ANALYSIS OF THE MULTIPLE INTERACTIONS BETWEEN IL-12 AND THE HIGH-AFFINITY IL-12 RECEPTOR COMPLEX

IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit , that exerts its biological effects by binding to specific cell surfa ce receptors, Two IL-12R proteins, designated human IL-12 (huIL-12) re ceptor beta 1 (huIL-12R beta 1) and huIL-12R beta 2, have been previou sly identified, These IL-12R individually bind huIL-12 with low affini ty 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 heterod imer-specific anti-huIL-12 mAb 20C2, which neutralizes huIL-12 bioacti vity but does not block I-125-huIL-12 binding to huIL-12R beta 1, bloc ked binding of huIL-12 to huIL-12R beta 2. In contrast, anti-huIL-12R beta 1 mAb 2B10 and mouse IL-12 p40 subunit homodimer (mo(p40)(2)) blo cked I-125-huIL-12 binding to huIL-12R beta 1, but not to huIL-12R bet a 2. Therefore, two classes of IL-12 inhibitors can he identified that differ in their ability to block huIL-12 interaction with either huIL -12R beta 1 or huIL-12R beta 2. Both mo(p40)(2) and 20C2 blocked high affinity binding to huIL-12R beta 1/beta 2-cotransfected COS-7 cells, although, as previously reported, mo(p40)(2) does not block high affin ity 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-gamma production, Therefore, IL-1 2, in binding to the high affinity IL-12R, interacts with IL-12R beta 1 primarily via regions on the IL-12 p40 subunit and with IL-12R beta 2 via 20C2-reactive, heterodimer-specific regions of IL-12 to which th e p35 and p10 subunits both contribute.