B-hCCR8 mice_Biocytogen Pharmaceuticals (Beijing) Co., Ltd._Biocytogen,drug development,antibody discovery

Strain Name	C57BL/6-Ccr8^tm1(CCR8)/Bcgen	Common Name	B-hCCR8 mice
Background	C57BL/6	Catalog number	110096
Related Genes	C-C motif chemokine receptor 8; CY6; TER1; CCR-8; CKRL1; CDw198; CMKBR8; GPRCY6; CMKBRL2; CC-CKR-8
NCBI Gene ID	12776

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 (+/+).

Protein expression analysis

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Strain specific analysis of CCR8 gene expression in B-hCCR8 mice by FACS. MC38 cells were inoculated into wild-type C57BL/6 (+/+) and homozygous B-hCCR8 mice (H/H). Tumors were harvested at the endpoint of experiment, and the TILs were analyzed by flow cytometry. Human CCR8 was both detectable on CD4+ T cells and Tregs in tumors of homozygous B-hCCR8 mice, and mouse CCR8 was detectable only in Wild-type mice.

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.

Analysis of leukocytes cell subpopulation in B-hCCR8 mice

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Analysis of thymus leukocyte subpopulations by FACS. Thymocytes were isolated from female C57BL/6 and B-hCCR8 mice (n=3, 8-week-old). Flow cytometry analysis of the thymocytes 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 thymus. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in B-hCCR8 mice

Analysis of blood leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hCCR8 mice (n=3, 8-week-old). Flow cytometry analysis of the blood 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 blood. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in B-hCCR8 mice

Analysis of T cell subpopulation in spleen, thymus and blood.

The lymphocytes were isolated from spleen, thymus and blood in C57BL/6 and B-hCCR8 mice (n=3, 8-week-old). The proportion of T cells subpopulation was tested by flow cytometry. There were no differences between C57BL/6 and B-hCCR8 mice, demonstrating that humanized of CCR8 does not change the overall development, differentiation or distribution of these T cell subtypes. Values are expressed as mean ± SEM (please confirm)

In vivo efficacy of anti-human CCR8 antibody

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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.