Basic Information
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mRNA expression analysis
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Strain specific analysis of PVRIG gene expression in WT and B-hPVRIG mice by RT-PCR. Mouse Pvrig mRNA was detectable in splenocytes of wild-type (+/+). Human PVRIG mRNA was detectable only in B-hPVRIG mice (H/H) but not in wild type mice.
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Protein expression analysis
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PVRIG expression analysis in homozygous B-hPVRIG mice by western blot. Heart collected from wild type and homozygous B-hPVRIG mice (H/H) and analyzed by western blot with anti-PVRIG antibody. Mouse PVRIG was detectable in wild-type mice. Human PVRIG was detectable in homozygous B-hPVRIG mice. Anti-PVRIG antibody is crossly reactive with PVRIG in human and mice.
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Analysis of immune cells
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Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from female C57BL/6 and B-hPVRIG 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-hPVRIG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPVRIG 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 flow cytometry. Splenocytes were isolated from female C57BL/6 and B-hPVRIG 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 CD4+ T cells, CD8+ T cells and Treg cells in homozygous B-hPVRIG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPVRIG 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 blood leukocyte subpopulations by flow cytometry. Blood cells were isolated from female C57BL/6 and B-hPVRIG mice (n=3, 8-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-hPVRIG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPVRIG 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 blood T cell subpopulations by flow cytometry. Blood cells were isolated from female C57BL/6 and B-hPVRIG mice (n=3, 8-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 CD3+ 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 Treg cells in homozygous B-hPVRIG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPVRIG 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 flow cytometry. Lymph nodes were isolated from female C57BL/6 and B-hPVRIG 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-hPVRIG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPVRIG 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. lymph node were isolated from female C57BL/6 and B-hPVRIG mice (n=3, 8-week-old). Flow cytometry analysis of the lymph node 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 CD4+ T cells, CD8+ T cells and Treg cells in homozygous B-hPVRIG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPVRIG 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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In vivo efficacy of anti-human PVRIG antibodies
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Antitumor activity of anti-human PVRIG antibodies in B-hPVRIG mice. (A) COM-701 (in house) and SRF-813 (in house) inhibited MC38 tumor growth in B-hPVRIG mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPVRIG mice (female, 6-week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with COM-701 (in house) and SRF-813 (in house) with doses and schedules in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human PVRIG antibodies were efficacious in controlling tumor growth in B-hPVRIG mice, demonstrating that the B-hPVRIG mice provide a powerful preclinical model for in vivo evaluation of anti-human PVRIG antibodies. Values are expressed as mean ± SEM.
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Summary
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mRNA expression analysis:
Mouse Pvrig mRNA was detectable in splenocytes of wild-type (+/+). Human PVRIG mRNA was detectable in B-hPVRIG mice (H/H) but not in wild-type mice.Protein expression analysis:
Mouse PVRIG was detectable in wild-type mice. Human PVRIG was detectable in homozygous B-hPVRIG mice.
Leukocytes cell subpopulation analysis：
B-hPVRIG mice have normal immune system and normal blood cell composition relative to the parental C57BL/6 strain.
In vivo efficacy：
Anti-human PVRIG antibodies were efficacious in controlling tumor growth in B-hPVRIG mice.
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Poster
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AACR 2022: Humanized PVRIG Mice: A Novel Tool for Evaluating Anti-PVRIG Immunotherapies
