Basic Information
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mRNA expression analysis
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Species-specific CXCR2 gene expression analysis in wild-type and humanized B-hCXCR2 mice by RT-PCR. Murine Cxcr2 mRNA was detected in splenocytes isolated from wild-type (+/+) mice, while human CXCR2 mRNA was exclusively detected in homozygous B-hCXCR2 (H/H) mice.
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Protein expression analysis in Gr-1+ cells
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Protein expression analysis in Gr-1+ splenic cells
Species-specific CXCR2 protein expression analysis in humanized B-hCXCR2 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hCXCR2 (H/H) mice, and analyzed by flow cytometry using species-specific anti-CXCR2 antibodies. Murine CXCR2 protein was detected in wild-type mice, while human CXCR2 protein was exclusively detected in B-hCXCR2 mice.
Protein expression analysis in Gr-1+ bone marrow cells
Species-specific CXCR2 protein expression analysis in humanized B-hCXCR2 mice. Bone marrow cells were isolated from wild-type C57BL/6 (+/+) and homozygous B-hCXCR2 (H/H) mice, and analyzed by flow cytometry using species-specific anti-CXCR2 antibodies. Murine CXCR2 protein was detected in wild-type mice, while human CXCR2 protein was exclusively detected in B-hCXCR2 mice.
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Analysis of T cell subpopulation in spleen
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Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCXCR2 mice (n=3, 7-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-hCXCR2 mice were similar to those in the C57BL/6 mice, demonstrating that CXCR2 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCXCR2 mice (n=3, 7-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 TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hCXCR2 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCXCR2 in place of its mouse counterpart 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（LNs）
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Analysis of LNs leukocyte subpopulations by FACS. LNs were isolated from female C57BL/6 and B-hCXCR2 mice (n=3, 7-week-old). Flow cytometry analysis of the LNs 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 and NK cells in homozygous B-hCXCR2 mice were similar to those in the C57BL/6 mice, demonstrating that CXCR2 humanized does not change the overall development, differentiation or distribution of these cell types in LNs. Values are expressed as mean ± SEM.
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Analysis of T cell subpopulation in lymph node（LNs）
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Analysis of LNs T cell subpopulations by FACS. LNs were isolated from female C57BL/6 and B-hCXCR2 mice (n=3, 7-week-old). Flow cytometry analysis of the LNs was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hCXCR2 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCXCR2 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in LNs. 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 B-hCXCR2 mice (n=3, 7-week-old). Flow cytometry analysis of the blood 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-hCXCR2 mice were similar to those in the C57BL/6 mice, demonstrating that CXCR2 humanized 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 B-hCXCR2 mice (n=3, 7-week-old). Flow cytometry analysis of the blood was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hCXCR2 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCXCR2 in place of its mouse counterpart 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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In vivo efficacy validation in IBD-induced B-hCXCR2 mice
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Drinking water supplemented with DSS induced inflammatory bowel disease (IBD) in humanized B-hCXCR2 mice. (A-C) B-hCXCR2 mice given DSS showed significantly decreased body weight compared to the vehicle-treated group, (D) while the disease activity index (DAI) was increased in DSS mice. However, when H3h9 (in house, anti-human CXCR2 Ab) was administered, body weight and disease severity were improved in DSS-B-hCXCR2 mice. Altogether, these results demonstrate that DSS induced IBD in B-hCXCR2 mice, and H3h9 treatment relieved the clinical symptoms associated with IBD.
