Basic Information
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Gene Targeting Strategy
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Exons 1~4 of the murine Trem1 gene, which encode the extracellular domain, were replaced by human TREM1 exons 1~4 in B-hTREM1 mice.
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mRNA Expression Analysis
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Strain specific analysis of TREM1 gene expression in wild type C57BL/6 (+/+) mice and homozygous B-hTREM1(H/H) mice by RT-qPCR.
The mRNA expression of hTREM1 in B-hTREM1(H/H) was similar to mTrem1 in the C57BL/6 (+/+), demonstrating that introduction of hTREM1 in place of its mouse counterpart does not change the expression level of TREM1 mRNA.
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Protein Expression Analysis
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Macrophage
Strain specific TREM1 expression analysis in homozygous B-hTREM1 mice by flow cytometry.
MC38 cells were inoculated into wild-type C57BL/6(+/+) and homozygous B-hTREM1 mice(H/H). Blood and tumor were harvested when tumor volume reached approximately 500 mm3, and analyzed by flow cytometry with species-specific anti-TREM1 antibody. Mouse TREM1 was exclusively detectable in blood and tumor macrophage of wild-type mice. Human TREM1 was exclusively detectable in blood and tumor macrophage of homozygous B-hTREM1 mice.
* It is not clear what population of cells was stained with specific anti-hTREM1 in tumors of C57BL/6 (Q2).
Monocyte(M-MDSC)
Strain specific TREM1 expression analysis in homozygous B-hTREM1 mice by flow cytometry.
MC38 cells were inoculated into wild-type C57BL/6(+/+) and homozygous B-hTREM1 mice(H/H). Blood and tumor were harvested when tumor volume reached approximately 500 mm3, and analyzed by flow cytometry with species-specific anti-TREM1 antibody. Mouse and human TREM1 were not detectable in blood monocyte of wild-type mice and homozygous B-hTREM1 mice. Human TREM1 was exclusively detectable in tumor M-MDSC of homozygous B-hTREM1 mice.
* It is not clear what population of cells was stained with specific anti-hTREM1 in tumors of C57BL/6 (Q2).
Neutrophil(G-MDSC)
Strain specific TREM1 expression analysis in homozygous B-hTREM1 mice by flow cytometry.
MC38 cells were inoculated into wild-type C57BL/6(+/+) and homozygous B-hTREM1 mice(H/H). Blood and tumor were harvested when tumor volume reached approximately 500 mm3, and analyzed by flow cytometry with species-specific anti-TREM1 antibody. Mouse TREM1 was exclusively detectable in blood and tumor neutrophil(G-MDSC) of wild-type mice. Human TREM1 was exclusively detectable in blood and tumor neutrophil(G-MDSC) of homozygous B-hTREM1 mice.
T cell
Strain specific TREM1 expression analysis in homozygous B-hTREM1 mice by flow cytometry.
MC38 cells were inoculated into wild-type C57BL/6(+/+) and homozygous B-hTREM1 mice(H/H). Blood and tumor were harvested when tumor volume reached approximately 500 mm3, and analyzed by flow cytometry with species-specific anti-TREM1 antibody. Mouse and human TREM1 were not detectable in blood and tumor T cells of wild-type mice and homozygous B-hTREM1 mice.
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Analysis of leukocytes cell subpopulation in spleen
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Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hTREM1 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 here. B. Results of FACS analysis. Percent of T cell, B cell, NK cell, monocyte, dendritic cell, granulocyte and macrophage in homozygous B-hTREM1 mice were similar to those in the C57BL/6 mice, demonstrating that TREM1 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
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Tumor Growth Curve and Body Weight Changes
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Subcutaneous homograft tumor growth of MC38 cells in B-hTREM1 mice. Wild-type murine colon cancer MC38 cells (5×105) were subcutaneously implanted into wild-type C57BL/6 mice and humanized B-hTREM1 mice (male, 8-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5 X long diameter X short diameter2. Shown in panel A, MC38 cells were able to establish tumors in B-hTREM1 mice, which can be used for in vivo efficacy studies.
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Combination therapy of an anti-mouse PD-1 antibody with an anti-human TREM1 antibody
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Antitumor activity of anti-mouse PD-1 antibody combined with anti-human TREM1 antibody in B-hTREM1 mice. (A) Anti-mouse PD-1 antibody combined with anti-human TREM1 antibody inhibited MC38 tumor growth in B-hTREM1 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hTREM1 mice (female, 9-week-old, n=5). Mice were grouped when tumor volume reached approximately 80-100 mm3, at which time they were treated with mPD-1 and hTREM1 antibodies. (B) Body weight changes during treatment. As shown in panel A, combination of anti-hTREM1 and anti-mPD-1 antibody shows more inhibitory effects than individual groups, demonstrating that the B-hTREM1 mice provide a powerful preclinical model for in vivo evaluation of anti-human TREM1 antibody. Values are expressed as mean ± SEM.