• 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.
• PD-L1 is a ligand for PD-1, mainly expressed on tumor cells, antigen-presenting cells, and some non-immune cells. By binding to PD-1 on T cells, PD-L1 can suppress the anti-tumor immune response, allowing tumor cells to evade immune surveillance.
• The B2M encodes a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. The protein has a predominantly beta-pleated sheet structure that can form amyloid fibrils in some pathological conditions. HLA-B belongs to the HLA class I heavy chain paralogues. The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. They are expressed in nearly all cells.
• The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/HLA-A11.1 plus mice. The exon 3 of mouse pdl1 gene that encodes the IgV domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/HLA-A11.1 plus mice. The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*1101 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains in B-hPD-1/hPD-L1/HLA-A11.1 plus mice. The mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.
• Human B2M, HLA, PD-1 and PD-L1 were detectable in homozygous B-hPD-1/hPD-L1/HLA-A11.1 plus mice but not in the wild-type C57BL/6JNifdc mice.
• Introduction of hB2M-HLA-A1101-H-2Db in place of mouse B2M may affected the development of CD8 + T cells, which in turn affected the proportion of T cell subtypes in spleen, lymph nodes and blood.
• Application: This product is used for pharmacodynamics and safety evaluation of vaccines for cancers.

Gene targeting strategy for B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

The exon 3 of mouse pdl1 gene that encodes the IgV domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*1101 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

The mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

Strain specific B2M and HLA-A expression analysis in wild-type mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (male, 8-week-old, n=1) or not. Protein expression was analyzed with anti-mouse B2M antibody (BD Biosciences, 744802), anti-hB2M antibody (Biolegend, 395712), anti-H-2D^b antibody (Biolegend, 111513) and anti-HLA-ABC antibody (Biolegend, 311406), by flow cytometry. Mouse B2M and H-2Db were detectable in wild-type C57BL/6JNifdc.

Strain specific B2M expression analysis in B-hPD-1/hPD-L1/HLA-A11.1 plus mice by flow cytometry. Splenocytes were collected from homozygous(HO) B-hPD-1/hPD-L1/HLA-A11.1 plus mice after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (male, 8-week-old, n=1) or not. Protein expression was analyzed with anti-mouse B2M antibody (BD Biosciences, 744802) and anti-hB2M antibody (Biolegend, 395712), by flow cytometry. Mouse B2M and human B2M were detectable in homozygous B-hPD-1/hPD-L1/HLA-A11.1 plus mice because the mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

Strain specific HLA-A expression analysis in B-hPD-1/hPD-L1/HLA-A11.1 plus mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1/hPD-L1/HLA-A11.1 plus mice after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (male, 8-week-old, n=1) or not. Protein expression was analyzed with anti-H-2D^b antibody (Biolegend, 111513) and anti-HLA-ABC antibody (Biolegend, 311406), by flow cytometry. Human HLA-A were detectable in B-hPD-1/hPD-L1/HLA-A11.1 plus mice but not in wild-type C57BL/6JNifdc.

Strain specific PD-1 and PD-L1 expression analysis in wild-type mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (male, 8-week-old, n=1) or not. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104), anti-human PD-1 antibody (Biolegend, 329904), anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) by flow cytometry. Mouse PD-1 and PD-L1 were detectable in wild-type C57BL/6JNifdc mice.

Strain specific PD-1 and PD-L1 expression analysis in B-hPD-1/hPD-L1/HLA-A11.1 plus mice by flow cytometry. Splenocytes were collected from homozygous(HO) B-hPD-1/hPD-L1/HLA-A11.1 plus mice after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (male, 8-week-old, n=1) or not. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104), anti-human PD-1 antibody (Biolegend, 329904), anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) by flow cytometry. Human PD-1 and PD-L1 were exclusively detectable in B-hPD-1/hPD-L1/HLA-A11.1 plus mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1/hPD-L1/HLA-A11.1 plus mice  (male, 8-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/hPD-L1/HLA-A11.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequency of T cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were lower than in C57BL/6JNifdc mice. Frequency of CD8+ T cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells B-hPD-1/hPD-L1/HLA-A11.1 plus mice were higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1/hPD-L1/HLA-A11.1 plus mice (male, 8-week-old, n=3). A. Flow cytometry analysis of the blood 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/hPD-L1/HLA-A11.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequency of CD8+ T cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Lymph node cells were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1/hPD-L1/HLA-A11.1 plus mice (male, 8-week-old, n=3). A. Flow cytometry analysis of the lymph node was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, and NK cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were similar to those in C57BL/6JNifdc mice. Frequency of CD8+ T cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-hPD-1/hPD-L1/HLA-A11.1 plus mice were higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.