Ebi3 Binding to IFN-γ and IL-10 Limits Their Function

EBV-induced gene 3 (Ebi3) is a (3 subunit within the IL-12 cytokine family that canonically binds to a subunits p19, p28, or p35 to form the heterodimeric cytokines IL-39, IL-27, and IL-35, respectively. In the last decade, the binding partners for Ebi3 have continued to expand to include IL-6 and the other IL-12 family (3 subunit p40, revealing the possibility that Ebi3 may be able to bind to other cytokines and have distinct functions. We first explored this possibility utilizing an in vivo mouse model of regulatory T cell- restricted deletions of the subunits composing the cytokine IL-35, p35, and Ebi3, and we observed a differential impact on CD8+ T cell inhibitory receptor expression despite comparable reduction in tumor growth. We then screened the ability of Ebi3 to bind to different cytokines with varying structural resemblance to the IL-12 family a subunits. These in vitro screens revealed extracellular binding of Ebi3 to both IFN-g and IL-10. Ebi3 bound to IFN-g and IL-10 abrogated signal transduction and downstream functions of both cytokines. Lastly, we validated that extracellular complex formation after mixing native proteins resulted in loss of function. These data suggest that secreted partnerless Ebi3 may bind to cytokines within the extracellular microenvironment and act as a cytokine sink, further expanding the potential immunological impact of Ebi3. The Journal of Immunology, 2024, 213: 1115-1124.