Basic Information
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Gene Targeting Strategy
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Gene targeting strategy for B-hIL27/hIL27RA mice. The mouse Il27 gene was replaced by full coding region gene sequence of human IL27 in B-hIL27/hIL27RA mice. The extracellular gene sequences of mouse Il27ra gene were replaced with human IL27RA counterpart gene in B-hIL27/hIL27RA mice.
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Protein Expression Analysis
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Strain specific IL27 expression analysis in homozygous B-hIL27/hIL27RA mice by ELISA. BMDC culture supernatant was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIL27/hIL27RA mice (H/H,H/H) stimulated with LPS（1μg/ml）, and analyzed by ELISA with species-specific IL27 ELISA kit. Mouse IL27 was exclusively detectable in wild-type mice. Human IL27 was detectable in homozygous B-hIL27/hIL27RA mice but not in wild-type mice.
Strain specific IL27RA expression analysis in homozygous B-hIL27/hIL27RA mice by flow cytometry. CD4+ T cells were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIL27/hIL27RA mice (H/H,H/H) stimulated with anti-CD3ε and anti-CD28 in vitro for 48h, and analyzed by flow cytometry with species-specific anti-IL27RA antibody. Mouse IL27RA was exclusively detectable in the wild-type mice. Human IL27RA was detectable in homozygous B-hIL27/hIL27RA mice but not in wild-type mice.
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Intracellular staining of pSTAT1 induced by IL27
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Intracellular pSTAT1 analysis in homozygous B-hIL27/hIL27RA mice by flow cytometry. T cells were isolated from spleen of wild-type mice (+/+) and homozygous B-hIL27/hIL27RA mice (H/H,H/H). CD4+ T cells were purified using magnetic beads then stimulated with mouse or human IL27 in vitro for 15min, and analyzed by flow cytometry with anti-p-STAT1 antibodies. Both mouse and human IL27 can induce STAT1, which means that IL27RA humanized didn’t affect the function of receptor.
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Analysis of Splenic Leukocyte Subpopulations in Humanized B-hIL27/hIL27RA Mice
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Analysis of splenic leukocyte subpopulations in humanized B-hIL27/hIL27RA mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL27/hIL27RA mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess splenic leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45 and used for further analysis as indicated. (B) Percent of T cells, B cells, NK cells, monocytes, dendritic cells, granulocytes and macrophages in homozygous B-hIL27/hIL27RA mice were similar to those in wild-type mice, demonstrating that hIL27/hIL27RA in place of their murine counterparts does not change the overall development, differentiation or distribution of splenic leukocytes. Values are expressed as mean ± SEM.
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Analysis of Splenic T Cell Subpopulations in Humanized B-hIL27/hIL27RA Mice
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Analysis of splenic T cell subpopulations in humanized B-hIL27/hIL27RA mice. Splenic lymphocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL27/hIL27RA mice (n=3, 6-week-old), and analyzed by flow cytometry to assess T cell subpopulations. There were no differences between wild-type and B-hIL27/hIL27RA mice, demonstrating that hIL27/hIL27RA in place of their murine counterparts does not change the overall development, differentiation or distribution of splenic T cell subtypes. Values are expressed as mean ± SEM.
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Analysis of Lymph Node Leukocyte Subpopulations in Humanized B-hIL27/hIL27RA Mice
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Analysis of lymph node leukocyte subpopulations in humanized B-hIL27/hIL27RA mice. Lymphocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL27/hIL27RA (H/H) mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess lymph node leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45 and used for further analysis as indicated. (B) Percent of T cells, B cells and NK cells in B-hIL27/hIL27RA mice were similar to those in wild-type mice, demonstrating that hIL27/hIL27RA in place of their murine counterparts does not change the overall development, differentiation or distribution of lymph node leukocytes. Values are expressed as mean ± SEM.
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Analysis of Lymph Node T Cell Subpopulations in Humanized B-hIL27/hIL27RA Mice
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Analysis of lymph node T cell subpopulations in humanized B-hIL27/hIL27RA mice. Lymphocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL27/hIL27RA (H/H) mice (n=3, 6-week-old), and analyzed by flow cytometry to assess T cell subpopulations. There were no differences between wild-type and B-hIL27/hIL27RA mice, demonstrating that hIL27/hIL27RA in place of their murine counterparts does not change the overall development, differentiation or distribution of lymph node T cell subtypes. Values are expressed as mean ± SEM.
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Analysis of Blood Leukocyte Subpopulations in Humanized B-hIL27/hIL27RA Mice
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Analysis of blood leukocyte subpopulations in humanized B-hIL27/hIL27RA mice. Blood cells were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL27/hIL27RA (H/H) mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess blood leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45 and used for further analysis as indicated. (B) Percent of T cells, B cells, NK cells, monocytes, dendritic cells, granulocytes and macrophages in homozygous B-hIL27/hIL27RA mice were similar to those in wild-type mice, demonstrating that hIL27/hIL27RA in place of their murine counterparts does not change the overall development, differentiation or distribution of blood leukocytes. Values are expressed as mean ± SEM.
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Analysis of Blood T Cell Subpopulations in Humanized B-hIL27/hIL27RA Mice
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Analysis of blood T cell subpopulations in humanized B-hIL27/hIL27RA mice. Blood cells were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL27/hIL27RA (H/H) mice (n=3, 6-week-old), and analyzed by flow cytometry to assess blood T cell subpopulations. There were no differences between wild-type and B-hIL27/hIL27RA mice, demonstrating that hIL27/hIL27RA in place of their murine counterparts does not change the overall development, differentiation or distribution of blood T cell subtypes. Values are expressed as mean ± SEM.
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In vivo efficacy of anti-IL27RA antibodies
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Antitumor activity of anti-IL27RA antibodies in B-hIL27/hIL27RA mice. (A) Anti-IL27RA antibodies inhibit MC38 tumor growth in B-hIL27/hIL27RA mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hIL27/hIL27RA mice (female, 6-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-IL27RA antibodies with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-IL27RA antibodies inhibit MC38 tumor growth in B-hIL27/hIL27RA mice. Values are expressed as mean ± SEM.
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Summary
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Protein expression analysis:
1.Human IL27 was detectable in homozygous B-hIL27/hIL27RA mice but not in wild-type mice.
2.Human IL27RA was detectable in homozygous B-hIL27/hIL27RA mice but not in wild-type mice.
Function analysis:
Both mouse and human IL27 can induce STAT1 and STAT3 activation, which means that IL27RA humanized didn’t affect the function of receptor.
Leukocytes cell subpopulation analysis：
IL27 and IL27RA humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, blood and lymph node.
In vivo efficacy:
Anti-IL27RA antibodies inhibit MC38 tumor growth in B-hIL27/hIL27RA mice.
