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.

Human IL-35 Inhibits the Bioactivity of IL-12 and Its Interaction with IL-12Rβ2

“…To further rule out the involvement of IL-12Rβ1, we repeated this experiment with p40 monomer. Consistent with prior literature ( 20 , 21 ), p40 monomer binding was specific to rIL-12Rβ1, not rIL-12Rβ2 ( Fig. 4D ); the addition of IL-35 did not disrupt p40 monomer binding to IL-12Rβ1 ( Fig.…”

“…The mechanism our data support, wherein IL-35 prevents IL-12 from associating with IL-12Rβ2, is reminiscent of another IL-12 family member that suppresses lymphocyte activity: the IL-12p40 homodimer [IL-12(p40) 2 ] ( 40 ). IL-12(p40) 2 competes with IL-12 for access to the IL-12Rβ1 chain ( 20 ) and (similar to IL-35) suppresses IL-12–elicited IFN-γ secretion ( 41 ). IL-12p40 monomer also ameliorates EAE, albeit via a slightly different mechanism involving prevention of IL-12Rβ1 internalization ( 42 ).…”

Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23

“…The two founding members of the family, IL-12 and IL-23, serve as pro-inflammatory and pro-stimulatory cytokines in the development of T h 1 and T h 17 subsets of helper T-cells, respectively 1,5 . Heterodimeric IL-12 and IL-23 share IL-12B (also, termed p40) as their β-subunit, which interacts with IL-12 receptor β1 (IL-12Rβ1) 6 , and are distinguished by their usage of their α-subunits IL-12A (p35) and IL-23A (p19) 7–9 , which interact with IL-12 receptor β2 (IL-12Rβ2) and IL23 receptor (IL-23R), respectively 6,10 (Figure 1a). Although the field has been greatly enriched by diverse functional and structural studies 11–15 , essential insights into the architecture of the complete extracellular receptor complexes mediated by IL-12 and IL-23 have eluded the field.…”

Evidence for increased expression of regulatory cytokine receptors interleukin-12R and interleukin-18R in common variable immunodeficiency

“…The ligand for IL-12 is the IL-12 receptor, which is composed of two subunits: the 73 000-MW IL-12R β 1 polypeptide chain (CD212), which binds IL-12, and the 130 000-MW IL-12R β 2 signal-transducing polypeptide chain. Although not always coexpressed on T cells, both of these subunits are necessary for a functional receptor [6][7][8]. Significantly, IL-12R β 2 is not expressed on naïve T cells, but is induced at low levels after antigen stimulation of the TCR on Th1, but not Th2, clones [9,10].…”