A novel spontaneous HLH mouse model by engrafting human HSCs into b-ndg MGMT3 mice

Abstract Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome with an incidence of 1-10 per million in children. Primary HLH results from genetic defects in cytotoxic function (e.g., PRF1, UNC13D), while secondary HLH is triggered by infections, malignancies, or autoimmune conditions. First-line therapy (etoposide + dexamethasone) has significant toxicity, and refractory cases require costly biologics like emapalumab (anti-IFNγ). Establishment of physiologically relevant animal models recapitulating human immune dysregulation is critical for developing new therapeutic approaches. Methods: We generated B-NDG MGMT3 mice by knock-in of human IL3, GM-CSF, CSF1, and THPO genes into B-NDG mice (severe immunodeficient, lacking mature T, B, NK cells). Newborn pups were engrafted with human umbilical cord hematopoietic stem cells (HSCs). Mice were monitored daily for survival. Peripheral blood was collected every 3 weeks post-engraftment (Week 6) for flow cytometry to quantify human immune cell subsets. At Week 13, complete blood counts (CBC) were analyzed, and livers/spleens were processed for H&E and Prussian blue staining, and immunohistochemistry (IHC) for human CD3 (T cells) and CD68 (macrophages) detection. B-NDG hIL15 mice served as controls. Results: Mortality occurred from Week 6 post-HSC engraftment, despite stable weight gain. At Week 12, human CD45+ leukocytes reached 70% chimerism, including T/B/NK cells, granulocytes, monocytes/macrophages, myeloid-derived suppressor cells (MDSCs), and conventional/plasmacytoid dendritic cells (cDCs/pDCs). CBC revealed significantly elevated leukocytes and reticulocytes (p<0.01) but reduced erythrocytes, hemoglobin, and platelets (p<0.001) vs. B-NDG hIL15 controls. H&E staining showed extensive mononuclear cell infiltration in livers/spleens. Prussian blue staining identified hemosiderin-laden hemophagocytic histiocytes (macrophages) in both organs. IHC confirmed widespread infiltration of human CD3+ T cells and CD68+ macrophages. Conclusions: The B-NDG MGMT3 mice exhibits hallmark HLH features: cytopenias (erythrocytes/platelets), reticulocytosis, tissue-infiltrating T cells/macrophages, and hemophagocytosis with hemosiderin deposition. This mouse model faithfully recapitulates human HLH pathophysiology and is ideal for evaluating novel therapeutics. Its flexibility enables modeling of secondary HLH (e.g., via EBV infection) to investigate context-specific mechanisms and treatments.