is achieved only when p40 is associated with a p35 subunit or another p40 subunit. When p40 is associated with a p35 subunit, th e heterodimer acts as an agonist mediating biologic activity. However, when p40 associates with another p40, the homodimer behaves as an ant agonist in vitro.