Basic Information
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Details
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Protein expression analysis
Strain specific IL4 expression analysis in homozygous B-hIL4/hIL4RA mice by ELISA.
Serum were collected from WT and homozygous B-hIL4/hIL4RA (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by ELISA with species-specific IL4 ELISA kit. Mouse Il4 was detectable in WT mice. Human IL4 was exclusively detectable in homozygous B-hIL4/hIL4RA mice but not WT mice.
Strain specific IL4RA expression analysis in homozygous B-hIL4/hIL4RA mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hIL4/hIL4RA (H/H) mice, and analyzed by flow cytometry with species-specific anti-IL4RA antibody. Mouse Il4ra was detectable in WT mice. Human IL4RA was exclusively detectable in homozygous B-hIL4/hIL4RA mice but not WT mice.
Phenotypic analysis
Analysis of spleen leukocytes cell subpopulations in B-hIL4/hIL4RA mice
Analysis of spleen leukocyte subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hIL4/hIL4RA mice (n=3, 10-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-hIL4/hIL4RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL4 and hIL4RA 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-hIL4/hIL4RA mice
Analysis of spleen T cell subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hIL4/hIL4RA mice (n=3, 10-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ T 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-hIL4/hIL4RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL4 and hIL4RA 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 Lymph node leukocyte subpopulations in B-hIL4/hIL4RA mice
Analysis of subpopulation of leukocytes in lymph node by FACS
Lymph node were isolated from female C57BL/6 and B-hIL4/hIL4RA mice (n=3, 10-week-old). Flow cytometry analysis of the lymph node was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ T cells were used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, and NK cells in homozygous B-hIL4/hIL4RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL4 and hIL4RA 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-hIL4/hIL4RA mice
Analysis of subpopulation of T cells in lymph node by FACS
Lymph node were isolated from female C57BL/6 and B-hIL4/hIL4RA mice (n=3, 10-week-old). Flow cytometry analysis of the lymph node was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ T cells were used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hIL4/hIL4RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL4 and hIL4RA 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.
IL-4 induced IgE secretion in B-hIL4/hIL4RA mice
(A) Splenic B cells from C57BL/6 and B-hIL4/hIL4RA mice were cultured with LPS alone or together with 50ng/mL mIL-4/hIL-4. Culture supplements were harvested on day 6 for quantification of IgE by ELISA.(B) Splenic B cells from B-hIL4/hIL4RA mice were incubated with increasing doses of dupilumab (in house) for half of an hour before adding LPS and hIL-4 (50 ng/mL). Culture supernatant was harvested on day 6 for quantification of IgE by ELISA. The result show that B cells from B-hIL4/hIL4RA mice responded well to LPS and IL-4 with IgE production, as similar to C57BL/6 mice. Dupilumab (in house) could effectively block the expression of IgE.
In vivo efficacy of anti-human IL4RA antibody with asthma mouse model
The number of BALF immune cells in mouse asthma model
The number of BALF Immune cells in mouse asthma model
BALF Immune cells were isolated from B-hIL4/hIL4RA mice (n=6). The number of eosinophils was analyzed by flow cytometry under the treatment of PBS/dupilumab (in house) before OVA challenge. After treatment of dupilumab (in house), the expression level of inflammatory cells was much lower than the positive control in homozygous B-hIL4/hIL4RA mice.
The number of BALF immune cells in acute mouse asthma model
The proportion of BALF Immune cells in mouse asthma model
BALF Immune cells were isolated from B-hIL4/hIL4RA mice (n=6). The proportion of eosinophils was analyzed by flow cytometry under the treatment of dupilumab (in house) before OVA challenge(G1 was not treatment with OVA). After treatment of dupilumab (in house), the expression level of inflammatory cells was much lower than the positive control in homozygous B-hIL4/hIL4RA mice.
OVA specific and total IgE production in serum and BALF of mouse asthma model
IgE production
Serum was collected at the study endpoint. IgE levels responded to OVA-specific antibody and total IgE levels were analyzed. The results show that the levels of IgE in mice treated with dupilumab (in house) is much lower than that in untreated mice.
H&E staining in asthma-like model in B-hIL4/hIL4RA mice
Airways from B-hIL4/hIL4RA mice exposed to PBS aerosols did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation both in C57BL/6 and B-hIL4/hIL4RA mice, as well as an increase in the level of mucus secretion. A reduction in inflammatory infiltrates and mucus secretion was observed in mice treated with dupilumab (in house).
Experimental schedule for Induction of AD-like skin lesions and in vivo efficacy of anti-human IL4RA antibody
Experimental schedule for Induction of AD-like skin lesions and in vivo efficacy of anti-human IL4R antibody.
OXZ was applied to dorsal and ear skin of mice on day 0, and then challenge to the same site of skin ten times from days 7 to 26. Anti-human IL4RA antibody dupilumab (in house) was administered by intraperitoneal injection twice a week on days 6 to 23. Serum, ear and dorsal skin were collected at the endpoint on day 26. AD: atopic dermatitis; OXZ: oxazolone.
In vivo efficacy of anti-human IL4RA antibody-Experiment 1 with AD model
Efficacy of anti-human IL4RA antibody in B-hIL4/hIL4RA mice. Mice in each group were treated with different dose of dupilumab produced in house. Doses are shown in legend. (A) Body weight changes during treatment. (B) Statistical analysis of ear thickness in each group. Epidermis of ear began to desquamate from day 18. So the ear thickness was decreased from day 18 as shown in figure. (C) Total IgE levels in serum. Total IgE levels were measured by ELISA on day 26. Ear thickness and concentrations of total serum IgE were negative related with the doses of antibody. (n = 5).
H&E staining of ear skin in atopic dermatitis model of B-hIL4/IL4RA mice
Effects of anti-human IL4RA antibody on inflammatory infiltration in ear skin of the AD mouse model. (A) Hematoxylin and eosin (H&E) staining. (B) Score of eosinophils infiltrated in ear epidermal skin (n=5). Infiltration scores of eosinophils in ear skin were negatively related to the doses of antibody, demonstrating that the B-hIL4/hIL4RA mice provide a powerful preclinical model for in vivo evaluation of anti-human IL4RA antibodies. Infiltration score of eosinophils: 1=slight; 2=mild; 3=moderate; 4=severe. AD: Atopic dermatitis; ND: Not detectable.
In vivo efficacy of anti-human IL4RA antibody-Experiment 2 with AD model
Efficacy of anti-human IL4RA antibody in B-hIL4/hIL4RA mice.
Mice in each group were treated with different dose of dupilumab produced in house. Doses are shown in legend. (A) Body weight changes during treatment. (B) Statistical analysis of ear thickness in each group. Epidermis of ear began to desquamate from day 18. So the ear thickness was decreased from day 18 as shown in figure. (C) Total IgE levels in serum. Total IgE levels were measured by ELISA on day 26. Ear thickness and concentrations of total serum IgE were negative related with the doses of antibody. (n = 5).
H&E staining of ear skin in atopic dermatitis model of B-hIL4/IL4RA mice
Effects of anti-human IL4RA antibody on inflammatory infiltration in ear skin of the AD mouse model. (A) Hematoxylin and eosin (H&E) staining. (B) Score of eosinophils infiltrated in ear epidermal skin (n=5). Infiltration scores of eosinophils in ear skin were significantly reduced after administration of the antibodies, demonstrating that the B-hIL4/hIL4RA mice provide a powerful preclinical model for in vivo evaluation of anti-human IL4RA antibodies. Infiltration score of eosinophils: 1=slight; 2=mild; 3=moderate; 4=severe. AD: Atopic dermatitis; ND: Not detectable.
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Publications using B-hIL4/hIL4RA mice
