B-hVSIG4 mice

Basic Information

Gene Targeting Strategy

Gene targeting strategy for B-hVSIG4 mice. Coding sequences of human VSIG4 and mouse 3’UTR were inserted into exon 2 of mouse Vsig4 gene. Mouse Vsig4 gene was replaced with human VSIG4 gene.

mRNA Expression Analysis

Strain specific analysis of VSIG4 gene expression in wild-type C57BL/6 mice and homozygous B-hVSIG4 mice by RT-PCR. Mouse Vsig4 mRNA was detectable in liver tissue of wild-type C57BL/6 mice (+/+). Human VSIG4 mRNA was detectable only in homozygous B-hVSIG4 mice but not in wild-type mice.

Protein Expression Analysis

Strain specific VSIG4 expression analysis in wild-type C57BL/6 mice and homozygous B-hVSIG4 mice by flow cytometry. PEMs (peritoneal exudate macrophages) were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hVSIG4 mice (H/H). Mouse VSIG4 was detectable only in wild-type mice. Human VSIG4 was detectable only in homozygous B-hVSIG4 mice (H/H).

Immune Cell Analysis

Analysis of leukocytes cell subpopulation in spleen

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hVSIG4 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-hVSIG4 mice were similar to those in the C57BL/6 mice, demonstrating that VSIG4 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in spleen

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hVSIG4 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-hVSIG4 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hVSIG4 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 leukocytes cell subpopulation in lymph node

Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hVSIG4 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, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hVSIG4 mice were similar to those in the C57BL/6 mice, demonstrating that VSIG4 humanized does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in lymph node

Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hVSIG4 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-hVSIG4 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hVSIG4 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 leukocytes cell subpopulation in blood

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hVSIG4 mice (n=3, 7-week-old). Flow cytometry analysis of the blood cells 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-hVSIG4 mice were similar to those in the C57BL/6 mice, demonstrating that VSIG4 humanized does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in blood

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hVSIG4 mice (n=3, 7-week-old). Flow cytometry analysis of the blood cells 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-hVSIG4 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hVSIG4 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.

In Vivo Efficacy Evaluation of an Anti-Human VSIG4 Antibody

Antitumor activity of anti-human VSIG4 antibody (hVSIG4 Ab-A, in house) in B-hVSIG4 mice. (A) Anti-human VSIG4 antibody inhibited MC38 tumor growth in B-hVSIG4 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hVSIG4 mice (female, 7-week-old, n=5). Mice were grouped when tumor volume reached approximately 100-150 mm3, at which time they were treated with anti-human VSIG4 antibody indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human VSIG4 antibody was efficacious in controlling tumor growth in B-hVSIG4 mice, demonstrating that the B-hVSIG4 mice provide a powerful preclinical model for in vivo evaluation of anti-human VSIG4 antibodies. Values are expressed as mean ± SEM.

Analysis of Tumor Infiltrating Lymphocytes

Flow cytometry analysis of tumor infiltrating lymphocytes (TILs). Tumor cells were harvested at the endpoint of experiment (n=5). Flow cytometry analysis of the lymphocytes were performed to assess cell number and proportion changes compared to the group treated with isotype antibody. Percentages of CD8+ T cells, CD4+ T cells, Tregs, M1 macrophages and M1/M2 ratio were increased, while percentages of MDSCs and M2 macrophages were decreased in the group treated with anti-VSIG4 antibody compared to the isotype antibody treated group. Values are expressed as mean ± SEM.

References

1. Katschke, K.J., Jr., et al. A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis. J Exp Med 204, 1319-1325 (2007).
2. Lieberman, L.A., et al. Complement receptor of the immunoglobulin superfamily reduces murine lupus nephritis and cutaneous disease. Clin Immunol 160, 286-291 (2015).
3. Li, J., et al. VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism. Nat Commun 8, 1322 (2017).
4. Liao, Y., et al. VSIG4 expression on macrophages facilitates lung cancer development. Lab Invest 94, 706-715 (2014).
5. Xu, T., et al. VSIG4 is highly expressed and correlated with poor prognosis of high-grade glioma patients. Am J Transl Res 7, 1172-1180 (2015).
6. Trouw, L.A., Pickering, M.C. & Blom, A.M. The complement system as a potential therapeutic target in rheumatic disease. Nat Rev Rheumatol 13, 538-547 (2017).
7. He, J.Q., Wiesmann, C. & van Lookeren Campagne, M. A role of macrophage complement receptor CRIg in immune clearance and inflammation. Mol Immunol 45, 4041-4047 (2008).
8. Small, A.G., et al., Complement receptor immunoglobulin: a control point in infection and immunity, inflammation and cancer. Swiss Med Wkly, 2016. 146: p. w14301.

Poster

AACR 2022: Evaluating In Vivo Efficacy of Anti-VSIG4 Antibodies in Humanized B-hVSIG4 Mice