Basic Information
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Targeting strategy
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Gene targeting strategy for B-hCCR8 mice. The exon 2 that encoding the full coding sequence of mouse Ccr8 gene was replaced by exon 2 of human CCR8 gene in B-hCCR8 mice.
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mRNA expression analysis
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Strain specific analysis of CCR8 gene expression in C57BL/6 and B-hCCR8 mice by RT-PCR. Mouse Ccr8 mRNA was detectable in thymocytes of wild-type mice (+/+) . Human CCR8 mRNA was detectable only in homozygous B-hCCR8 mice (H/H) but not in wild-type mice (+/+).
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Protein expression analysis of mouse and human CCR8 in spleen
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Strain specific analysis of CCR8 gene expression in B-hCCR8 mice by FACS. Spleen was isolated from the mice, and analyzed by flow cytometry. Human and mouse CCR8 were not expressed neither in splenocytes of wild-type mice nor B-hCCR8 mice separately.
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Protein expression analysis of mouse and human CCR8 in thymus
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Strain specific analysis of CCR8 gene expression in B-hCCR8 mice by FACS. Thymus was isolated from the mice, and analyzed by flow cytometry. Human and mouse CCR8 were not expressed neither in thymocytes of wild-type mice nor B-hCCR8 mice separately.
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Protein expression analysis of mouse and human CCR8 in blood
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Strain specific analysis of CCR8 gene expression in B-hCCR8 mice by FACS. Blood were harvested from the mice, and analyzed by flow cytometry. Human and mouse CCR8 were not expressed neither in blood cells of wild-type mice nor B-hCCR8 mice separately.
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Analysis of leukocytes cell subpopulation in B-hCCR8 mice
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Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCCR8 mice (n=3, 8-week-old). Flow cytometry analysis of the splenocytes were 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 cell, B cell, NK cell, monocyte, dendritic cell, granulocyte and macrophage in homozygous B-hCCR8 mice were similar to those in the C57BL/6 mice, demonstrating that CCR8 humanized 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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Summary for protein expression
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Strain specific analysis of CCR8 gene expression in B-hCCR8 mice by FACS. Human CCR8 was both detectable on CD4+ T cells and Treg cells in tumors of homozygous B-hCCR8 mice, but not in splenocytes and blood cells. Mouse CCR8 was detectable in tumors and slightly expressed in splenocytes of wild-type mice, but not in blood cells.
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In vivo efficacy of anti-human CCR8 antibody
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Antitumor activity of anti-human CCR8 antibody (in house) in B-hCCR8 mice. (A) Anti-human CCR8 antibody inhibited MC38 tumor growth in B-hCCR8 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCCR8 mice (male, 6-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human CCR8 antibodies indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human CCR8 antibodies were efficacious in controlling tumor growth in B-hCCR8 mice but the experimental conditions need to be optimized. B-hCCR8 mice provide a powerful preclinical model for in vivo evaluation of anti-human CCR8 antibodies. Values are expressed as mean ± SEM.
Antitumor activity of anti-human CCR8 antibody (in house) in B-hCCR8 mice. (A) Anti-human CCR8 antibody inhibited MC38 tumor growth in B-hCCR8 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCCR8 mice (female, 8 week-old, n=8). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human CCR8 antibody indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human CCR8 antibody were efficacious in controlling tumor growth in B-hCCR8 mice. B-hCCR8 mice provide a powerful preclinical model for in vivo evaluation of anti-human CCR8 antibody. Values are expressed as mean ± SEM.
Antitumor activity of anti-human CCR8 antibody in B-hCCR8 mice. (A) Anti-human CCR8 antibody get from cooperation company inhibited MC38 tumor growth in B-hCCR8 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCCR8 mice (female, 8 week-old, n=7). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human CCR8 antibody. (B) Body weight changes during treatment. As shown in panel A, anti-human CCR8 antibody were efficacious in controlling tumor growth in B-hCCR8 mice. B-hCCR8 mice provide a powerful preclinical model for in vivo evaluation of anti-human CCR8 antibody. Values are expressed as mean ± SEM.
Antitumor activity of anti-human CCR8 antibody in B-hCCR8 mice. (A) Anti-human CCR8 antibody get from cooperation company inhibited MC38 tumor growth in B-hCCR8 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCCR8 mice (female, 8 week-old, n=7). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human CCR8 antibody. (B) Body weight changes during treatment. As shown in panel A, anti-human CCR8 antibody were efficacious in controlling tumor growth in B-hCCR8 mice. B-hCCR8 mice provide a powerful preclinical model for in vivo evaluation of anti-human CCR8 antibody. Values are expressed as mean ± SEM.
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Leukocytes subpopulations in spleen
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Analysis of leukocytes subpopulations in spleen by FACS.
Splenocytes were harvested at the endpoint of the experiment and flow cytometry analysis was performed to assess the leukocyte subpopulations. The percent of CCR8+ Treg cells were significant decrease in anti-human CCR8 antibody treatment group (G2). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)
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Poster
