Basic Information

Strain Name
C57BL/6-CD3etm2(CD3e)Bcgen/Bcgen
Stock Number
110008
Common Name
B-hCD3e Mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
CD3e (CD3e molecule)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

The CD3e molecule, epsilon encoded by CD3e gene is a 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. The CD3 complex, a common surface marker on T cells, has important functions not only as an essential component in forming the T cell receptor (TCR)-CD3 complex, but also as an external signal transducer; therefore, the CD3 complex is one of the target molecules to modulate T cell functions. The epsilon polypeptide plays an essential role in T-cell development.

Targeting Strategy

B-hCD3e-Mice-targeting-strategy

Details

Phenotype

Protein expression analysis

B-hCD3e-Mice-details-protein-expression-analysis

Splenocytes from both wild type (WT)C57BL/6 and homozygous B-hCD3e mice were analyzed by flow cytometry. Results: mCD3+ cells were detectable in the WT C57BL/6 mice, while hCD3+ cells were detectable in homozygous B-hCD3e mice.

Thymus and spleen weights of WT C57BL/6 and B-hCD3e mice

B-hCD3e-Mice-details-Thymus-and-spleen-weights-of-WT-C57BL6-and-B-hCD3e-mice

Thymus and spleen were collected from both WT C57BL/6 and homozygous B-hCD3e mice (n=6), and their weights were measured for comparison. There was no detectable obvious between spleen weight of WT and that of B-hCD3e mice. However, thymus weight of B-hCD3e mice was lower compared to that of WT mice.

B-hCD3e-Mice-details-Analysis-of-lymphocyte-subpopulation-in-B-hCD3e-mice

Analysis of lymphocyte subpopulation in thymus in B-hCD3e mice Thymocytes were isolated from C57BL/6 and B-hCD3e mice(n=4). The proportion of lymphocyte subpopulation was tested by flow cytometry. As a result, the expression profile of lymphocyte subpopulation in homozygous B-hCD3e mice is similar to that in the C57BL/6 mice, indicating that T cell differentiation is not affected by the humanization of CD3e.

B-hCD3e-Mice-details-Analysis-of-lymphocyte-subpopulation-in-B-hCD3e-mice2

Analysis of lymphocyte subpopulation in spleen in B-hCD3e mice. Splenocytes were isolated from C57BL/6 and B-hCD3e mice(n=4). The proportion of lymphocyte subpopulation was tested by flow cytometry. As a result, the expression profile of lymphocyte subpopulation in homozygous B-hCD3e mice is similar to that in the C57BL/6 mice, indicating that differentiation of T cell, B cell and Treg is not affected by the humanization of CD3e.

Analysis of lymphocyte subpopulation in B-hCD3e mice

B-hCD3e-Mice-details-Analysis-of-lymphocyte-subpopulation-in-B-hCD3e-mice3

Lymphocytes were isolated from spleen, peripheral blood and lymph node in C57BL/6 and B-hCD3e mice (n=4). The proportion of lymphocyte subpopulation was tested by flow cytometry. As a result, the expression profile of lymphocyte subpopulation in homozygous B-hCD3e mice is similar to that in the C57BL/6 mice, indicating that T cell differentiation is not affected by the humanization of CD3e.

Analysis of T cell activation stimulated with anti-CD3e antibody in vitro

B-hCD3e-Mice-details-Analysis-of-T-cell-activation-stimulated-with-anti-CD3e-antibody-in-vitro

T cells (2.5×106) were isolated from C57BL/6 and B-hCD3e mice (n=4) splenocytes, and incubated in the presence of anti-CD3 antibody (C57BL/6, anti-mCD3; B-hCD3e, anti-hCD3) with anti-mouse CD28 for 24h, 48h and 72h. T cell proliferation were tested using flow cytometry. As a result, the T cell activation in B-hCD3e mice is specifically up-regulated by anti-hCD3 antibody, similar to the activation level as shown in the C57BL/6 treated with anti-mCD3 antibody.

B-hCD3e-Mice-details-Analysis-of-T-cell-activation-stimulated-with-anti-CD3e-antibody-in-vitro2

T cells (2.5×10 6 ) were isolated from C57BL/6 and B-hCD3e mice (n=4) splenocytes, and incubated in the presence of anti-CD3 antibody (C57BL/6, anti-mCD3; B- hCD3e, anti-hCD3) with anti-mouse CD28 for 24h, 48h and72h. IFN-γ and IL-2 production were then tested using ELISA method. As a result, elevated secretion of IFN-γ and IL-2 was detected in B-hCD3e mice after anti-hCD3 antibody treatment and C57BL/6 mice after anti-mCD3 antibody treatment.

Analysis of T cell activation stimulated with anti-CD3 antibody in vivo

B-hCD3e-Mice-details-Analysis-of-T-cell-activation-stimulated-with-anti-CD3-antibody-in-vivo

Lymphocytes were isolated from spleen in C57BL/6 and B-hCD3e mice (n=5) after stimulation (24h). The proportion of lymphocyte subpopulation was tested by flow cytometry.

B-hCD3e-Mice-details-Analysis-of-T-cell-activation-stimulated-with-anti-CD3-antibody-in-vivo2

Lymphocytes were isolated from spleen in C57BL/6 and B-hCD3e mice (n=5) after stimulation (48h). The proportion of lymphocyte subpopulation was tested by flow cytometry.

Application

CD3 Abs efficacy evaluation

B-hCD3e-Mice-details-CD3-Abs-efficacy-evaluation

Murine colon cancer MC38 cells were subcutaneously implanted into C57BL/6 (A) and B-hCD3e (B) mice. Mice were grouped when the tumor size was approximately 150±50 mm3 (n=5).
In the humanized mouse model, mPD-1 antibody significantly inhibited tumor growth, indicating normal T cell function. More aggressive tumor growth after anti-hCD3 antibody treatment was observed. This may have result from activation induced cell death (AICD). As a result, the B-hCD3e mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy studies.

T cell activation in CD3 Abs efficacy evaluation

B-hCD3e-Mice-details-T-cell-activation-in-CD3-Abs-efficacy-evaluation

The ratio of B cells and T cells in the blood of mice was detected by flow cytometry. Lymphocytes were isolated from peripheral blood at the 48 hours. In the anti-hCD3 antibody group, the proportion of T cells was significantly decreased due to the activation-induced cell death (AICD) effect caused by CD3E antibody. However, the proportion of T cells in humanized CD3e mice did no change after either anti-mPD-1 or anti-mCD3 antibody treatment.

Percentage of CD4+ and CD8+ cells after hCD3 antibody treatment

B-hCD3e-Mice-details-percentage-of-CD4+-and-CD8+-cells-after-hCD3-antibody-treatment

Compared with hIgG Ab, CD4 % and CD8 % decreased after hCD3 Ab treatment in blood.

hCD3 Abs efficacy evaluation with two doses

Murine colon cancer MC38 cells were subcutaneously implanted into C57BL/6 (Fig. A) and B-hCD3E (Fig. B) mice. Mice were grouped when the tumor size was approximately 150±50mm3 (n=5).
The mPD-1 antibody significantly inhibited tumor growth, whereas the specific CD3 mouse antibody made the tumor larger. As a result, the B-hCD3 mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy studies.

Dose-dependent T cell depletion caused by hCD3 Ab treatment

B-hCD3e-Mice-details-Dose-dependent-T-cell-depletion-caused-by-hCD3-Ab-treatment

The ratio of B cells and T cells in the blood of mice was detected by flow cytometry. Lymphocytes were isolated from peripheral blood at the experimental end point. In the treatment group, the proportion of T cells was significantly decreased due to the activation induced cell death (AICD) effect caused by CD3E antibody.

Blinatumomab efficacy evaluation

B-hCD3e-Mice-details-Blinatumomab-efficacy-evaluation

Murine colon cancer MC38-hCD19 cells were subcutaneously implanted into B- hCD3E mice. Mice were divided into control and treatment group (n=6) when the tumor size was approximately 100±20 mm3. High doses of hCD3E antibodies (Blinatumomab) can significantly inhibit tumor growth confirming that the B-hCD3E mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy study.

AICD analysis after antibody treatment in B-hCD3e mice

B-hCD3e-Mice-details-AICD-analysis-after antibody-treatment-in-B-hCD3e-mice

B-hCD3e-Mice-details-AICD-analysis-after antibody-treatment-in-B-hCD3e-mice2

The ratio of T cell in spleen and blood were analyzed at 24h 72h and 168h by flow cytometry.

Reference

1. Sci Transl Med. 2011 Feb 2;3(68):68ra10. doi: 10.1126/scitranslmed.3001830.
2. Sci Rep. 2018 Mar 19;8:46960. doi: 10.1038/srep46960.

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