The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides 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.
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Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDX) in immunodeficient NSG mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin-CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.
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