Basic Information

Strain Name
C57BL/6-Lrrc32tm1(LRRC32)/Bcgen
Stock Number
110102
Common Name
B-hGARP mice
Background
C57BL/6
Related Genes
LRRC32 (Leucine-Rich Repeat-Containing Protein 32), GARP (Glycoprotein-A repetitions predominant)
Targeting Strategies
The exon 1~2 of mouse Lrrc32 gene that encode the extracellular domain were replaced by human LRRC32 exon 1~2 in B-hGARP mice.

Description

GARP (glycoprotein-A repetitions predominant) is a type I transmembrane cell surface docking receptor for latent transforming growth factor-β (TGF-β) that is abundantly expressed on regulatory T lymphocytes and platelets. GARP regulates the availability of membrane-bound latent TGF-β and modulates its activation. The function of GARP has been extensively studied on regulatory T lymphocytes (Tregs), where it complexes with αVβ8 integrins to release active TGF-β from the surface of the cells. Via this function, GARP was shown to be involved in enhancing the suppressive phenotype of Tregs and in maintaining Treg-mediated peripheral tolerance. A strong connection between GARP and cancer by describing the pro-tumorigenic function of this protein in several human malignancies and the unexpected role of platelet GARP in immune evasion and the cancer progression. Moreover, GARP expression has been recently described on human B cells in response to B cell receptor activation and Toll-like receptor (TLR) 9 ligation. Several drugs against GARP are also in development or in clinical trials. Thus, there is a need for pre-clinical models to evaluate the safety and efficacy of GARP-targeted therapeutics before they enter human trials. Biocytogen has generated a humanized GARP mouse for in vitro functional and in vivo efficacy evaluation of GARP antagonists.

Details

mRNA expression analysis

Strain specific analysis of GARP gene expression in WT and B-hGARP mice by RT-PCR. Mouse Garp mRNA was detectable in splenocytes of wild-type (+/+) mice. Human GARP mRNA was detectable only in H/H, but not in +/+ mice.

Protein expression analysis in Treg cells

Strain specific GARP expression analysis in homozygous B-hGARP mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hGARP (H/H) mice, and analyzed by flow cytometry with species-specific GARP antibody. Mouse GARP was detectable in WT mice. Human GARP was exclusively detectable in homozygous B-hGARP but not WT mice.

Analysis of spleen leukocyte subpopulations in B-hGARP mice

Analysis of splenic leukocyte subpopulations by FACS.Splenocytes were isolated from female C57BL/6 and B-hGARP mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocyte/macrophages, and DC were similar in homozygous B-hGARP mice and C57BL/6 mice, demonstrating that introduction of hGARP in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in spleen. Similar results were obtained for blood leukocyte analysis. Values are expressed as mean ± SEM.


Analysis of splenic T cell subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hGARP mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on CD3+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hGARP and C57BL/6 mice, demonstrating that introduction of hGARP 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 in B-hGARP mice



Analysis of blood leukocyte subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hGARP mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocyte/macrophages, and DC were similar in homozygous B-hGARP mice and C57BL/6 mice, demonstrating that introduction of hGARP in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in blood. Values are expressed as mean ± SEM.


Analysis of blood T cell subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hGARP mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on CD3+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hGARP and C57BL/6 mice, demonstrating that introduction of hGARP 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.

Combination therapy of anti-mouse PD-1 antibody and anti-human GARP/latent-TGFβ1 antibody


Antitumor activity of anti-mouse PD-1 antibody combined with anti-human GARP/latent-TGFβ1 antibody in B-hGARP mice. (A) Anti-mouse PD-1 antibody combined with anti-human GARP/latent-TGFβ1 antibody (in house) inhibited MC38 tumor growth in B-hGARP mice. Murine colon cancer MC38 cells (5E5) were subcutaneously implanted into homozygous B-hGARP mice (female, 7-week-old, n=6). Mice were grouped when tumor volume reached approximately 50~70 mm3, at which time they were treated with anti-mouse PD-1 antibody and anti-human GARP/latent-TGFβ1 antibody with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, combination of anti-mPD-1 antibody and anti-human GARP/latent-TGFβ1 antibody were efficacious in controlling tumor growth in B-hGARP, demonstrating that the B-hGARP mice provide a powerful preclinical model for in vivo evaluation of anti-human GARP antibodies. Values are expressed as mean ± SEM.