Analysis of the Multiple Interactions Between IL-12 and the High Affinity IL-12 Receptor Complex

Abstract

IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, that exerts its biological effects by binding to specific cell surface receptors. Two IL-12R proteins, designated human IL-12 (huIL-12) receptor β1 (huIL-12Rβ1) and huIL-12Rβ2, have been previously identified. These IL-12R individually bind huIL-12 with low affinity and in combination bind huIL-12 with high affinity and confer IL-12 responsiveness. In this study the interactions of huIL-12 with these two identified human IL-12R protein subunits are examined. The heterodimer-specific anti-huIL-12 mAb 20C2, which neutralizes huIL-12 bioactivity but does not block 125I-huIL-12 binding to huIL-12Rβ1, blocked binding of huIL-12 to huIL-12Rβ2. In contrast, anti-huIL-12Rβ1 mAb 2B10 and mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to huIL-12Rβ1, but not to huIL-12Rβ2. Therefore, two classes of IL-12 inhibitors can be identified that differ in their ability to block huIL-12 interaction with either huIL-12Rβ1 or huIL-12Rβ2. Both mo(p40)2 and 20C2 blocked high affinity binding to huIL-12Rβ1/β2-cotransfected COS-7 cells, although, as previously reported, mo(p40)2 does not block high affinity binding to IL-12R on PHA-activated human lymphoblasts. Furthermore, these two classes of IL-12 inhibitors synergistically decreased huIL-12-stimulated proliferation and IFN-γ production. Therefore, IL-12, in binding to the high affinity IL-12R, interacts with IL-12Rβ1 primarily via regions on the IL-12 p40 subunit and with IL-12Rβ2 via 20C2-reactive, heterodimer-specific regions of IL-12 to which the p35 and p40 subunits both contribute.