Basic Information

Strain Name
C57BL/6-Cd28tm1(CD28)Bcgen/Bcgen
Stock Number
110007
Common Name
B-hCD28 Mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
Cd28 (CD28 antigen)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. T cell stimulation through CD28 in addition to the T-cell receptor (TCR) can provide a potent signal for the production of various interleukins (IL-2 and IL-6 in particular). CD28 is the only B7 receptor constitutively expressed on naïve T cells. Association of the TCR of a naive T cell with MHC: antigen complex without CD28: B7 interaction results in a T cell that is anergic. Agonistic antibodies targeting CD28 have entered clinical trials.

Targeting Strategy

Gene targeting strategy for B-hCD28 mice. The exons 2~3 of mouse Cd28 gene that encode the extracellular domain were replaced by human CD28 exons 2~3 in B-hCD28 mice.

Details

Protein expression analysis

Strain specific CD28 expression analysis in heterozygous B-hCD28 mice by flow cytometry. Splenocytes were collected from WT and heterozygous B-hCD28 (H/+) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CD28 antibody. Mouse CD28 was detectable in WT mice. Human CD28 was exclusively detectable in heterozygous B-hCD28 but not WT mice.

Strain specific CD28 expression analysis in homozygous B-hCD28 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCD28 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CD28 antibody. Mouse CD28 was detectable in WT mice. Human CD28 was exclusively detectable in homozygous B-hCD28 but not WT mice.

Analysis of spleen leukocytes cell subpopulations in B-hCD28 mice

Analysis of spleen leukocyte subpopulations by FACS Splenocytes were isolated from female C57BL/6 and B-hCD28 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 CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK, Monocyte, DC and macrophage cells in homozygous B-hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of spleen T cell subpopulations in B-hCD28 mice

Analysis of spleen T cell subpopulations by FACS Splenocytes were isolated from female C57BL/6 and B-hCD28 mice (n=3, 6 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. Percent of CD8, CD4, and Treg cells in homozygous B-hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocytes cell subpopulations in B-hCD28 mice

Analysis of blood leukocyte subpopulations by FACS Blood were isolated from female C57BL/6 and B-hCD28 mice (n=3, 6 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. Percent of T, B, NK, Monocyte, DC and macrophage cells in homozygous B-hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood.

Analysis of blood T cell subpopulations in B-hCD28 mice

Analysis of blood T cell subpopulations by FACS Blood were isolated from female C57BL/6 and B-hCD28 mice (n=3, 6 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. Percent of CD8, CD4, and Treg cells in homozygous B-hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in blood. Values are expressed as mean ± SEM.

Analysis of T cell activation stimulated with anti-CD3 and anti-CD28 antibody in vitro

Analysis of T cell activation stimulated with anti-CD3 and anti-CD28 antibody in vitro. Homozygous B-hCD28 mice were treated with anti-mCD3e antibody and anti-mCD28 antibody or anti-hCD28 antibody as indicated on the graph (female, 6 week-old, n=3). T cells were analyzed at 24, 48 and 72 hours after treatment. Anti-mCD28 antibody resulted in an increased activation of T cells in WT mice, and anti-hCD28 antibody resulted in an increased activation of T cells in B-hCD28 mice. Values are expressed as mean ± SEM.

Analysis of T cell proliferation stimulated with anti-CD3 and anti-CD28 antibody in vitro

Analysis of T cell proliferaion stimulated with anti-CD3 and anti-CD28 antibody in vitro. Homozygous B-hCD28 mice were treated with anti-mCD3e antibody and anti-mCD28 antibody or anti-hCD28 antibody as indicated on the graph (female, 6 week-old, n=3). T cells were analyzed at 48 and 72 hours after treatment. Anti-mCD28 antibody resulted in an increased proliferation of T cells in WT mice, and anti-hCD28 antibody resulted in an increased proliferation of T cells in B-hCD28 mice. Values are expressed as mean ± SEM.

Anti human CD28 therapies led to changed cytokine profile in B-hCD28 mice

T cells from WT, and Homozygous hCD28 mice were treated with anti-mCD3e antibody and anti-mCD28 antibody or anti-hCD28 antibody in vitro. IFN-γ and IL-2 expression levels were analyzed.

Anti-human CD28 therapies led to changed cytokine profile in B-hCD28 mice. Homozygous B-hCD28 mice were treated with anti-mCD3e antibody and anti-mCD28 antibody or anti-hCD28 antibody as indicated on the graph (female, 6 week-old, n=3). Serum cytokine levels were analyzed at 24, 48 and 72 hours after treatment. Anti-mCD28 antibody resulted in an increase of IFN-γ, IL-2 in WT mice, and anti-hCD28 antibodies resulted in an increase of IFN-γ, IL-2 in B-hCD28 mice. Values are expressed as mean ± SEM.

hPD-L1 recombination protein immunization led to unchanged IgG profile in B-hCD28 mice

hPD-L1 recombination protein immunization led to unchanged IgG profile in B-hCD28 mice. C57BL/6 and B-hCD28 mice (female, 6 week-old, n=5) were immunized by hPD-L1 recombination protein with CFA. No difference was found in serum antibody titer in WT and hCD28 mice, indicating that the B7-1/CD28 co-stimulating signaling pathway was normal in hCD28 mice.

In vivo efficacy of anti human CD28 antibodies

Antitumor activity of anti-human CD28 antibodies in B-hCD28 mice. (A) Anti-human CD28 antibodies inhibited MC38 tumor growth in B-hCD28 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCD28 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 anti-human CD28 antibodies with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, two anti-human CD28 antibodies were efficacious in controlling tumor growth in B-hCD28 mice, demonstrating that the B-hCD28 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD28 antibodies. Values are expressed as mean ± SEM.

References

1. FEBS J. 2016 Sep;283(18):3325-34. doi: 10.1111/febs.13754. Epub 2016 Jun 6.
2. Cytokine Growth Factor Rev. 2016 Apr;28:11-9. doi: 10.1016/j.cytogfr.2016.02.004. Epub 2016 Mar 4.
3. Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):E6437-E6446. Epub 2016 Oct 5.

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