Development of a low-dose PBMC humanized mouse model using CD47;Rag2;IL2rγ triple KO mice: Enhanced leukocyte reconstitution and extended experimental window

Abstract Humanized mice (hu-mice), which recapitulate the human immune system, have become increasingly important for preclinical immunotherapy studies. Among these models, the human peripheral blood mononuclear cells (PBMC)-engrafted hu-mice model is the simplest and fastest. However, its utility is hindered by the development of lethal graft-versus-host disease (GvHD) and the insufficient reconstitution of human leukocytes. To address these limitations, we developed PBMC hu-mice models using a novel strain, NOD-CD47nullRag2nullIL-2rγnull (RTKO) focusing on the immunological defects of the NOD strain and the immunotolerance provided by CD47 deficiency. Six-week-old female NOD-Rag2nullIL-2rγnull (RID) and RTKO mice were intravenously injected with three different PBMC doses (3×106, 5×106, and 1×107 cells). At standard doses (5×106 and 1×107 cells), RTKO mice exhibited enhanced engraftment of human leukocytes, though GvHD was more severe compared to the RID strain, resulting in a limited experimental window. However, in a subsequent trial using a lower dose of PBMCs (3 × 106 cells), RTKO mice demonstrated notable advantages, including stable reconstitution of human leukocytes, milder GvHD symptoms without life-threatening lesions, and a markedly prolonged experimental window. Considering the difficulties in generating hematopoietic stem cell (HSC)-engrafted hu-mice, the extended experimental window provided by this model, which is comparable to HSC hu-mice, is a significant improvement. Moreover, the radiation tolerance conferred by the Rag gene mutation in this model offers another advantage for radiotherapy research. Consequently, the low-dose PBMC RTKO model serves as a versatile and valuable platform for a broad spectrum of immunotherapy studies, especially in the field of immuno-oncology. Competing Interest Statement The authors have declared no competing interest.