Basic Information
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Description
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B-hPD-1/hPD-L1/hSIRPA/hCD47,Tg(hLILRB2/hLILRB3) mice obtained by mating B-hPD-1/hPD-L1, Tg(hLILRB2/hLILRB3) mice (111954) and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice (140577).
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Targeting strategy
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B-hPD-1/hPD-L1/hSIRPA/hCD47,Tg(hLILRB2/hLILRB3) mice obtained by mating B-hPD-1/hPD-L1, Tg(hLILRB2/hLILRB3) mice (111954) and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice (140577). The exon 2 of mouse Pd-1 gene that encode the IgV domain was replaced by human PD-1 exon 2. The exon 3 of mouse Pdl1 gene that encode the IgV domain was replaced by human PD-L1 exon 3. The exon 2 of mouse Sirpα gene that encode the IgV domain was replaced by human SIRPα exon 2. The exon 2 of mouse Cd47 gene that encode the IgV domain was replaced by human CD47 exon 2. Bacterial artificial chromosome (BAC) clones for both the human LILRB2 and LILRB2 genes were integrated into the mouse genome randomly.
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Protein expression analysis-Spleen
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Protein expression analysis-Spleen T cells
Strain specific PD-1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908). Mouse PD-1 was only detectable in wild-type mice. Human PD-1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
Protein expression analysis-Spleen T cells-Anti-CD3ε-treated
Strain specific PD-1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs, and analyzed by flow cytometry with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908). Mouse PD-1 was only detectable in wild-type mice. Human PD-1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.
Protein expression analysis-Spleen T cells
Strain specific PD-L1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706). Mouse PD-L1 was only detectable in wild-type mice. Human PD-L1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
Protein expression analysis-Spleen T cells-Anti-CD3
Strain specific PD-L1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs, and analyzed by flow cytometry with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706). Mouse PD-L1 was only detectable in wild-type mice. Human PD-L1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.
Protein expression analysis-Spleen T cells
Strain specific CD47 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human CD47 antibody (Biolegend, 323108). Mouse CD47 was only detectable in wild-type mice. Human CD47 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
Protein expression analysis-Spleen T cells-Anti-CD3
Strain specific CD47 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs, and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human CD47 antibody (Biolegend, 323108). Mouse CD47 was only detectable in wild-type mice. Human CD47 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.
Protein expression analysis-Spleen leukocytes
Strain specific SIRPA expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with cross-recognized both human and mouse SIRPa antibody (Biolegend, 144011) and anti-human SIRPa antibody (Biolegend, 323810). Human SIRPa was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.
Protein expression analysis-Spleen-T cells
Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was not detectable in T cells of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in T cells of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
Protein expression analysis-Spleen-B cells
Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was not detectable in B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in B cells of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
Protein expression analysis-Spleen-Macrophages
Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was only detectable in macrophages of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in macrophages of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
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Protein expression analysis-Blood-Monocytes
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Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Blood cells were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was only detectable in monocytes of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in monocytes of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.
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Protein expression analysis-RBC
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Strain specific CD47 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Red blood cells were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human CD47 antibody (Biolegend, 323108). Mouse CD47 was only detectable in wild-type mice. Human CD47 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.
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Frequency of leukocyte subpopulations in spleen
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Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old). 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, DCs, granulocytes, monocytes, macrophages, CD4+ T cells, CD8a+ T cells and Tregs in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice were similar to those in C57BL/6N mice, demonstrating that humanization of hPD-1, hPD-L1, hSIRPA, hCD47 and the transgene of LILRB2 and LILRB3 do not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM. The frequency of leukocyte subpopulations in lymph node of B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice were also comparable to wild-type C57BL/6N mice (Data not shown).
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Tumor growth curve & body weight changes
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Subcutaneous homograft tumor growth of B-hPD-L1 plus/hCD47 MC38 cells. B-hPD-L1 plus/hCD47 MC38 cells (5×105) were subcutaneously implanted into B-hPD-1/hPD-L1/hSIRPA/hCD47,Tg(hLILRB2/hLILRB3) mice (female, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5 × long diameter × short diameter2. As shown in panel A, B-hPD-L1 plus/hCD47 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
