• Human PBMC-engrafted humanized mice are attractive models for in vivo analysis of human immune responses. We previously reported the successful engraftment of human PBMCs in B-NDG mice. However, due to the severe xenograft versus host disease (xeno-GvHD) in these mice, the experimental window is limited. The pathogenesis of GvHD is directly related to human T cell implantation, and the mismatch between human and mouse MHCs is the main cause of GvHD after human PBMCs engraftment. It has been shown that knocking out MHC I and/or II molecules in mice can reduce GvHD and extend the experimental window after engraftment of human PBMCs.
• MHC class I molecules are composed of two subunits, α and β chains, and the α chain in NOD background strain of mice is encoded by H2-K1, H2-D1 gene. Knocking out H2-K1 and H2-D1 gene in mice makes it unable to form a complete MHC Class I molecule. MHC class II molecules are also composed of two subunits, α and β chains, and the β chain is encoded by H2-Ab1 gene. Knocking out H2-Ab1 gene in mice makes it unable to form a complete MHC Class II molecule. These incomplete MHC Class I and MHC Class II molecules will not be able to effectively express on the cell surface and perform antigen-presenting functions, thereby potentially reducing the occurrence of GvHD.
• Application: This model can be used to study the mechanism of xeno-GvHD in vivo, the pathogenesis of immune-related diseases such as tumor, autoimmune and metabolic diseases, and to evaluate the efficacy of novel drugs .

Gene targeting strategy for B-NDG MHC I/II DKO mice ad. The exons 2-8 of mouse H2-K1 and H2-D1 gene, the exons 2-4 of mouse H2-Ab1 gene were knocked out in B-NDG MHC I/II DKO mice ad. So the transcription and translation of mouse H2-K1, H2-D1, H2-Ab1 genes will be disrupted.

The elimination of human IgG1 antibody in the plasma of B-NDG MHC I/II DKO mice ad was significantly slower than that in B-NDG mice. BALB/c mice (n=5), B-NDG mice (n=4) B-NDG MHC I/II DKO mice ad (n=4) were treated with YH001 (anti-human CTLA4 antibody). Blood samples were collected at different time point for the PK assay. Results showed that B-NDG MHC I/II DKO mice ad can significantly extend the half-life of antibody drugs in mice.

B-NDG MHC I/II DKO mice ad has a long life span and reduced severity of GvHD when engrafted with human PBMCs. B-NDG MHC I/II DKO mice ad were engrafted intravenously with human PBMCs (1×10⁷) from two donors (Donor 1, Donor 2) on day 0 (n=6/8). Survival rates of the mice were analyzed with Kaplan Meier survival curves. Body weight was measured twice a week. Clinical signs of GvHD were scored once a week. Euthanasia was implemented when the body weight decreased more than 20%. Meanwhile, the GvHD score of the mouse was recorded as 10. The results showed that at 8 weeks after PBMC engraftment, the mice did not exhibit significant symptoms of GvHD and their body weight continued to increase. Values were expressed as mean ± SEM.

Human PBMCs were successfully reconstituted in B-NDG MHC I/II DKO mice ad. B-NDG MHC I/II DKO mice ad were engrafted intravenously with human PBMCs (1×10⁷) from two donors on day 0 (n=6/8). Peripheral blood was taken to analyze the reconstitution level of human immune cells. A. Frequency of reconstituted human immune cells; B. Absolute cell number of reconstituted human immune cells. The results showed that two weeks after the reconstitution of human PBMCs in B-NDG MHC I/II DKO mice ad, the frequency and absolute cell number of CD45+ cells in peripheral blood began to increase. The frequency of reconstituted human T cells exceeded 90% from three weeks and continued to rise, eventually reaching nearly 100%. Reconstituted human T cells include CD4+T cells, CD8+T cells and Tregs. A small amount of DCs can also be detected. This indicates that B-NDG MHC I/II DKO mice ad is a powerful immunodeficient mouse model for reconstitution of the human T cells using human PBMCs.