Subunit engineering enables the generation of human interleukin-39 as a proinflammatory cytokine

Interleukins are signaling proteins that control immune responses. Defining the range of human interleukins, their associated receptors, signaling pathways, and functions is thus indispensable for understanding and modulating protective and pathological immunity. Interleukin-39 (IL-39), a heterodimer composed of the subunits IL-23α and EBI3, has been described as a proinflammatory cytokine in mice, but its existence in the human interleukin repertoire has remained controversial. Here, we showed that IL-39 did not readily form from IL-23α and EBI3 in human cells. A combined computer modeling and experimental approach identified structural features that destabilize the human IL-23α:EBI3 heterodimer. Using these insights, we rationally engineered human IL-23α and EBI3 to form disulfide-linked heterodimers that were secreted by cells. This engineered cytokine engaged the receptor subunits IL-23R and gp130 on primary human immune cells to stimulate phosphorylation of the transcriptional regulators STAT1 and STAT3, resulting in proinflammatory cytokine secretion. Together, our findings define limitations in the biogenesis of human IL-39 and show that these can be overcome by molecular engineering. This work highlights the potential of rational cytokine engineering for generating active immune signaling proteins and lays the foundation for further defining possible roles of IL-39 in human immunity.