B-hA2AR mice

Basic Information

mRNA expression analysis

 

 

 

 

 

Species-specific A2AR gene expression analysis in wild-type and humanized B-hA2AR mice by RT-PCR. Murine A2ar mRNA was detected in splenocytes isolated from wild-type (+/+) mice, while human A2AR mRNA was exclusively detected in homozygous B-hA2AR mice.

 

Protein expression analysis

Protein expression analysis in T cells

Species-specific A2AR protein expression analysis in humanized B-hA2AR mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hA2AR (H/H) mice stimulated with anti-CD3ε (7.5 μg/mice, stimulation for 24 hours, i.p.) in vivo, and analyzed by flow cytometry using anti-m/hA2AR antibodies. Murine and human A2AR proteins were detected in wild-type and B-hA2AR mice.

Protein expression analysis in CD4+ T cells

Species-specific A2AR protein expression analysis in humanized B-hA2AR mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hA2AR (H/H) mice stimulated with anti-CD3ε (7.5 μg/mice, stimulation for 24 hours, i.p.) in vivo, and analyzed by flow cytometry using anti-m/hA2AR antibodies. Murine and human A2AR proteins were detected in wild-type and B-hA2AR mice.

Functional analysis

 

 

 

 

 

 

 

 

 

cAMP measurements in spleen and bone of wild-type mice and B-hA2AR mice incubated with different concentration of CGS21680 (an A2AR agonist), and analyzed by LANCE® Ultra cAMP Kit (PerkinElmer). The cAMP levels of spleen cells and bone marrow cells in B-hA2AR mice were higher than those in wild-type C57BL/6 mice at 500μM CGS21680. It was speculated that B-hA2AR mice were more sensitive to the stimulation of CGS201680 than wild-type mice. At the background level, there was no significant difference in A2AR mediated cAMP pathway between humanized B-hA2AR and wild-type mice. Compared to wild-type mice, B-hA2AR mice effectively mediated the A2AR-dependent cAMP pathway under strong stimulation.

Analysis of immune cells

Analysis of spleen leukocyte subpopulations in B-hA2AR mice

Analysis of splenic leukocyte subpopulations by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 and humanized B-hA2AR mice (female, n=3, 6 weeks-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots gated on single live CD45+ cells for analysis as indicated. (B) Percent of T, B, NK cells, monocytes/macrophages, and DC were similar between B-hA2AR mice and C57BL/6 mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these spleen cell subtypes. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations in B-hA2AR mice

Analysis of splenic T cell subpopulations by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 and humanized B-hA2AR mice (female, n=3, 6 weeks-old), and analyzed by flow cytometry to assess T cell subsets. (A) Representative flow cytometry plots gated on TCRβ+ T cells and further analyzed. (B) Percent of CD8+ T cells, CD4+ T cells, and Tregs were similar between B-hA2AR mice and wild-type mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these spleen T cell subtypes. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations in B-hA2AR mice

Analysis of blood leukocyte subpopulations by flow cytometry. Blood was isolated from wild-type C57BL/6 and humanized B-hA2AR mice (female, n=3, 6 weeks-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots gated on single live CD45+ cells for further analysis. (B) Percent of T, B, NK cells, monocytes/macrophages, and DC were similar between B-hA2AR mice and wild-type mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these blood cell subtypes. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations in B-hA2AR mice

Analysis of blood T cell subpopulations by flow cytometry. Blood was isolated from wild-type C57BL/6 and humanized B-hA2AR mice (female, n=3, 6 weeks-old), and analyzed by flow cytometry to assess T cell subsets. (A) Representative flow cytometry plots gated on TCRβ+ T cells and further analyzed. (B) Percent of CD8+ T cells, CD4+ T cells, and Tregs were similar between homozygous B-hA2AR mice and wild-type mice, demonstrating that introduction of hA2AR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these blood T cell subtypes. Values are expressed as mean ± SEM.

In vivo efficacy of A2AR small molecule drugs

Antitumor activity of A2AR small molecule drugs in humanized B-hA2AR mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hA2AR mice (female, 8-9 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with different doses of  an A2AR small molecule drug. (B) Body weight changes during treatment. A2AR small molecule drugs were effective at controlling MC38 tumor growth in B-hA2AR mice, demonstrating that B-hA2AR mice provide a powerful preclinical model for in vivo efficacy evaluation of A2AR small molecule drugs. Values are expressed as mean ± SEM.