Background: 

• CD3 is a key component of the T cell receptor (TCR) complex, composed of γ, δ, ε, and ζ chains that form dimers (e.g., CD3γε, ζζ). Its intracellular domains contain ITAMs essential for signal transduction. When the TCR engages an MHC–peptide complex, CD3 transmits the signal into the cell, initiating cascades that drive T cell activation, proliferation, and effector functions. The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. 
• The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains.  

Targeting strategy:

• The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human CD3E exons 2-9 in B-hCD3EDG/hCD8 mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/hCD8 mice, respectively. 
• The exons 1-3 and partial exon 4 of mouse Cd8a gene that encode the extracellular domain were replaced by human CD8A exons 1-3 and partial exon 4 in B-hCD3EDG/hCD8 mice. The exons 1-3 and partial exon 4 of mouse Cd8b1 gene that encode the extracellular domain were replaced by human CD8B exons 1-3 and partial exon 4 in B-hCD3EDG/hCD8 mice.  

Validation:

• Mouse CD3E was detectable on T cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice. Human CD3E was detectable on T cells of homozygous B-hCD3EDG/hCD8 mice.
• Mouse CD8A was detectable on T, NKT, and TCRβ+CD4- T cells from the spleen and blood of C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were detectable on T, NKT, and TCRβ+CD4- T cells from the spleen and blood of homozygous B-hCD3EDG/hCD8 mice.
• Humanization of CD3E,CD3D, CD3G and CD8 does not change the overall frequency or distribution of immune cell types in the spleen, blood, lymph nodes and thymus.

Application: This product is used for the pharmacological and safety evaluation of hCD3EDG/hCD8 bispecific antibodies.

Gene targeting strategy for B-hCD3EDG/hCD8 mice. 

• The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human CD3E exons 2-9 in B-hCD3EDG/hCD8 mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/hCD8 mice, respectively.
• The exons 1-3 and partial exon 4 of mouse Cd8a gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-hCD3EDG/hCD8 mice. The genomic region of mouse Cd8a gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD8A expression is driven by endogenous mouse CD8a promoter, while mouse Cd8a gene transcription and translation will be disrupted.
• The exons 1-3 and partial exon 4 of mouse Cd8b1 gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-hCD3EDG/hCD8 mice. The genomic region of mouse Cd8b1 gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD8B1 expression is driven by endogenous mouse CD8a promoter, while mouse Cd8b1 gene transcription and translation will be disrupted.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (BD Pharmingen, 562426). Mouse CD3E was detectable in wild-type mice, but not in B-hCD3EDG/hCD8 mice. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hCD8 mice.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (BD Pharmingen, 562426). Mouse CD3E was detectable in wild-type mice, but not in B-hCD3EDG/hCD8 mice. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in spleen T cells of B-hCD3EDG/hCD8 mice.  

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen TCRβ+CD4- T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in spleen TCRβ+CD4- T cells of B-hCD3EDG/hCD8 mice.  

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen NKT cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in spleen NKT cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in blood T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in blood T cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in blood TCRβ+CD4- T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in blood TCRβ+CD4- cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in blood NKT cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in blood NKT cells of B-hCD3EDG/hCD8 mice.  

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (male, n=3, 9-week-old), homozygous B-hCD3EDG/hCD8 mice (male, n=3, 9-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequency of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hCD8 mice were similar to those in C57BL/6JNifdc, demonstrating that humanization of CD3E, CD3D,CD3G and CD8 does not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in blood, lymph nodes and thymus of B-hCD3EDG/hCD8 mice were also comparable to wild-type C57BL/6 mice (Data not shown).Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.