(P-BT-13) Conditional Depletion of Macrophages in a New Line of Immunodeficient Mice Improves Human Cell Circulation After Blood Transfusion

Background/Case Studies: Immunodeficient mice, such as the NOD-SCID-Gamma (NSG) strain of mice, are important for the study of human blood cells. Xenogeneic cells can engraft these mice because of their lack of lymphocytes, functional hemolytic complement factor 5 (C5), and macrophage defects. Nonetheless, cellular barriers remain that limit engraftment of foreign cells, which is exemplified by low levels of peripheral blood cell chimerism in humanized mice. Mouse macrophages are one of the barriers. Accordingly, we have created a line of mice that allow for depletion of macrophages by crossing NSG mice with macrophage Fas-induced apoptosis (MaFIA) mice resulting in a stable line of NSG-MaFIA mice.

Study

Design/Methods: NSG-MaFIA mice were generated by crossing NSG and MaFIA mice, with the hybrids backcrossed for 9 generations to NSG mice. Flow cytometry was used to show the presence of the MaFIA gene construct, using a fluorescent marker, among peripheral blood, peritoneal, bone marrow, and spleen cells. Macrophage apoptosis was achieved through administration of the dimerizer. The phenotypic characteristics of NSG mice were confirmed in NSG-MaFIA mice by flow cytometry, CBC analysis, testing of radiation sensitivity, sequencing the C5 gene, and transfusion of human red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs).

Results/Findings: The MaFIA transgene was introgressed into NSG mice as exhibited by expression of a fluorescent marker. Functional MaFIA expression was evidenced by weight loss and decreased fluorescent marker expression after dimerizer treatment. NSG-MaFIA mice are lymphopenic, sensitive to X-ray irradiation, and carry a mutated C5 gene. Transfusion of human RBCs resulted in similar clearance in NSG and NSG-MaFIA mice (n = 5 mice each group), indicating a similar innate immune response. However, transfusion of RBCs after dimerizer depletion of monocytic cells in NSG-MaFIA mice (n = 10) led to prolonged circulation of RBCs (2-way ANOVA, group effect P = 0.0174). Area under the curve analysis over 24 hours indicated a 1.75-fold increase in circulating RBCs in the NSG-MaFIA mice. Dimerizer treatment also made NSG-MaFIA mice more amenable to engraftment by human PBMCs, with standard doses of cells leading to rapid onset of lethal graft versus host disease. With reduced graft sizes, NSG-MaFIA mice (n = 10) had higher circulating levels of human blood cells compared with NSG mice (n = 10) after 14 and 20 days.

Conclusions: NSG-MaFIA mice were generated through breeding of the MaFIA transgene into the NSG mice. Selective depletion of monocytic cells occurred with dimerizer treatment. NSG-MaFIA mice shared the major phenotypic characteristics associated with NSG mice. Transfusion of human RBCs and PBMCs into macrophage depleted NSG-MaFIA mice increased the persistence of the human cells in the circulation, indicating a role for monocytic cells in the removal of foreign blood cells from NSG mice.