• CD3 Molecule: As a vital component of the T-cell receptor (TCR) complex, CD3 is responsible for transducing antigen recognition signals into the cell interior. The engagement and ligation of CD3 can directly induce T-cell activation. 
• CD8 Molecule: CD8 Binding of MHC-peptide complexes in cis or trans Regulates CD8+ T-cell Responses. The coreceptor CD8αβ can greatly promote activation of T cells by strengthening T-cell receptor (TCR) binding to cognate peptide-MHC complexes (pMHC) on antigen presenting cells and by bringing p56Lck to TCR/CD3. CD8 can also bind to pMHC on the T cell (in cis) and that this inhibits their activation. 
• T-Cell Engagement (TCE): CD3 redirection bispecific antibodies bind tumor associated antigens (TAA) on cancer cells and CD3 on recruited T cells to elicit cancer cell cytotoxicity independent of T cell receptor specificity. Cancer cell cytotoxicity occurs through perforin and granzyme B release resulting in apoptosis through caspase pathway induction. 
• CD3/CD8/TAA Antibody: To circumvent the unintended activation of regulatory T cells (Tregs), which are typically CD4+, the antibody is engineered with a preferential affinity for CD8+ cells. This selectivity enhances cytotoxic efficacy while simultaneously mitigating immunosuppressive effects.

CD20: A B-cell surface marker modulating calcium influx and activation. 

• Gene Information: Located on chromosome 11q12, the MS4A1 gene encodes CD20, a highly conserved hydrophobic transmembrane protein belonging to the membrane-spanning 4-domain family A member 1. 
• Protein Expression: D20 is expressed exclusively on the surface of B cells, spanning from the late pro-B stage through mature cells, but is notably absent on pluripotential stem cells and mature plasma cells. 
• Signaling Pathway: Upon activation, CD20 functions as or regulates a store-operated calcium channel, altering intracellular calcium concentrations to modulate B-cell activation, differentiation, and cell-cycle progression. 
• Therapeutic Inhibition: Monoclonal antibodies like rituximab target CD20 to deplete pathogenic B cells via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8/hCD20 mice by flow cytometry. Splenocytes and blood cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hCD20 mice, and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (Biolegend, 317344).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8/hCD20 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD8/CD20 mice (H/H), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100730; anti-human CD8B, BD, 742392). -

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8/hCD20 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD8/CD20 mice (H/H), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100730; anti-human CD8B, BD, 742392). 

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8/hCD20 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD8/CD20 mice (H/H), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100730; anti-human CD8B, BD, 742392).

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8/hCD20 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD8/CD20 mice (H/H), and analyzed by flow cytometry with species-specific anti-CD8 antibody (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100730; anti-human CD8B, BD, 742392).

Strain specific CD20 expression analysis in homozygous B-hCD3EDG/hCD8/hCD20 mice by flow cytometry. Splenocytes and blood cells were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD8/hCD20 mice, and analyzed by flow cytometry with species-specific anti-mouse CD20 antibody (Biolegend, 152108) and anti-human CD20 antibody (Biolegend, 302306).