Basic Information
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mRNA expression analysis
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Species-specific NKG2D gene expression analysis in humanized B-hNKG2D mice by RT-PCR. Murine Nkg2d mRNA was detected in splenocytes isolated from wild-type (+/+) mice, while human NKG2D mRNA was exclusively detected in homozygous B-hNKG2D (H/H) mice.
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Protein expression analysis in NK cells
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Species-specific NKG2D protein expression analysis in homozygous B-hNKG2D mice by flow cytometry. Splenocytes were collected from wild-type (WT) mice (+/+) and homozygous B-hNKG2D mice (H/H). Mouse NKG2D was only detectable in NK cells from WT mice. Human NKG2D was only detectable in NK cells from homozygous B-hNKG2D mice but not in NK cells from WT mice.
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Protein expression analysis in mCD8+ T cells
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Species-specific NKG2D protein expression analysis in homozygous B-hNKG2D mice by flow cytometry. Splenocytes were collected from wild-type (WT) mice (+/+) and homozygous B-hNKG2D mice (H/H). Spleen cells were stimulated for the indicated number of days coated with 5 μg/ml anti-TCRβ mAb before analysis of NKG2D surface expression on gated CD8+ T cells. Mouse NKG2D was only detectable in activated CD8+ T cells from WT mice. Human NKG2D was only detectable in activated CD8+ T cells from homozygous B-hNKG2D mice but not in CD8+ T cells from WT mice.
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Analysis of splenic immune cells
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Analysis of splenic leukocyte subpopulations by flow cytometry. Splenocytes were isolated from C57BL/6 and homozygous B-hNKG2D mice (female, n=3, 8-week-old), and analyzed by flow cytometry 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-hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKG2D in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
Analysis of splenic T cell subpopulations by flow cytometry. Splenocytes were isolated from C57BL/6 and homozygous B-hNKG2D mice (female, n=3, 8-week-old), and analyzed by flow cytometry 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+ T cells, CD4+ T cells, and Tregs in homozygous B-hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKG2D 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 lymph node immune cells
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Analysis of lymph node leukocyte subpopulations by flow cytometry. Leukocytes were isolated from C57BL/6 and homozygous B-hNKG2D mice (female, n=3, 8-week-old), and analyzed by flow cytometry 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 cells, B cells and NK cells in homozygous B-hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKG2D in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.
Analysis of lymph node T cell subpopulations by flow cytometry. Leukocytes were isolated from C57BL/6 and homozygous B-hNKG2D mice (female, n=3, 8-week-old), and analyzed by flow cytometry 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 CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKG2D in place of its mouse counterpart 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 blood immune cells
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Analysis of blood leukocyte subpopulations by flow cytometry. Blood was isolated from C57BL/6 and homozygous B-hNKG2D mice (female, n=3, 8-week-old), and analyzed by flow cytometry 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-hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKG2D in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
Analysis of blood T cell subpopulations by flow cytometry. Blood was isolated from C57BL/6 and homozygous B-hNKG2D mice (female, n=3, 8-week-old), and analyzed by flow cytometry 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+ T cells, CD4+ T cells, and Tregs in homozygous B-hNKG2D mice were similar to those in C57BL/6 mice, demonstrating that introduction of hNKG2D 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.
