• CD3 consists of four protein chains (CD3E, CD3D, CD3G and CD3Z), which are important biological markers on the T cell membrane. CD3 can form a TCR/CD3 complex with the T cell receptor, participating in the regulation of T cell antigen recognition, signal transduction and T cell development. The B2M encodes a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. The protein has a predominantly beta-pleated sheet structure that can form amyloid fibrils in some pathological conditions. HLA-A belongs to the HLA class I heavy chain paralogues. The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. They are expressed in nearly all cells. Another physiological function of B2M is to form a complex with FcRn, which can prolong the half-life of antibodies, and B2M/FcRn has been reconstituted in B-hCD3EDG/HLA-A2.1 plus mice.
• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/HLA-A2.1 plus mice. The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains in B-hCD3EDG/HLA-A2.1 plus mice. The mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hCD3EDG/HLA-A2.1 plus mice.
• Human CD3E, B2M, and HLA were detectable in homozygous B-hCD3EDG/HLA-A2.1 plus mice but not in the wild-type C57BL/6JNifdc mice.
• Introduction of hB2M-HLA-A0201-H-2Db in place of mouse B2M affected the development of CD8 + T cells, which in turn affected the proportion of T cell subtypes in spleen, lymph nodes and blood.
• Application: This product is used for the pharmacological and safety evalsuation of antibodies in oncology.

Gene targeting strategy for B-hCD3EDG/HLA-A2.1 plus mice.

The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/HLA-A2.1 plus mice.

The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains in B-hCD3EDG/HLA-A2.1 plus mice.

The mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hCD3EDG/HLA-A2.1 plus mice.

Strain specific B2M expression analysis in wild-type mice (WT) and homozygous (HO) B-hCD3EDG/HLA-A2.1 plus mice by flow cytometry. Splenocytes were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). Protein expression was analyzed with anti-mouse B2M antibody (BD Biosesciences, 744802), and anti-hB2M antibody (Biolegend, 395712) by flow cytometry. Mouse B2M was detectable in both C57BL/6JNifdc mice and B-hCD3EDG/HLA-A2.1 plus mice because the mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hCD3EDG/HLA-A2.1 plus mice. Human B2M was only detectable in B-hCD3EDG/HLA-A2.1 plus mice.

Strain specific B2M expression analysis in wild-type mice (WT) and homozygous (HO) B-hCD3EDG/HLA-A2.1 plus mice by flow cytometry. Blood cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). Protein expression was analyzed with anti-mouse B2M antibody (BD Biosesciences, 744802), and anti-hB2M antibody (Biolegend, 395712) by flow cytometry. Mouse B2M was detectable in both C57BL/6JNifdc mice and B-hCD3EDG/HLA-A2.1 plus mice because the mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hCD3EDG/HLA-A2.1 plus mice. Human B2M was only detectable in B-hCD3EDG/HLA-A2.1 plus mice.

Strain specific B2M expression analysis in wild-type mice (WT) and homozygous (HO) B-hCD3EDG/HLA-A2.1 plus mice by flow cytometry. Splenocytes and blood cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). Protein expression was analyzed with anti-H-2Db antibody (Biolegend, 111513) and anti-HLA-ABC antibody (Biolegend, 311406) by flow cytometry. Mouse H-2Db was detectable in C57BL/6JNifdc mice. Human HLA-A was only detectable in B-hCD3EDG/HLA-A2.1 plus mice but not in wild-type C57BL/6JNifdc.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils, and Tregs in B-hCD3EDG/HLA-A2.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequencies of T cells and CD8+ T cells in B-hCD3EDG/HLA-A2.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-hCD3EDG/HLA-A2.1 plus mice was higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the blood was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils, and Tregs in B-hCD3EDG/HLA-A2.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequency of CD8+ T cells in B-hCD3EDG/HLA-A2.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-hCD3EDG/HLA-A2.1 plus mice was higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Lymph node cells were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the lymph node was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, and NK cells in B-hCD3EDG/HLA-A2.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequency of CD8+ T cells in B-hCD3EDG/HLA-A2.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-hCD3EDG/HLA-A2.1 plus mice was higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Thymocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/HLA-A2.1 plus mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the lymph node was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, and NK cells in B-hCD3EDG/HLA-A2.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequency of CD8+ T cells in B-hCD3EDG/HLA-A2.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-hCD3EDG/HLA-A2.1 plus mice was higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.