Strain specific NKP46 expression analysis in wild-type and B-hNKP46 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 and homozygous B-hNKP46 mice (H/H), and analyzed by flow cytometry with species-specific NKP46 antibodies. Mouse NKP46 was detectable in wild-type mice. Human NKP46 was exclusively detectable in homozygous B-hNKP46 mice.

Strain specific NKP46 expression analysis in wild-type and B-hNKP46 mice by flow cytometry. Blood was collected from wild-type C57BL/6 and homozygous B-hNKP46 mice (H/H), and analyzed by flow cytometry with species-specific NKP46 antibodies. Mouse NKP46 was detectable in wild-type mice. Human NKP46 was exclusively detectable in homozygous B-hNKP46 mice.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-hNKP46 mice (n=3, 6-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. B. Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in homozygous B-hNKP46 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKP46 in place of its murine counterpart does not change the overall development, differentiation or distribution of these cell types in the 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-hNKP46 mice (n=3, 6-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. B. Results of FACS analysis. The percentages of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hNKP46 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKP46 in place of its murine counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in the 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-hNKP46 mice (n=3, 6-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. B. Results of FACS analysis. The percentages of T cells, B cells, and NK cells in homozygous B-hNKP46 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKP46 in place of its murine counterpart does not change the overall development, differentiation or distribution of these cell types in the 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-hNKP46 mice (n=3, 6-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. B. Results of FACS analysis. The percentages of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hNKP46 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKP46 in place of its murine counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in the lymph node. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and homozygous B-hNKP46 mice (n=3, 6-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. B. Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in homozygous B-hNKP46 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKP46 in place of its murine counterpart does not change the overall development, differentiation or distribution of these cell types in the blood. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and homozygous B-hNKP46 mice (n=3, 6-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. B. Results of FACS analysis. The percentages of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hNKP46 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hNKP46 in place of its murine counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in the blood. Values are expressed as mean ± SEM.

Antitumor activity of anti-human NKP46-based Ab in B-hNKP46 mice. (A) Anti-human NKP46-based Ab inhibited MC38 tumor growth in B-hNKP46 mice. Murine colon cancer MC38 cells (5E5) were subcutaneously implanted into B-hNKP46 mice (female, 7-8 week-old, n=7). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with anti-human NKP46-based Ab with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human NKP46-based Ab was efficacious in controlling tumor growth in B-hNKP46 mice. Values are expressed as mean ± SEM. (All antibodies were provided by the clients)