Protein expression analysis

Strain specific H-2Kb/H-2Db (MHC-I) and I-Ak (MHC-II) expression analysis in B-NDG mice, B-NDG B2m KO plus mice and B-NDG MHC I/II DKO mice plus by flow cytometry.  Splenocytes were collected from the three mice and analyzed by flow cytometry. Mouse H-2Kb/H-2Db was only detectable in B-NDG mice but not in B-NDG B2m KO plus mice and B-NDG MHC I/II DKO mice plus. Mouse I-Ak was only detectable in B-NDG mice and B-NDG B2m KO plus mice but not in B-NDG MHC I/II DKO mice plus.

Frequency of leukocyte subpopulations in spleen，blood and bone marrow 

Frequency of leukocyte subpopulations in spleen, blood and bone marrow by flow cytometry. Blood, spleen and bone marrow were collected from B-NDG mice and B-NDG MHC I/II DKO mice plus (male, 9-week-old, n=3). Leukocyte subpopulations were analyzed by flow cytometry analysis. Results showed that T cells, B cells and NK cells were not detectable in all tissues of B-NDG mice and B-NDG MHC I/II DKO mice plus. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001. 

Absolute number of reconstituted human immune cells  

Human PBMCs were successfully reconstituted in B-NDG MHC I/II DKO mice plus. B-NDG MHC I/II DKO mice plus were engrafted intravenously with human PBMCs (1×10⁷) on day 0 (n=6). Peripheral blood was taken weekly to analyze the reconstitution level of human immune cells. The experiment was ended 112 days (16 weeks) after engraftment. 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 plus, the frequency and absolute cell number of CD45+ cells in peripheral blood began to increase, and a relatively stable level of reconstitution was maintained until the end point at 16 week. The frequency of reconstituted human T cells exceeded 90% from two 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. Human PD-1 is widely expressed on CD4+T cells and CD8+T cells. This indicates that B-NDG MHC I/II DKO mice plus is a powerful immunodeficient mouse model for reconstitution of the human immune system using human PBMCs.

Successfully establishing CDX model in B-NDG MHC I/II DKO mice plus engrafted with human PBMCs and verifying the efficacy of bispecific antibody