Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfid e-bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40)(2), has been shown to be a potent IL-12 antagonist. To study the interaction between (p40)(2) and the known IL-12 receptor (IL-12R) subunits, IL-12R beta 1 and IL-12R beta 2, we directly measured the binding activity of mouse (p40), to ConA-activated l ymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These r esults demonstrated the presence of both high (K-d about 5 pM) and low affi nity (K-d about 15 nM) binding sites for mouse (125)l-labeled (p40)(2). To elucidate which of the IL-12R subunits binds mouse (p40)(2), binding studie s of mouse (125)l-labeled (p40)(2) to Ba/F3 cells expressing recombinant mo use IL-12R beta 1 and/or mouse IL-12 beta 2 were carried out. Mouse IL-12R beta 1 bound mouse (125)l-labeled (p40)(2) with high and low affinities, co mparable to that observed on Con A blasts and B cells. In contrast, mouse I L-12R beta 2 bound mouse (125)l-labeled (p40)(2) very poorly. These data de monstrate that similar to IL-12, mouse (p40)(2) binds with both high and lo w affinity to Con A blasts and B cells, and that IL-12R beta 1 is responsib le for mediating the specific binding of mouse (p40)(2).