Strain specific analysis of NKG2D gene expression in wild-type (WT) mice (+/+) and B-hNKG2D mice (H/H) by RT-PCR. Mouse Nkg2d mRNA was detectable only in splenocytes of WT mice (+/+). Human NKG2D mRNA was detectable only in homozygous B-hNKG2D mice (H/H) but not in WT mice (+/+).

Strain specific NKG2D 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.

Strain specific NKG2D 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.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-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-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 spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-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-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.

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and homozygous B-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-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 FACS. Leukocytes were isolated from female C57BL/6 and homozygous B-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-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.

Analysis of blood leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and homozygous B-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-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 FACS. Blood were isolated from female C57BL/6 and homozygous B-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-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 blood. Values are expressed as mean ± SEM.