• 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.
• TGFBR2, the protein encoded by this gene, is a transmembrane protein containing a protein kinase domain. It associates with TGF-beta receptor type 1 to form a heterodimeric complex and is capable of binding TGF-beta. Upon ligand binding, this receptor complex phosphorylates downstream proteins; these activated proteins then translocate to the nucleus, where they modulate the transcription of genes involved in cell proliferation, cell cycle arrest, wound healing, immunosuppression, and tumorigenesis.
• Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 plus/hTGFBR2 mice. A chimeric CDS that encodes mouse TGFBR2 signal peptide, human extracellular domain, mouse Tgfbr2 transmembrane and cytoplasmic domain, followed by mouse 3’UTR-STOP is inserted right after mouse the exon 2 of Tgfbr2 to replace the exon 2 of mouse Tgfbr2 gene. The chimeric TGFBR2 protein expression will be driven by endogenous mouse Tgfbr2 promoter, while mouse Tgfbr2 gene transcription and translation will be disrupted.
• Human PD-1 and TGFBR2 were detectable in homozygous B-hPD-1 plus/hTGFBR2 mice but not in the wild-type C57BL/6JNifdc mice.
• Humanization of PD-1 and TGFBR2 does not change the overall frequency or distribution of immune cell types in spleen, blood, and lymph nodes.
• Application: This product is used for pharmacodynamics and safety evalsuation of PD-1/TGFBR2-targeted antibodies.

Gene targeting strategy for B-hPD-1 plus/hTGFBR2 mice.

Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 plus/hTGFBR2 mice.

A chimeric CDS that encodes mouse TGFBR2 signal peptide, human extracellular domain, mouse Tgfbr2 transmembrane and cytoplasmic domain, followed by mouse 3’UTR-STOP is inserted right after mouse the exon 2 of Tgfbr2 to replace the exon 2 of mouse Tgfbr2 gene. The chimeric TGFBR2 protein expression will be driven by endogenous mouse Tgfbr2 promoter, while mouse Tgfbr2 gene transcription and translation will be disrupted.

Strain specific PD-1 expression analysis in wild-type mice (WT) and homozygous (HO) B-hPD-1 plus/hTGFBR2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hPD-1 plus/hTGFBR2 mice after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 10-week-old, n=3) or not. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104), and anti-human PD-1 antibody (Biolegend, 329904), by flow cytometry. Mouse PD-1 was detectable in wild-type C57BL/6JNifdc mice, but not in the homozygous B-hPD-1 plus/hTGFBR2 mice. Human PD-1 was exclusively detectable in B-hPD-1 plus/hTGFBR2 mice.

Strain specific TGFBR2 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hPD-1 plus/hTGFBR2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hPD-1 plus/hTGFBR2 mice (female, 10-week-old, n=3). Protein expression was analyzed with anti-mouse TGFBR2 antibody (R&D, FAB532P), and anti-human TGFBR2 antibody (Biolegend, 399705) by flow cytometry. Mouse TGFBR2 was detectable in wild-type C57BL/6JNifdc mice, but not in the homozygous B-hPD-1 plus/hTGFBR2 mice. Human TGFBR2 was exclusively detectable in B-hPD-1 plus/hTGFBR2 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hTGFBR2 mice (female, 10-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, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and Tregs in B-hPD-1 plus/hTGFBR2 mice were similar to those in C57BL/6JNifdc mice. The frequency of leukocyte subpopulations in blood and lymph nodes of B-hPD-1 plus/hTGFBR2 mice were also comparable to wild-type C57BL/6JNifdc mice (Data not shown). Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.