Strain specific analysis of B7-H4 gene expression in wild type and B-hB7-H4 mice by RT-PCR. 

Mouse B7-h4 mRNA was detectable only in ovary of wild type mice (+/+). Human B7-H4 mRNA was detectable only in homozygous B-hB7-H4 mice (H/H) but not in wild type mice.

Strain specific B7-H4 expression analysis in homozygous B-hB7-H4 mice by western blot. 

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hB7-H4 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-hB7-H4 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hB7-H4 in place of its mouse counterpart dose 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-hB7-H4 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-hB7-H4 mice were similar to those in the C57BL/6 mice, demonstrating that B7-H4 humanized 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 blood leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hB7-H4 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-hB7-H4 mice were similar to those in the C57BL/6 mice, demonstrating that B7-H4 humanized 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 were isolated from female C57BL/6 and B-hB7-H4 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-hB7-H4 mice were similar to those in the C57BL/6 mice, demonstrating that B7-H4 humanized does not change the overall development, differentiation or distribution of these T cell subtypes in the blood. Values are expressed as mean ± SEM.

Analysis of lymph nodes leukocyte subpopulations by FACS. lymph nodes were isolated from female C57BL/6 and B-hB7-H4 mice (n=3, 7-week-old). Flow cytometry analysis of the leukocytes 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 in homozygous B-hB7-H4 mice were similar to those in the C57BL/6 mice, demonstrating that B7-H4 humanized 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 nodes T cell subpopulations by FACS. lymph nodes were isolated from female C57BL/6 and B-hB7-H4 mice (n=3, 7-week-old). Flow cytometry analysis of the leukocytes 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-hB7-H4 mice were similar to those in the C57BL/6 mice, demonstrating that B7-H4 humanized does not change the overall development, differentiation or distribution of these T cell subtypes in the lymph node. Values are expressed as mean ± SEM.

Antitumor activity of anti-human B7-H4 antibody in B-hB7-H4 mice. (A) FPA-150 (in house) inhibited B-hB7-H4 MC38 tumor growth in B-hB7-H4 mice. B-hB7-H4 MC38 cells (3ⅹ10⁶) were subcutaneously implanted into homozygous B-hB7-H4 mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with FPA-150 (in house) with doses and schedules in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human B7-H4 antibody was efficacious in controlling tumor growth in B-hB7-H4 mice, demonstrating that the B-hB7-H4 mice provide a powerful preclinical model for in vivo evaluation of anti-human B7-H4 antibodies. Values are expressed as mean ± SEM.