B-NDG B2m KO mice plus

Basic Information

Strain Name
B-NDG-B2mtm1Fcrntm1(mB2m)/Bcgen
Common Name
B-NDG B2m KO mice plus
Background
B-NDG
Catalog number
110601
Related Genes
B2m (beta-2 microglobulin) ; Fcgrt (Fc receptor, IgG, alpha chain transporter,as known as Fcrn)

Description

Human PBMC-engrafted humanized mice are attractive models for in vivo analysis of human immune responses. We previously reported that human PBMCs could be engrafted in B-NDG mice successfully.However, because of severe xenogeneic graft versus host disease (xeno-GVHD) in these mice, there is a limited window for experimentation. This makes it difficult to analyze human immune responses precisely. So knockout MHC class I β2-microglobulin (β2m) is an effective way to solve this problem. But it has been reported that FcRn is a heterodimer of an α-chain and β2m and differs from other IgG Fc receptors in that it is structurally related to MHC class I molecules. It has been shown that several functions attributed to FcRn are affected in β2m-deficient mice, such as newborn β2m-deficient pups show lower IgG serum levels at birth and accumulate less IgG before weaning than normal littermates [1,2]. So Biocytogen developed B-NDG B2m KO mice plus, this mouse expresses the B2m gene fused in the FcRn gene while knock out the murine B2m gene. This mouse combines the B-NDG mouse background with the absence of the MHC class I molecule β2m and shows no difference in the metabolism of IgG drugs in mice compared with wild-type mice.It is effective against GVHD effects. This model can be useful for studying the in vivo mechanism of xenograft versus host disease and for therapeutic drug efficacy evaluation.

Details

Flow cytometric analysis of MHC class l expression

Blood cells, splenocyte, Bone marrow cells were collected from B-NDG B2m KO plus, B-NDG, NOD-scid mice (n=5) and analyzed by flow cytometry with MHC class I(H-2 Kd). MHC class I were exclusively detectable in B-NDG, NOD-scid mice, B-NDG B2m KO plus not detectable MHC class I.

Protein expression analysis(MHC class lI expression in DC)

Flow cytometric analysis of MHC class lI expression. Blood cells, splenocyte, Peritoneal lavage fluids were collected from B-NDG B2m KO plus, B-NDG, NOD-scid mice (n=3) and analyzed by flow cytometry with MHC class II(mIA-IE). Single live cells were gated for DC (CD45+CD11b+CD11c+) population and used for further analysis as indicated here. MHC class II were detectable in B-NDG, NOD-scid mice, B-NDG B2m KO plus DC subpopulations.

Protein expression analysis(MHC class lI expression in Macrophages)

Flow cytometric analysis of MHC class lI expression. Blood cells, splenocyte, Peritoneal lavage fluids were collected from B-NDG B2m KO plus, B-NDG, NOD-scid mice (n=3) and analyzed by flow cytometry with MHC class II(mIA-IE). Single live cells were gated for macrophages (CD45+CD11b+F4/80+) population and used for further analysis as indicated here. MHC class II were detectable in B-NDG, NOD-scid mice, B-NDG B2m KO plus macrophage subpopulations.

Flow cytometric analysis of T , B and NK cells

Blood cells, splenocyte, cells were collected from B-NDG B2m KO plus, B-NDG, NOD-scid and C57BL/6 mice (n=5) and analyzed by flow cytometry with T, B and NK cells. T, B and NK cells were not detectable in B-NDG B2m KO plus and B-NDG mice. T, B cells were undetectable and NK cells were detectable in NOD scid mice. T, B and NK cells were detectable in C57BL/6 mice.

Flow cytometric analysis of Macrophages

Flow cytometric analysis of DC cells and monocytes

Histopatholoical sections of tissues from B-NDG B2M plus and control mice

(A) Spleen from C57BL/6 has normal structure with well-defined follicles. (B) Spleen from NOD-scid show hypoplasia of white pulp. (C, D) Spleen from both B-NDG and B-NDG B2M plus show complete loss of follicular structure. (E) Thymic lobes from C57BL/6 have normal structure with a well-defined cortex. (G) Thymic lobes from NOD scid mouse are hypoplastic and lack a defined cortex.(H) Thymic lobes from both B-NDG 2 mouse are severely hypoplastic,lack a defined cortex. (F) B-NDG B2M plus show no thymic lobes on the normal anatomy location.

Determination of plasma concentrations of human IgG in different mice

Homozygous B-NDG, B-NDG B2m KO mice,B-NDG B2m KO mice plus and C57BL/6 mice were treated with human IgG (n=5). Blood samples were collected at different time point for the PK assay.The results showed that the PK results of B-NDG and B-NDG B2m KO mice plus groups were basically in line with the pharmacokinetic characteristics, with no difference compared with wild-type mice, and the drug concentration of B-NDG B2m KO mice group could not be measured at the time point 2 days later.

B-NDG B2m KO mice plus vs B-NDG mice in human PBMC induced GvHD model

Conclusions: B2M/FcRn mice significantly extend survival compared with B-NDG mice in the human PBMC induced GvHD model. B2M/FcRn mice delay the onset and reduce the severity of GvHD.

Methods: B2M/FcRn (11-week-old female, n=5) and B-NDG (10-week-old female, n=6) mice were engrafted i.v. with 20M human PBMC from three healthy donors (D1-3) on day 0. Clinical signs of GvHD were scored approximately twice a week according to the table below. Data was analyzed in Prism. Values were expressed as mean ± SEM. **: P<0.01.

B-NDG B2m KO mice plus are well suited for human immune system engraftment

Human PBMC (5E6) were intravenous implanted into homozygote B-NDG B2m KO mice plus and B-NDG mice (female, 7-week-old, n=6). Representative flow cytometric analysis of PBMCs from mice after engraftment with human PBMC. B-NDG B2m KO mice plus show a little change in body weight and exhibit longer survival compared with B-NDG. The results showed that the human PBMC engrafted humanized mice model was successfully constructed.

Anti-tumor efficacy in PBMC RKO model in B-NDG B2m KO mice plus (preliminary)

Conclusions: Check point inhibitors inhibited RKO tumor growth in PBMC engrafted B-NDG B2m KO mice plus. B-NDG B2m KO mice plus appeared to show less GvHD symptoms as indicated by maintenance of body weight.

Method: B-NDG B2m KO mice plus (B-NDG background) mice (n=7/8), engrafted with human PBMC and implanted with human RKO tumor, were treated with pembro/ipi (200 ug) BIW, ip.

CAR-T in vivo efficacy study using NCI-H226 CDX Tumor model in B-NDG B2m KO mice plus

Antitumor activity of CAR-T therapy in B-NDG B2m KO mice plus. Human NCI-H226(A) (1E7) were implanted into B-NDG B2m KO mice plus. (A) Mice were grouped when tumor size was approximately 150±50 mm3. at which time they were treated with CAR-T cells (5E5) with schedules indicated in panel. (B) Body weight changes during treatment. The results showed that CAR-T cells differently inhibited tumor growth in B-NDG B2m KO mice plus. B-NDG is a powerful model for human CAR-T cells efficacy study. Values are expressed as mean ± SEM.

 

 

Summary

1.  Flow cytometric analysis of MHC class l and Il expression.  MHC class I were not detectable in B-NDG B2m KO mice plus .

2.  Flow cytometric analysis using specific markers for T, B and NK cells. T, B and NK cells were not detectable in B-NDG B2m KO plus.

3.  Determination of plasma concentrations of human IgG in different mice .The results showed that the PK results of B-NDG and B-NDG B2m KO mice plus groups were basically in line with the pharmacokinetic characteristics, with no difference compared with wild-type mice Physiological function.

4.  B-NDG B2m KO mice plus are well suited for human immune system engraftment. Human PBMC (5E6) were intravenous implanted into homozygote B-NDG B2m KO mice plus and B-NDG mice (female, 7-week-old, n=6). Representative flow cytometric analysis of PBMCs from mice after engraftment with human PBMC. B-NDG B2m KO mice plus show a little change in body weight and exhibit longer survival compared with B-NDG.

Back to top