B-hPD-1/hLAG3 mice

Basic Information

Strain Name
C57BL/6-Pdcd1 tm1(PDCD ) Lag3 tm1(LAG3) /Bcgen
Stock Number
120520
Common Name
B-hPD-1/hLAG3 mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
PD-1(Programmed death-1); LAG3(Lymphocyte activation gene 3,CD223)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

PD-L1 (Programmed cell death ligand-1), also known as B7-H1 and CD274, is mainly expressed in antigen-presenting cells (APCs) and activated T cells, and is one of the two ligands of PD-1. The interaction between PD1 and PD-L1 plays an important role in the negative regulation of the immune response. PD-L1 is highly expressed in a variety of solid tumors. PD-1 and PD-L1 interactions can reduce T cell activation and promote tumor immune escape. The PD-1/PD-L1 signaling pathway can be blocked and antitumor immune response can be restored by using by anti-PD-1 or anti-PD-L1 antibodies to block the binding of PD1 to PD-L1.

LAG3 (Lymphocyte activation gene 3, CD223) is a lymphocyte activation gene and a member of the Ig family. It is mainly expressed in activated T cells, NK cells, B cells and plasmacytoid DCs. LAG3 is a negative regulator of immunity and mainly binds to MHC class Ⅱ molecules to regulate the function of dendritic cells. The expression of LAG3 is associated with the negative immunoregulatory function of specific T cells. Inhibition of LAG3 function enhances the antitumor effect of specific CD8+ T cells, therefore LAG3 is a potential target for tumor immunotherapy.

Targeting Strategy

Gene targeting strategy for B-hPD-1/hLAG3 mice. The exon 2 of mouse Pd-1 gene that encodes the extracellular domain was replaced by human PD-1 exon 2 in B-hPD-1/hLAG3 mice. The exons 2-3 of mouse Lag3 gene that encode the extracellular domain were replaced by human LAG3 exons 2-3 in B-hPD-1/hLAG3 mice.

Details

Phenotype

Protein Expression Analysis

Strain specific PD-1 and LAG3 expression analysis in homozygous B-hPD-1/hLAG3 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hLAG3 (H/H) mice, and analyzed by flow cytometry with species-specific anti-PD-1 and anti-LAG3 antibody. Mouse PD-1 and LAG3 were detectable in WT mice. Human PD-1 and LAG3 were exclusively detectable in homozygous B-hPD-1/hLAG3 but not WT mice.

Strain specific PD-1 and LAG3 expression analysis in homozygous B-hPD-1/hLAG3 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hLAG3 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-1 and anti-LAG3 antibody. Mouse PD-1 and LAG3 were detectable in WT mice while mouse LAG3 was also detectable in homozygous B-hPD-1/hLAG3, due to the anti-mouse LAG3 antibody cross-reacts with human LAG3. Human PD-1 and LAG3 were exclusively detectable in homozygous B-hPD-1/hLAG3 but not WT mice.

Strain specific mPD-1 and mLAG3 expression analysis in homozygous B-hPD-1/hLAG3 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hLAG3 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-mouse PD-1 and LAG3 antibody. Mouse PD-1 and LAG3 were both detectable in WT mice while mouse LAG3 was also detectable in homozygous B-hPD-1/hLAG3, due to the anti-mouse LAG3 antibody cross-reacts with human LAG3.

Strain specific hPD-1 and hLAG3 expression analysis in homozygous B-hPD-1/hLAG3 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hLAG3 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-human PD-1 and LAG3 antibody. Human PD-1 and LAG3 were both detectable in homozygous B-hPD-1/hLAG3.

Analysis of spleen leukocytes cell subpopulations in B-hPD-1/hLAG3 mice

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/hLAG3 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 CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK, Monocyte, DC and macrophage cells in homozygous B-hPD-1/hLAG3 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1/hLAG3 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of spleen T cell subpopulations in B-hPD-1/hLAG3 mice

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/hLAG3 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. Percent of CD8, CD4, and Treg cells in homozygous B-hPD-1/hLAG3 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1/hLAG3 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocytes cell subpopulations in B-hPD-1/hLAG3 mice

Analysis of blood leukocyte subpopulations by FACS .Blood were isolated from female C57BL/6 and B-hPD-1/hLAG3 mice (n=3, 6 week-old) Flow cytometry analysis of the blood 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. Percent of T, B, NK, Monocyte, DC and macrophage cells in homozygous B-hPD-1/hLAG3 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1/hLAG3 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of blood T cell subpopulations in B-hPD-1/hLAG3 mice

Analysis of blood T cell subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hPD-1/hLAG3 mice (n=3, 6 week-old). Flow cytometry analysis of the blood leukocytes 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. Percent of CD8, CD4, and Treg cells in homozygous B-hPD-1/hLAG3 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1/hLAG3 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.

Blood routine test in B-hPD-1/hLAG3 mice

Complete blood count (CBC). Blood from female C57BL/6 and B-hPD-1/hLAG3 mice (n=3, 6 week-old) was collected and analyzed for CBC. There was no differences among any measurement between C57BL/6 and B-hPD-1/hLAG3 mice, indicating that introduction of hPD-1/hLAG3 in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood chemistry of B-hPD-1/hLAG3 mice

Blood chemistry tests of B-hPD-1/hLAG3 mice. Serum from the C57BL/6 and B-hPD-1/hLAG3 mice (n=3, 6 week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hPD-1/hLAG3 mice, indicating that introduction of hPD-1/hLAG3 in place of its mouse counterpart does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.

Combination therapy of anti-human PD-1 antibody and anti-human LAG3 antibody

Antitumor activity of anti-human PD-1 antibody combined with anti-human LAG3 antibody in B-hPD-1/hLAG3 mice. (A) Anti-human PD-1 antibody combined with anti-human LAG3 antibody inhibited MC38 tumor growth in B-hPD-1/hLAG3 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hLAG3 mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with pembrolizumab and anti-human LAG3 antibody with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, combination of pembrolizumab and anti-hLAG3 antibody shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/hLAG3 mice provides a powerful preclinical model for in vivo evaluating combination therapy efficacy of hPD-1 antibodies and hLAG3 antibodies. Values are expressed as mean ± SEM.

References

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