Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo–electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rβ, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rβ binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

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Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

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