Dendritic cell derived IL-12p40 binds extracellular proteins to make heterodimeric cytokines

Abstract IL-12 and IL-23 are heterodimeric cytokines which share a common subunit, p40. Binding of p40 to p35 results in IL-12, while binding to p19 generates IL-23. Assembly of these cytokines is thought to occur only when both relevant subunits are expressed inside a dendritic cell (DC) allowing for covalent linkage and secretion. Intriguingly, the p40 subunit is also released from DC as a monomer with unknown function. We previously hypothesized that monomeric p40 can bind p35 (or p19) released by another cell in the extracellular space. Consistent with this, recombinant p40 and p35 were shown to assemble in cell free extracts. To test this hypothesis in a physiological setting, in vitro and in vivo assays were performed by mixing p40−/− and p35−/− cells. In these assays, since no one cell can make both subunits, the only way IL-12 can be present is through extracellular assembly. In vitro bioassays for IL-12 function showed proliferation and IFNγ production in response to mixing. In vivo, mixed bone marrow chimeras infected with Leishmania major were able to induce IFNγ production by T cells. These results suggest that functional IL-12 assembly can occur in the extracellular milieu from component peptides. Importantly, this mechanism may allow even non-hematopoietic tissues to direct T cell differentiation by releasing proteins capable of binding to DC-derived p40. In order to identify novel heterodimeric cytokines capable of fulfilling that paradigm, we have used a panel of proteins that can bind p40, identified from an unbiased mass spectrometry based analysis. Recombinant versions of these proteins expressed as dimers with p40 are being evaluated for functional activity.