Western blot analysis of B7-H3 protein expression in homozygous B-hB7-H3/hNKG2D mice. Tissues were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hB7-H3/hNKG2D mice (H/H), and analyzed by western blot with species-specific anti-B7-H3 antibody. Human B7-H3 was detectable in homozygous B-hB7-H3/hNKG2D mice but not in wild-type C57BL/6 mice.

Strain specific NKG2D expression analysis in wild-type and B-hB7-H3/hNKG2D mice by flow cytometry. Splenocytes were collected from wild-type (WT) mice (+/+) and homozygous B-hB7-H3/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-hB7-H3/hNKG2D mice but not in NK cells from WT mice.

Strain specific NKG2D expression analysis in wild-type and B-hB7-H3/hNKG2D mice by flow cytometry. Splenocytes were collected from wild-type (WT) mice (+/+) and homozygous B-hB7-H3/hNKG2D mice (H/H). Spleen cells were stimulated for the indicated number of days coated with 10 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-hB7-H3/hNKG2D mice but not in CD8+ T cells from WT mice.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-hB7-H3/hNKG2D mice (n=3, 8-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-hB7-H3/hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H3 and hNKG2D in place of their mouse counterparts 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 homozygous B-hB7-H3/hNKG2D mice (n=3, 8-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. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hB7-H3/hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H3 and hNKG2D in place of their mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and homozygous B-hB7-H3/hNKG2D mice (n=3, 8-week-old). Flow cytometry analysis of the 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. The percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hB7-H3/hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H3 and hNKG2D in place of their mouse counterparts 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 FACS. Blood were isolated from female C57BL/6 and homozygous B-hB7-H3/hNKG2D mice (n=3, 8-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. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hB7-H3/hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H3 and hNKG2D in place of their mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and homozygous B-hB7-H3/hNKG2D mice (n=3, 8-week-old). Flow cytometry analysis of the 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. The percent of T cells, B cells and NK cells in homozygous B-hB7-H3/hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H3 and hNKG2D in place of their mouse counterparts 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 FACS. Leukocytes were isolated from female C57BL/6 and homozygous B-hB7-H3/hNKG2D mice (n=3, 8-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. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hB7-H3/hNKG2D mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H3 and hNKG2D in place of their mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.