Neonatal NOD/SCID/IL2rg-Null Mice Support the Functional Development of Human Hematopoietic and Immune Systems.
Authors: Ishikawa Fumihiko , Yasukawa Masaki , Lyons Bonnie , Yoshida Shuro , Shultz Leonard D. , Harada Mine 
Published: April 2020

(Purpose) We aimed to develop a new model for studying the development of a human hematopoietic and immune systems in vivo.

(Methods) In order to establish a new model of xenogeneic transplantation, we establish an immune-compromised strain, NOD.Cg-PrkdcscidIL2rgtmlWjl/Sz (NOD/SCID/IL2rg-null) mice by backcrossing a complete null mutation of the IL2 receptor common gamma chain (IL2rg) onto the NOD/SCID background. 1 x 105 human CB-derived lineage antigen negative (Lin−) CD34+ cells were intravenously transplanted into newborn NOD/SCID/IL2rg-null mice following 100cGy irradiation. At 3 months post-transplantation, the engraftment of human cells was evaluated by flow cytometric analysis, immunostaining, and functional assays for production of human immunoglobulin and T-cell cytotoxicity against allogeneic cells.

(Results) NOD/SCID/IL2rg-null mice showed extremely low activity of NK cells along with the complete lack of mature B cells and T cells. During post-natal development of the NOD/SCID/IL2rg-null mice, a human hematopoietic system was developed following injection of human CB-derived Lin-CD34+ cells. In BM of the recipient mice, human glycophorin A+ erythroid cells were present at 9.5 +/− 6.2% (n=5), and human CD41+ megakaryocytes were present at 1.64 +/− 0.42% (n=5). Human CB-derived Lin−CD34+ cells generated multi-lineage leukocytes, CD33+ myeloid cells, CD19+ B cells, and CD3+ T cells. The engraftment level of human CD45+ cells in peripheral blood was significantly higher (68.9 +/− 11.6%, n=5) in NOD/SCID/IL2rg-null mice than that in NOD/SCID/b2mnull mice (12.4 +/− 5.9%, n=4). Mature erythrocytes and platelets were identified in peripheral blood. The xenogeneic environment supported the systemic development of a human immune system, containing each stage of B cells and T cells in primary and secondary lymphoid tissues. CD34+CD19+ pro-B cells, CD10+CD19+ B cells, and CD19+CD20hi mature B cells were identified in the BM and spleen. Immature CD4+CD8+ double positive T cells were the major cell populations in the thymus, while spleen contained abundant single positive T cells at 1.39 +/− 0.61 (n=5) CD4/CD8 ratio, suggesting that human CB stem/progenitor-derived T cells underwent the maturation and proliferation similarly as identified in human body. Transplanted human stem cells reconstituted mucosal immunity in intestinal tracts as evidenced by human IgA+ B cells and CD3+ T cells. Adaptive human immune system cooperatively functioned in xenogeneic environment to produce antigen-specific human IgM and IgG antibodies, when engrafted mice were immunized with ovalbumin. Furthermore, human CD4+ T cells as well as CD8+ T cells generated in the xenogeneic host exerted cytotoxicity against allogeneic target cells.

(Conclusion) The neonatal NOD/SCID/IL2rg-null model will facilitate studying post-natal development of the human hematopoietic and immune systems and for studying of human immune surveillance in vivo against exogenous antigens.