C57BL/6-Il4tm2(IL4)BcgenIl4ratm1(IL4RA)Bcgen/Bcgen • 120551
Biocytogen's B-hIL4/hIL4RA mice are double humanized IL-4/IL-4Rα mouse models engineered for preclinical research on human cytokine signaling, particularly in allergic inflammation. In these models, exons 1–4 of the mouse Il4 gene and exons 4–7 of the Il4ra gene are replaced with human IL4 and IL4RA counterparts, resulting in exclusive expression of human IL-4 and IL-4Rα proteins.
These mice maintain normal immune cell profiles and enable functional studies of human immune responses. Splenic B cells respond to LPS and human IL-4 stimulation by producing IgE—a response effectively blocked by the IL-4Rα antagonist dupilumab. The B-hIL4/hIL4RA mice are ideal for evaluating therapeutic antibodies targeting the human IL-4/IL-4Rα axis in asthma, atopic dermatitis, and other Th2-mediated diseases.
Gene targeting strategy of IL4/IL4RA humanized mice.
In IL4/L4RA humanized mice, the exons 1–4 of the mouse Il4 gene, which encode the full-length coding sequence, were replaced with the corresponding human IL4 exons 1–4. Similarly, the exons 4–7 of the mouse Il4ra gene, which encode the extracellular region, were replaced with human IL4RA exons 4–7. This targeted replacement strategy ensures strain-specific expression of human IL4 and IL4RA, providing a valuable mouse model for immunology and cytokine research.
Strain-specific IL4 and IL4RA gene expression analysis in wild-type and IL4/IL4RA humanized mice by RT-PCR. Mouse Il4 and Il4ra mRNA were exclusively detectable in splenocytes of wild-type C57BL/6 mice, but not in homozygous B-hIL4/hIL4RA mice. In contrast, human IL4 and IL4RA mRNA were exclusively detectable in homozygous IL4/IL4RA humanized mice, but absent in wild-type controls. This confirms successful humanization and strain-specific expression of the IL4/IL4RA genes.
Strain-specific IL4 expression analysis in IL4/IL4RA humanized mice by ELISA. Serum samples were collected from wild-type C57BL/6 mice (+/+) and homozygous IL4/IL4RA humanized mice (H/H) following in vivo stimulation with anti-mouse CD3ε antibody (37.5 μg/mL, 200 μL/mouse, i.p.) for 1.5 hours (female, 6–8 weeks old, n=3). Protein levels were measured using anti-mouse IL4 antibody (BioLegend, 431104) and anti-human IL4 antibody (BioLegend, 430307) by ELISA. Mouse IL4 was detectable only in wild-type C57BL/6 mice, while human IL4 was exclusively detectable in IL4/IL4RA humanized mice and absent in wild-type controls. ND: Not detected.
Strain-specific IL4RA expression analysis in IL4/IL4RA humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous IL4/IL4RA humanized mice (H/H) after in vivo stimulation with anti-mouse CD3ε antibody (37.5 μg/mL, 200 μL/mouse, i.p.) for 1.5 hours (female, 8-week-old, n=2). Protein expression was assessed by flow cytometry using anti-mouse IL4RA antibody (BioLegend, 144804) and anti-human IL4RA antibody (BioLegend, 355003). Mouse IL4RA expression was detectable only in wild-type C57BL/6 mice, while human IL4RA expression was exclusively detectable in IL4/IL4RA humanized mice and absent in wild-type controls.
Flow cytometry analysis of spleen leukocyte subpopulations in IL4/IL4RA humanized mice. Splenocytes were isolated from female wild-type C57BL/6 mice and homozygous IL4/IL4RA humanized mice (n=3, 10-week-old). Flow cytometry (FACS) was performed to assess major leukocyte subpopulations. A. Representative FACS plots: Single live cells were gated for CD45⁺ leukocytes and further analyzed as indicated. B. Quantitative results: The percentages of T cells, B cells, NK cells, monocytes, dendritic cells (DCs), and macrophages in IL4/IL4RA humanized mice were comparable to those in wild-type C57BL/6 mice. These results demonstrate that replacement of mouse Il4 and Il4ra with their human counterparts does not alter spleen leukocyte development, differentiation, or distribution.
Flow cytometry analysis of spleen T cell subpopulations in IL4/IL4RA humanized mice. Splenocytes were isolated from female wild-type C57BL/6 mice and homozygous IL4/IL4RA humanized mice (n=3, 10-week-old). Flow cytometry (FACS) was performed to assess T cell subpopulations. A. Representative plots: Single live CD45⁺ cells were gated for CD3⁺ T cells and further analyzed. B. Quantitative results: The percentages of CD8⁺, CD4⁺, and regulatory T cells (Tregs) in IL4/IL4RA humanized mice were comparable to those in wild-type C57BL/6 mice, demonstrating that replacement of mouse Il4 and Il4ra with human IL4 and IL4RA does not alter spleen T cell development, differentiation, or distribution. Data are presented as mean ± SEM.
Flow cytometry analysis of lymph node leukocyte subpopulations in IL4/IL4RA humanized mice.
Lymph nodes were isolated from female wild-type C57BL/6 mice and homozygous IL4/IL4RA humanized mice (n=3, 10-week-old). Flow cytometry (FACS) was performed to assess leukocyte subpopulations. A. Representative plots: Single live CD45⁺ cells were gated for further analysis. B. Quantitative results: The percentages of T cells, B cells, and NK cells in IL4/IL4RA humanized mice were comparable to those in wild-type C57BL/6 mice, indicating that replacement of mouse Il4 and Il4ra with human IL4 and IL4RA does not alter lymph node leukocyte development, differentiation, or distribution. Data are presented as mean ± SEM.
Flow cytometry analysis of lymph node T cell subpopulations in IL4/IL4RA humanized mice. Lymph nodes were isolated from female wild-type C57BL/6 mice and homozygous IL4/IL4RA humanized mice (n=3, 10-week-old). Flow cytometry (FACS) was performed to assess T cell subpopulations. A. Representative plots: Single live CD45⁺ T cells were gated for further analysis. B. Quantitative results: The percentages of CD8⁺, CD4⁺, and regulatory T cells (Tregs) in IL4/IL4RA humanized mice were comparable to those in wild-type C57BL/6 mice, indicating that replacement of mouse Il4 and Il4ra with human IL4 and IL4RA does not alter lymph node T cell development, differentiation, or distribution. Data are presented as mean ± SEM.
Dupilumab inhibits IL4-induced IgE secretion in IL4/IL4RA humanized mice.
(A) Splenic B cells from wild-type C57BL/6 mice and homozygous IL4/IL4RA humanized mice were cultured with LPS alone or in combination with 50 ng/mL mouse IL4 (mIL4) or human IL4 (hIL4). Supernatants were collected on day 6 and IgE levels were quantified by ELISA. (B) Splenic B cells from IL4/IL4RA humanized mice were pretreated with increasing doses of dupilumab (in-house) for 30 minutes before stimulation with LPS and hIL4 (50 ng/mL). Supernatants were collected on day 6 for IgE quantification by ELISA. Results demonstrate that B cells from IL4/IL4RA humanized mice responded to LPS and IL4 with IgE production similar to C57BL/6 mice, and that dupilumab effectively blocked IL4-driven IgE secretion.
Dupilumab inhibits hIL4-induced B cell activation in IL4/IL4RA humanized mice. Splenocytes were isolated from IL4/IL4RA humanized mice and incubated with varying concentrations of human IgG4 control or dupilumab for 30 minutes, followed by stimulation with human IL4 (50 pM) for 72 hours. B cell activation was assessed by flow cytometry (FACS). Dupilumab effectively blocked B cell activation in a dose-dependent manner, whereas the control IgG4 antibody showed no inhibitory effect.
Dupilumab reduces BALF immune cell infiltration in IL4/IL4RA humanized mouse asthma model. Bronchoalveolar lavage fluid (BALF) immune cells were isolated from homozygous IL4/IL4RA humanized mice (n=4–5) in an OVA-induced asthma model. Flow cytometry (FACS) was used to analyze the number and proportion of eosinophils. Treatment groups included PBS, control hIgG4 (25 mg/kg, i.p., twice weekly), and dupilumab (25 mg/kg, i.p., twice weekly). Dupilumab treatment significantly reduced the number of CD45⁺ cells and eosinophils compared with the positive control, demonstrating effective inhibition of airway inflammation in IL4/IL4RA humanized mice.
Dupilumab reduces OVA-specific and total IgE production in IL4/IL4RA humanized mouse asthma model. OVA-specific and total IgE levels were measured in bronchoalveolar lavage fluid (BALF) and serum collected from homozygous IL4/IL4RA humanized mice in an OVA-induced asthma model. Mice were treated with PBS, control hIgG4 (25 mg/kg, i.p., twice weekly), or dupilumab (25 mg/kg, i.p., twice weekly).
H&E staining reveals lung inflammation and dupilumab efficacy in IL4/IL4RA humanized mouse asthma model. Lung tissues were collected at the study endpoint from homozygous IL4/IL4RA humanized mice. Mice treated with PBS aerosols (G1) showed no signs of inflammation. OVA exposure (G2) induced significant peribronchial and perivascular inflammation, mucus production, and infiltration of inflammatory cells, including eosinophils. In contrast, dupilumab treatment (G3, in-house) markedly reduced eosinophil infiltration and airway inflammation compared with OVA-challenged controls.
The asthma model was established in IL4/IL4RA humanized mice by HDM sensitization and challenge. Bronchoalveolar lavage fluid (BALF) was analyzed by flow cytometry. (A) Number of CD45⁺ cells. (B) Number of eosinophils. (C) Proportion of eosinophils among CD45⁺ cells. HDM exposure (G2) significantly increased leukocyte infiltration and eosinophil counts compared with PBS controls (G1), confirming successful model establishment. Treatment with dupilumab (25 mg/kg, in-house, G3) markedly reduced CD45⁺ cells and eosinophil infiltration compared with the HDM model group, demonstrating effective suppression of airway inflammation.
Dupilumab reduces total serum IgE in HDM-induced asthma model using IL4/IL4RA humanized mice. Serum samples were collected at the study endpoint from IL4/IL4RA humanized mice in an HDM-induced asthma model. Total IgE levels were quantified by ELISA. HDM exposure (G2) significantly increased serum IgE compared with PBS controls (G1), confirming successful model establishment. Dupilumab treatment (25 mg/kg, in-house, G3) markedly reduced serum IgE levels compared with the HDM group, demonstrating effective inhibition of IL4/IL4RA-mediated IgE production.
Experimental schedule for induction of AD-like skin lesions and dupilumab efficacy in IL4/IL4RA humanized mice. An AD-like skin inflammation model was established in IL4/IL4RA humanized mice using oxazolone (OXZ). On day 0, mice were sensitized with 0.8% OXZ applied to dorsal and ear skin, followed by repeated challenges with 0.4% OXZ (25 μL/ear or dorsal) from days 7 to 26. Dupilumab (anti-human IL4RA antibody, in-house) was administered intraperitoneally twice weekly from days 6 to 23. Serum, ear, and dorsal skin were collected at the endpoint (day 26) for analysis.
The in vivo efficacy of dupilumab (anti-human IL4RA antibody, in-house) was evaluated in an oxazolone (OXZ)-induced atopic dermatitis (AD) model using IL4/IL4RA humanized mice. Mice received different doses of dupilumab or control hIgG4 (n=5 per group). (A) Body weight changes during treatment. (B) Ear thickness measurements showed OXZ-induced inflammation, with desquamation occurring from day 18 onward. (C) Total serum IgE levels were quantified by ELISA on day 26. Dupilumab treatment reduced ear thickness and total IgE levels in a dose-dependent manner, demonstrating its therapeutic efficacy in the AD-like skin inflammation model.
Dupilumab reduces eosinophil infiltration in ear skin of IL4/IL4RA humanized mouse AD model. The effects of dupilumab (anti-human IL4RA antibody, in-house) were evaluated in an oxazolone (OXZ)-induced AD model using IL4/IL4RA humanized mice (n=5 per group). (A) Hematoxylin and eosin (H&E) staining of ear skin. (B) Eosinophil infiltration scores in the ear epidermis. Eosinophil infiltration was significantly increased in OXZ-treated mice (G2) compared with controls (G1). Dupilumab treatment reduced eosinophil infiltration in a dose-dependent manner, confirming the efficacy of IL4RA blockade in suppressing AD-like skin inflammation. Infiltration scoring: 1 = slight, 2 = mild, 3 = moderate, 4 = severe.
Dupilumab reduces ear inflammation and serum IgE in IL4/IL4RA humanized mouse AD model. The in vivo efficacy of dupilumab (anti-human IL4RA antibody, in-house) was evaluated in an oxazolone (OXZ)-induced AD model using IL4/IL4RA humanized mice (n=5 per group). (A) Body weight changes during treatment. (B) Ear thickness analysis showed OXZ-induced swelling, with desquamation observed after day 18. (C) Total serum IgE levels measured by ELISA on day 26. Dupilumab treatment significantly reduced ear thickness and IgE levels compared with the OXZ + hIgG4 control group, demonstrating effective suppression of AD-like skin inflammation.
Dupilumab reduces eosinophil infiltration in ear skin of IL4/IL4RA humanized mouse AD model. The effects of dupilumab (anti-human IL4RA antibody, in-house) were evaluated in an oxazolone (OXZ)-induced AD model using IL4/IL4RA humanized mice (n=5 per group). (A) Hematoxylin and eosin (H&E) staining of ear skin sections. (B) Quantification of eosinophil infiltration scores in ear epidermis. OXZ challenge (G2) significantly increased eosinophil infiltration compared with vehicle controls (G1). Dupilumab treatment (G3) markedly reduced eosinophil infiltration, confirming its therapeutic efficacy in suppressing AD-like skin inflammation. Infiltration scoring scale: 1 = slight, 2 = mild, 3 = moderate, 4 = severe.
Q1: What are B-hIL4/hIL4RA mice?
A1: These humanized mice express human IL-4 and IL-4RA, making them ideal for studying IL-4/IL-4Rα signaling in allergy, asthma, and immunotherapy research.
Q2: Why use B-hIL4/hIL4RA mice instead of wild-type mice?
A2: Human IL-4–targeting drugs often don’t bind to mouse proteins. B-hIL4/hIL4RA mice enable accurate in vivo testing of antibodies and biologics.
Q3: Do B-hIL4/hIL4RA mice maintain normal immune function?
A3: Yes. They show normal development and distribution of major immune cells, including T cells, B cells, NK cells, and macrophages.
Q4: What research areas benefit from B-hIL4/hIL4RA mice?
A4: These mice are valuable for evaluating anti-IL-4/IL-4Rα therapies, IgE response studies, Th2 immunity, and preclinical testing in asthma and allergy models.
