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B-hCD3EDG mice
Strain Name C57BL/6-Cd3etm1(CD3E)Cd3dtm1(CD3D)Cd3gtm1(CD3G)/Bcgen Common Name  B-hCD3EDG mice
Background C57BL/6 Catalog number  110039
Related Genes 
CD3E(CD3e molecule); CD3D(CD3d molecule);  CD3G(CD3g molecule)

Gene Description


The protein encoded by CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The genes encoding the epsilon, gamma and delta polypeptides are located in the same cluster on chromosome 11. The epsilon polypeptide plays an essential role in T-cell development. T-cell surface glycoprotein CD3 delta chain is a protein that in humans is encoded by the CD3D gene. CD3D has been shown to interact with CD8A. T-cell surface glycoprotein CD3 gamma chain is a protein that in humans is encoded by the CD3G gene, defects in CD3G are associated with T cell immunodeficiency.


mRNA expression analysis

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Strain specific analysis of CD3EDG gene expression in WT and homozygous B-hCD3EDG mice by RT-PCR. Mouse Cd3edg mRNA was detectable only in thymocytes of wild type (+/+). Human CD3EDG mRNA was detectable only in H/H, but not in +/+ mice. 

Protein expression analysis


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Strain specific CD3E expression analysis in heterozygous B-hCD3EDG mice by flow cytometry.
Splenocytes were collected from WT and heterozygous B-hCD3EDG mice, and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse CD3E was detectable in WT mice and heterozygous B-hCD3EDG mice. Human CD3E were exclusively detectable in heterozygous B-hCD3EDG mice but not WT mice.

Weight of spleen and the total cell number of splenocytes in homozygous B-hCD3EDG mice 

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(A) Size of spleen from C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). (B) The weight of spleen in C57BL/6 and  B-hCD3EDG mice was similar. (C) The number of splenocytes in C57BL/6 and B-hCD3EDG mice was similar.


Weight of thymus and the total cell number of thymocytes in homozygous B-hCD3EDG mice 


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(A) Size of thymus from C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). (B) The weight of thymus in C57BL/6 and  B-hCD3EDG mice was similar, but the weight of thymus in B-hCD3EDG mice was lower than that in C57BL/6 mice.(C) The number of thymocytes in C57BL/6 and B-hCD3EDG mice was similar, but the number of thymocytes in B-hCD3EDG mice was lower than that in C57BL/6.


Analysis of thymus leukocytes cell subpopulations in homozygous B-hCD3EDG mice

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Analysis of thymus leukocyte subpopulations by FACS

Thymocytes were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old) . Flow cytometry analysis of the thymocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD4/CD8 cells, B cells in homozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in thymus.


Analysis of thymus T cell subpopulations in homozygous B-hCD3EDG mice

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Analysis of thymus T cell subpopulations by FACS

Thymocytes were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). Flow cytometry analysis of the thymocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in thymus. Values are expressed as mean ± SEM.


Analysis of spleen leukocytes cell subpopulations in homozygous B-hCD3EDG mice


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Analysis of spleen leukocyte subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK, DC, Granulocyte, Monocyte, and macrophage cells in heterozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.


Analysis of spleen T cell subpopulations in homozygous B-hCD3EDG mice


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Analysis of spleen T cell subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.


Analysis of lymph node leukocytes cell subpopulations in homozygous B-hCD3EDG mice

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Analysis of spleen leukocyte subpopulations by FACS

Lymph nodes were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). Flow cytometry analysis was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK in homozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph nodes.


Analysis of lymph node T cell subpopulations in homozygous B-hCD3EDG mice


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Analysis of spleen T cell subpopulations by FACS
Lymph node were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node . Values are expressed as mean ± SEM.

Analysis of T cell activation stimulated with anti-CD3E antibody in vitro

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T cells (2×105) were isolated from splenocytes of C57BL/6 and B-hCD3EDG mice (n=3,16 week-old), and were incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG mice was significantly up-regulated by anti-hCD3E antibody, similar to the activation level shown in C57BL/6 mice treated with anti-mCD3E antibody.


Analysis of T cell activation stimulated with anti-CD3E antibody in vitro

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T cells (2×105) were isolated from splenocytes of C57BL/6 and B-hCD3EDG mice (n=3,16 week-old), and were incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 48h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG mice was significantly up-regulated by anti-hCD3E antibody, similar to the activation level shown in C57BL/6 mice treated with anti-mCD3E antibody.

Analysis of T cell activation stimulated with anti-CD3E antibody in vitro


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T cells (2×105) were isolated from splenocytes of C57BL/6 and B-hCD3EDG mice (n=3,16 week-old), and were incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 72h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG mice was significantly up-regulated by anti-hCD3E antibody, similar to the activation level shown in C57BL/6 mice treated with anti-mCD3E antibody.


Analysis of T cell activation stimulated with anti-CD3E antibody in vitro


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T cells (2×105) were isolated from the splenocytes of C57BL/6 and B-hCD3EDG mice (n=3, 16 week-old), and incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h, 48h and 72h. T cell proliferations were measured by flow cytometry. As a result, the T cell activation in B-hCD3EDG mice was specifically up-regulated by anti-hCD3E antibody, similar to the level of activation in the anti-mCD3E antibody-treated C57BL/6 mice. 


Analysis of T cell activation stimulated with anti-CD3E antibody in vitro


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T cells (2×105) were isolated from the splenocytes of C57BL/6 and B-hCD3EDG mice (n=3, 16 week-old), and incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h, 48h and 72h. T cell proliferations were measured by flow cytometry. As a result, the T cell activation in B-hCD3EDG mice was specifically up-regulated by anti-hCD3E antibody, similar to the level of activation in the anti-mCD3E antibody-treated C57BL/6 mice. 

Analysis of T cell activation stimulated with anti-CD3E antibody in vitro

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T cells (2×105) were isolated from the splenocytes of C57BL/6 and B-hCD3EDG mice (n=3, 16 week-old), incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method. As a result, there is no significant difference regarding the production of IFN-γ and IL-2 between B-hCD3EDG mice and C57BL/6 mice. ND: not detectable. 


Cytotoxicity evaluation of CD3/BCMA bispecific antibody in vitro


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Splenocytes of B-hCD3EDG mice were mixed with B-hBCMA MC38 and various concentrations of CD3/BCMA bispecific antibodies provided by client were added. The killing activity was detected after 48 hours. When the ratio of effector cells to target cells  (E:T) were 20:1 and 40:1, the killing effect of the test sample was detected, but the killing effect was more obvious at 40:1.
Serum titers of OVA-specific antibodies 

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B-hCD3EDG mice (n=5, 6 week-old) were immunized three times with OVA, 2 weeks apart. Blood samples were collected a week after immunization. (A) Quantification of serum subtypes of mice before immunization. (B) Serum titer test of mice after the second and third immunizations. The levels of OVA-specific antibodies titers of B-hCD3EDG mice before immunization were similar to those in C57BL/6 mice, and the specific antibody titers in the serum of each mouse were significantly increased after the third immunizations, demonstrating that introduction of hCD3EDG instead of its mouse counterpart did not affect the humoral immune response of mice. Values are expressed as mean ± SEM.


In vivo efficacy of anti-hCD3E antibody and anti-mPD-1 antibody in B-hCD3EDG mice

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Antitumor activity of anti-hCD3E antibody and anti-mPD-1 antibody in B-hCD3EDG mice. 
Murine colon cancer MC38 cells were subcutaneously implanted into B-hCD3EDG mice (female, 8 week-old, n=5). Mice were grouped when the tumor size was approximately 100±50mm3 , at which time they were treated with different antibodies with doses and schedules indicated in panel. (A) Tumor volume changes during treatment. (B) Body weight changes during treatment. As shown in panel A, anti-mPD-1 antibody significantly inhibited tumor growth in B-hCD3EDG mice, indicating their T cells function normally. However, in B-hCD3EDG mice, tumor growth was faster after anti-hCD3E antibody treatment, which may be caused by activation induced cell death (AICD). As a result, the B-hCD3EDG mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy studies. Values are expressed as mean ± SEM.

In vivo efficacy of anti-hCD3E antibody and anti-mPD-1 antibody in B-hCD3EDG mice

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T cells and B cells activation in vivo efficacy .
Lymphocytes were isolated from peripheral blood at 48 hours after treatment. In the anti-hCD3E antibody treatment group, the proportion of T cells was significantly decreased due to the activation induced cell death (AICD) effect caused by anti-hCD3E antibody treatment. (A) The percentage of TCR-β + cells in total CD45+ cells after treatment.  (B) The percentage of CD19+ cells in total CD45+ cells after treatment. 

In vivo efficacy of anti-hCD3E antibody and anti-mPD-1 antibody in B-hCD3EDG mice

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T cells and B cells activation in vivo efficacy .
Lymphocytes were isolated from tumor at 48 hours after treatment. In the anti-hCD3E antibody treatment group, the proportion of T cells was significantly decreased due to the activation induced cell death (AICD) effect caused by anti-hCD3E antibody treatment. (A) The percentage of TCR-β + cells in total CD45+ cells after treatment.  (B) The percentage of CD19+ cells in total CD45+ cells after treatment. 


In vivo efficacy of T cell bispecific antibody 


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Antitumor activity of antibody X in B-hCD3EDG mice. 
B-hBCMA MC38 cells were subcutaneously implanted into B-hCD3EDG mice (female, 6 week-old, n=5). Mice were grouped when the tumor size was approximately 100mm3, at which time they were treated with antibody X (provided by client) with doses and schedules indicated in panel A. (A) Tumor volume changes during treatment. (B) Body weight changes during treatment. As shown in panel A, antibodies X with different doses were efficacious in controlling tumor growth in B-hCD3EDG mice, demonstrating that the B-hCD3EDG mouse model is a powerful tool for in vivo efficacy study of T cell bispecific antibody. Values are expressed as mean ± SEM.