Basic Information

Strain Name
C57BL/6-Btlatm1(BTLA)Bcgen/Bcgen
Stock Number
110005
Common Name
B-hBTLA Mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
Btla (B and T lymphocyte associated)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

BTLA (B and T lymphocyte associated) is a B cell and T cell attenuator, another immune checkpoint negative regulator of the lg superfamily. It is structurally similar to CTLA4 and PD-1, and is expressed in B cells, T cells, NK cells, dendritic cells and macrophages. BTLA binds to its ligand HVEM (HVEM is a member of the tumor necrosis factor receptor superfamily) to transmit a co-inhibition signal in the body’s anti-tumor immune response , and is associated with the immune escape mechanism of the tumor. BTLA inhibitors enhance the TCR signaling pathway, restore T cell function, and is a potential novel target for tumor biotherapy.

Targeting Strategy

Gene targeting strategy for B-hBTLA mice. The exon 2 of mouse Btla gene that encodes the extracellular domain was replaced by human BTLA exon 2 in B-hBTLA mice.

Details

Phenotype

mRNA Expression Analysis

RT-PCR analysis of B-hBTLA gene. The hBTLA, but not mBTLA, mRNA was detectable in splenocytes of the homozygous B-hBTLA mice.

Protein Expression Analysis

Splenocytes from both wild type (WT) C57BL/6 and homozygous B-hBTLA mice were analyzed by flow cytometry. Mouse BTLA+ B cells were detectable in the WT C57BL/6 mice, while human BTLA+ B cells were detectable in the homozygous B-hBTLA mice.

Analysis of splenic leukocyte subpopulations in B-hBTLA mice

Analysis of splenic leukocyte subpopulations by FACS Splenocytes were isolated from female C57BL/6 and B-hBTLA 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-hBTLA mice and C57BL/6 mice, demonstrating that introduction of hBTLA 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 splenic T cell subpopulations in B-hBTLA mice

Analysis of splenic T cell subpopulations by FACS Splenocytes were isolated from female C57BL/6 and B-hBTLA 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-hBTLA and C57BL/6 mice, demonstrating that introduction of hBTLA 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 in B-hBTLA mice

Analysis of lymph node leukocyte subpopulations by FACS Leukocytes were isolated from female C57BL/6 and B-hBTLA mice (n=3, 6 week-old) Flow cytometry analysis of the leukocytes was performed 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 and NK cells were similar in homozygous B-hBTLA mice and C57BL/6 mice, demonstrating that introduction of hBTLA 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 in B-hBTLA mice

Analysis of lymph node T cell subpopulations by FACS Leukocytes were isolated from female C57BL/6 and B-hBTLA mice (n=3, 6 week-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-hBTLA and C57BL/6 mice, demonstrating that introduction of hBTLA 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.

T cells in B-hBTLA mice bind anti-human BTLA antibody

Analysis of splenocytes of B-hBTLA mice by FACS. Splenocytes were isolated from homozygous B-hBTLA mice. Flow cytometry analysis of the splenocytes was performed to assess anti-human BTLA Ab1 binding with T cells. Single live cells were gated for CD45 population and used for further analysis as indicated here. T cell in homozygous B-hBTLA mice binds well with hBTLA Ab1 (in house) vs isotype control.

Application

In vivo efficacy of anti-human BTLA antibodies

Antitumor activity of anti-human BTLA antibodies in B-hBTLA mice. (A) Anti-human BTLA antibodies inhibited MC38 tumor growth in B-hBTLA mice. Murine colon cancer hHVEM MC38 cells were subcutaneously implanted into homozygous B-hBTLA mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with two anti-human BTLA antibodies with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, anti-human BTLA antibodies were efficacious in controlling tumor growth in B-hBTLA mice, demonstrating that the B-hBTLA mice provide a powerful preclinical model for in vivo evaluation of anti-human BTLA antibodies. Values are expressed as mean ± SEM.

Antitumor activity of anti-human BTLA antibodies in B-hBTLA mice. (A) Anti-human BTLA antibodies inhibited MC38 tumor growth in B-hBTLA mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hBTLA mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with two anti-human BTLA antibodies with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, anti-human BTLA antibodies were efficacious in controlling tumor growth in B-hBTLA mice, demonstrating that the B-hBTLA mice provide a powerful preclinical model for in vivo evaluation of anti-human BTLA antibodies. Values are expressed as mean ± SEM.

 

 

References

1. J Immunol. 2005 Mar 15;174(6):3377-85.
2. Graefes Arch Clin Exp Ophthalmol. 2012 Feb;250(2):289-95. doi: 10.1007/s00417-011-1695-8. Epub 2011 Jul 22.
3. J Leukoc Biol. 2010 Feb;87(2):223-35. doi: 10.1189/jlb.0809590. Epub 2009
Dec 9.

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