Single-cell proteomics and transcriptomics resolve the development of humand and murine eosinophils and the impact of IL-5 neutralization on the expansion of their lineage

Biological therapies targeting eosinophils through the eosinopoietin Interleukin-5 (IL-5), such as mepolizumab and reslizumab, or its receptor IL5a, such as benralizumab, are widely used in the treatment of severe eosinophilic asthma. This calls for a refined understanding of the ontogeny and mechanisms of expansion of the eosinophil lineage. In a translational study, we combined single-cell proteomics and transcriptomics and generated transgenic IL5Ra reporter mice to comprehensively resolve eosinophilopoiesis. Our results reconcile human and murine eosinophilopoiesis by showing that eosinophils arise from a conserved pathway of myelopoiesis also generating basophils and mast cells but no neutrophils. Using high dimension screening in flow cytometry, we also characterized major immunophenotypes along the continuum of eosinophil maturation in both human and mice. We designed simple flow cytometric methods for fully capturing eosinophilopoiesis, and obtained extensive cell surface immunophenotyping and transcriptomes. Using these resources, we were able to show that eosinophil lineage expansion during eosinophilia relies on IL-5-dependent transit amplification. Conversely, and contradicting a long-held belief, we observed that deletion or neutralization of IL-5 using monoclonal antibodies downregulates transit amplification but does not block or compromise eosinophil maturation. This work will provide resources, methods and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics and the regulation of eosinophil development and numbers in health and disease.