• PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily. PD-1 is expressed on activated T cells, B cells, and myeloid cells. Its main function is to regulate the immune response and prevent excessive immune activation. When PD-1 binds to its ligands (PD-L1 and PD-L2), it sends inhibitory signals to T cells, dampening their activity.
• 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.
• Of the immunoglobulin superfamily, Lymphocyte-activation protein 3 (LAG3) features four extracellular immunoglobulin-like domains. Its encoding gene comprises eight exons, and analyses of its sequence, exon-intron structure, and chromosomal location all point to a strong evolutionary and structural link with CD4.
• The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PD-1 exon 2 in B-hPD-1/hLAG3 plus/hCD8 mice. The exons 2-7 of mouse Lag3 gene that encode the extracellular domain were replaced by human LAG3 exons 2-7 in B-hPD-1/hLAG3 plus/hCD8 mice. 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-hPD-1/hLAG3 plus/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-hPD-1/hLAG3 plus/hCD8 mice.
• Human PD-1, LAG3, and CD8 were detectable in homozygous B-hPD-1/hLAG3 plus/hCD8 mice but not in the wild-type C57BL/6JNifdc mice.
• Humanization of PD-1, LAG3, and CD8 does not change the overall frequency or distribution of immune cell types in spleen, blood, bone marrow and thymus.
• Application: This product is used for pharmacodynamics and safety evaluation of PD-1/LAG3-targeting therapeutic agents.

Gene targeting strategy for B-hPD-1/hLAG3 plus/hCD8 mice.

The exon 2 of mouse Pdcd1 gene encodes the IgV domain is replaced by human counterparts in B-hPD-1/hLAG3 plus/hCD8 mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The human PD-1 expression is driven by endogenous mouse Pdcd1 promoter, while mouse Pdcd1 gene transcription and translation will be disrupted.

The exons 2-7 of mouse Lag3 gene that encode extracellular domain are replaced by human counterparts in B-hPD-1/hLAG3 plus/hCD8 mice. The genomic region of mouse Lag3 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 LAG3 expression is driven by endogenous mouse Lag3 promoter, while mouse Lag3 gene transcription and translation will be disrupted.

The exons 1-3 and partial exon 4 of mouse Cd8a gene that encode extracellular domain are replaced by human counterparts in B-hPD-1/hLAG3 plus/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 extracellular domain are replaced by human counterparts in B-hPD-1/hLAG3 plus/hCD8 mice. The genomic region of mouse Cd8b 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 CD8B expression is driven by endogenous mouse Cd8b1 promoter, while mouse Cd8b1 gene transcription and translation will be disrupted.

Strain specific PD-1 expression analysis in wild-type mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice after stimulated with or without anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 12-week-old, n=3). Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 135252), and anti-human PD-1 antibody (Biolegend, 329928) by flow cytometry. Mouse PD-1 was only detectable in wild-type C57BL/6JNifdc mice.

Strain specific PD-1 expression analysis in homozygous B-hPD-1/hLAG3 plus/hCD8 mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1/hLAG3 plus/hCD8 mice after stimulated with or without anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 12-week-old, n=3). Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 135252), and anti-human PD-1 antibody (Biolegend, 329928) by flow cytometry. Human PD-1 was only detectable in homozygous B-hPD-1/hLAG3 plus/hCD8 mice.

Strain specific LAG3 expression analysis in wild-type mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice after stimulated with or without anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 12-week-old, n=3). Protein expression was analyzed with anti-mouse LAG3 antibody (Biolegend, 125242), and anti-human LAG3 antibody (Biolegend, 369306) by flow cytometry. Mouse LAG3 was only detectable in wild-type C57BL/6JNifdc mice.

Strain specific LAG3 expression analysis in homozygous B-hPD-1/hLAG3 plus/hCD8 mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1/hLAG3 plus/hCD8 mice after stimulated with or without anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 12-week-old, n=3). Protein expression was analyzed with anti-mouse LAG3 antibody (Biolegend, 125242), and anti-human LAG3 antibody (Biolegend, 369306) by flow cytometry. Human LAG3 was only detectable in B-hPD-1/hLAG3 plus/hCD8 mice.

Strain specific CD8 expression analysis in wild-type mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice after stimulated with or without anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 12-week-old, n=3). Protein expression was analyzed with anti-mouse LAG3 antibody (Invitrogen, MCD0830), and anti-human CD8a antibody (Biolegend, 300920) by flow cytometry. Mouse CD8a was only detectable in wild-type C57BL/6JNifdc mice.

Strain specific CD8 expression analysis in wild-type mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1/hLAG3 plus/hCD8 mice after stimulated with or without anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 12-week-old, n=3). Protein expression was analyzed with anti-mouse LAG3 antibody (Invitrogen, MCD0830), and anti-human CD8a antibody (Biolegend, 300920) by flow cytometry. Human CD8a was only detectable in detectable in B-hPD-1/hLAG3 plus/hCD8 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous in B-hPD-1/hLAG3 plus/hCD8 mice (female, 12-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 T cells, B cells, NK cells, dendritic cells, myeloid cells, granulocytes, CD4+ T cells, CD8+ T cells, and Tregs in B-hPD-1/hLAG3 plus/hCD8 mice were similar to those in C57BL/6JNifdc mice, demonstrating that humanization of PD-1, LAG3, and CD8 does not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in blood, bone marrow and thymus of B-hPD-1/hLAG3 plus/hCD8 mice were also comparable to wild-type C57BL/6 mice (Data not shown).