Basic Information
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Targeting strategy
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Gene targeting strategy for B-hPD-1/hPD-L1/hCD73 mice. The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PDCD1 exon 2 in B-hPD-1/hPD-L1/hCD73(v2) mice. The exon 3 of mouse Pd-l1 gene that encodes the extracellular domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/hCD73(v2) mice. The human NT5E whole coding sequence was inserted following the 5’UTR of the mouse Nt5e in B-hCD73 mice.
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Protein expression analysis
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Strain specific PD-1, PD-L1 and CD73 expression analysis in homozygous B-hPD-1/hPD-L1/hCD73 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/hCD73 mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-1, anti-PD-L1 and anti-CD73 antibody. Mouse PD-1, PD-L1 and CD73 were detectable in WT mice. Human PD-1, PD-L1 and CD73 were exclusively detectable in homozygous B-hPD-1/hPD-L1/hCD73 plus but not WT mice.
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Analysis of leukocytes cell subpopulation in spleen
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Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD73 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hPD-1/hPD-L1/hCD73 mice were similar to those in the C57BL/6 mice, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
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Analysis of T cell subpopulation in spleen
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Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD73 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hPD-1/hPD-L1/hCD73 mice were similar to those in the C57BL/6 mice, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.
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Analysis of leukocytes cell subpopulation in lymph node
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Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD73 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of T cells, B cells and NK cells in homozygous B-hPD-1/hPD-L1/hCD73 mice were similar to those in the C57BL/6 mice, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.
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Analysis of T cell subpopulation in lymph node
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Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD73 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hPD-1/hPD-L1/hCD73 mice were similar to those in the C57BL/6 mice, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.
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Analysis of leukocytes cell subpopulation in blood
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Analysis of blood leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD73 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hPD-1/hPD-L1/hCD73 mice were similar to those in the C57BL/6 mice, demonstrating the humanized mouse does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
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Analysis of T cell subpopulation in blood
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Analysis of blood T cell subpopulations by FACS. Blood were isolated from female C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD73 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hPD-1/hPD-L1/hCD73 mice were similar to those in the C57BL/6 mice, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.
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Summary
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- Protein expression analysis:
Human PD-1、PD-L1、CD73 were exclusively detectable in homozygous B-hPD-1/hPD-L1/hCD73 mice but not wild-type mice, and mouse PD-1、PD-L1、CD73 were detectable in wild-type mice.
- Leukocytes profile:
Percentages of T, B, NK cells, monocyte/macrophages, and DC were similar in homozygous B-hPD-1/hPD-L1/hCD73 mice and wild-type mice, demonstrating that the humanized mouse does not change the overall development, differentiation, or distribution of these cell types in spleen, lymph node leukocytes and blood.
- In vivo efficacious：
Anti-CD73 antibodies were efficacious in controlling tumor growth in B-hPD-1/hPD-L1/hCD73 mice.
