• IL33 is an epithelial alarmin cytokine that promotes type 2 inflammation and is strongly associated with asthma, allergic inflammation, airway remodeling, and epithelial barrier injury.
• TSLP and TSLPR form a key upstream cytokine-receptor pathway that activates dendritic cells, innate lymphoid cells, and Th2 immune responses in allergic disease.
• IL33/hTSLP/hTSLPR humanized mice (B-hIL33/hTSLP/hTSLPR mice) are triple target-humanized mice carrying human IL33, human TSLP, and a chimeric humanized TSLPR receptor in a C57BL/6 background.
• IL33/hTSLP/hTSLPR humanized mice (B-hIL33/hTSLP/hTSLPR mice) enable in vivo evaluation of anti-human IL33 antibodies, anti-human TSLP antibodies, and combination therapies in inflammation and asthma models.
• Human IL33, human TSLP, and human TSLPR expression were validated by ELISA and flow cytometry, and therapeutic efficacy was assessed in an OVA/hIL33/hTSLP-induced asthma model.

Key Advantages

• Human IL33 and human TSLP expression confirmed after MC903 stimulation
• Human TSLPR expression validated in bone marrow-derived dendritic cells/macrophage-lineage cultures
• Supports anti-human IL33 antibody and anti-human TSLP antibody efficacy studies
• OVA/hIL33/hTSLP-induced asthma model enables BALF, serum IgE, and lung histopathology readouts
• Useful for allergic inflammation, asthma, atopic dermatitis, type 2 inflammation, cytokine biology, and combination therapy studies

Validation

• IL33/TSLP Protein Validation:
  Human IL33 and human TSLP were detectable in homozygous IL33/hTSLP/hTSLPR humanized mice (B-hIL33/hTSLP/hTSLPR mice) after MC903 ear stimulation, while mouse IL33 and mouse TSLP were detected in wild-type controls.
• TSLPR Protein Validation:
  Human TSLPR was detected in homozygous IL33/hTSLP/hTSLPR humanized mice (B-hIL33/hTSLP/hTSLPR mice) by flow cytometry, while mouse TSLPR was detectable in wild-type mice.
• Efficacy Validation:
  Itepekimab analog and Tezepelumab analog reduced asthma-related BALF immune-cell infiltration, serum total IgE, and lung inflammation in OVA/hIL33/hTSLP-induced IL33/hTSLP/hTSLPR humanized mice (B-hIL33/hTSLP/hTSLPR mice).
• Histopathology Validation:
  Lung H&E staining showed reduced inflammatory infiltration and mucus secretion after anti-human IL33 and anti-human TSLP antibody treatment.

Application

• Anti-human IL33 antibody efficacy evaluation
• Anti-human TSLP antibody and TSLPR-pathway studies
• Combination therapy research targeting IL33 and TSLP pathways
• OVA-induced asthma and allergic airway inflammation studies
• Type 2 inflammation, allergic disease, and epithelial cytokine biology research

IL33

• Exons 2-8 of mouse IL33 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in IL33/hTSLP/hTSLPR humanized mice (B-hIL33/hTSLP/hTSLPR mice).
• The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. allowing human IL33 expression to be driven by the native mouse IL33 promoter, while endogenous mouse IL33 transcription and translation are abolished.

TSLP

• Exons 1–5 of the mouse Tslp gene, which encode the entire protein (from ATG to stop codon), are replaced with the corresponding human sequences.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human TSLP expression to be driven by the native mouse Tslp promoter, while endogenous mouse Tslp transcription and translation are abolished.

TSLPR

• A chimeric CDS encoding the human TSLPR extracellular and transmembrane domains fused to the mouse TSLPR cytoplasmic domain, followed by the mouse 3′ UTR and stop codon, is inserted immediately downstream of the mouse Tslpr signal peptide to replace part of exon 2 of the endogenous Tslpr gene.
• Expression of the chimeric TSLPR protein is driven by the native mouse Tslpr promoter, while endogenous mouse Tslpr transcription and translation are disrupted.

• Mouse IL33 was detected exclusively in wild-type C57BL/6 mice
• Human IL33 was detected in homozygous IL33/TSLP/TSLPR humanized mice, but not in wild-type mice.

Strain-specific analysis of mouse and human IL33 protein expression in wild-type C57BL/6 mice and IL33/TSLP/TSLPR humanized mice by ELISA. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6 mice and homozygous IL33/TSLP/TSLPR humanized mice (male, 6 weeks old, n = 3) for 7 consecutive days. Mouse and human IL33 protein levels in ear tissue homogenates were quantified by ELISA using species-specific mouse IL33 (R&D Systems, M3300) and human IL33 (R&D Systems, D3300B) ELISA kits, respectively.

• Mouse TSLP was detected exclusively in wild-type C57BL/6 mice
• Human TSLP was detected in homozygous IL33/TSLP/TSLPR humanized mice, but not in wild-type mice.

Strain-specific analysis of mouse and human TSLP protein expression in wild-type C57BL/6 mice and IL33/TSLP/TSLPR humanized mice by ELISA. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6 mice and homozygous IL33/TSLP/TSLPR humanized mice (male, 6 weeks old, n = 3) for 7 consecutive days. Mouse and human TSLP protein levels in ear tissue homogenates were quantified by ELISA using species-specific mouse TSLP (BioLegend, 434107) and human TSLP (BioLegend, 434207) ELISA kits, respectively.

• Mouse TSLPR was exclusively detectable in wild-type C57BL/6 mice, but not in homozygous IL33/TSLP/TSLPR humanized mice.
• Human TSLPR was exclusively detectable in homozygous IL33/TSLP/TSLPR humanized mice, but not in wild-type C57BL/6 mice.

Flow cytometry analysis of mouse and human TSLPR protein expression in bone marrow cells from wild-type C57BL/6 mice and IL33/TSLP/TSLPR humanized mice. Bone marrow cells were collected from wild-type C57BL/6 mice and homozygous IL33/TSLP/TSLPR humanized mice. Mouse and human TSLPR protein expression on bone marrow cells was analyzed by flow cytometry using species-specific anti-mouse TSLPR (BioLegend, 300312) and anti-human TSLPR (BioLegend, 100312) antibodies.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous IL33/TSLP/TSLPR humanized mice were similar to those in C57BL/6JNifdc.
• Humanization of IL33, TSLP and TSLPR does not affect normal immune cell development or splenic distribution.

Flow cytometry analysis of leukocyte subpopulations in immune organs and peripheral blood of IL33/TSLP/TSLPR humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female wild-type C57BL/6JNifdc mice and IL33/TSLP/TSLPR humanized mice (9 weeks old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous IL33/TSLP/TSLPR humanized mice were comparable to those in C57BL/6JNifdc.
• Humanization of TSLP, TSLPR, and IL7R does not affect normal T cell development, differentiation, or splenic distribution.

Flow cytometry analysis of T-cell subpopulations in immune organs and peripheral blood of IL33/TSLP/TSLPR humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female wild-type C57BL/6JNifdc mice and IL33/TSLP/TSLPR humanized mice (9 weeks old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Growth curve of IL33/TSLP/TSLPR humanized mice. Eight-week-old mice were grouped by sex (10 males and 10 females). Body weight was measured weekly for 12 weeks on the same day each week. The minimum and maximum body weights shown in the table were calculated from the mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of IL33/TSLP/TSLPR humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of IL33/TSLP/TSLPR humanized mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

The organs of female C57BL/6JNifdc and IL33/TSLP/TSLPR humanized mice (32-week-old, n=10).

• No abnormalities were observed.

The organs of male C57BL/6JNifdc and IL33/TSLP/TSLPR humanized mice (32-week-old, n=10).

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, ovary, uterus).

Histopathological analysis of organs in IL33/TSLP/TSLPR humanized mice. Major organs from IL33/TSLP/TSLPR humanized mice were collected at 32 weeks of age and analyzed by H&E staining (female, n = 10).

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, testis).

Histopathological analysis of organs in IL33/TSLP/TSLPR humanized mice. Major organs from IL33/TSLP/TSLPR humanized mice were collected at 32 weeks of age and analyzed by H&E staining (male, n = 10).

Experimental schedule for OVA-induced asthma and in vivo efficacy evaluation of anti-human IL33 and anti-human TSLP antibodies in IL33/TSLP/TSLPR humanized mice. IL33/TSLP/TSLPR humanized mice were administered ovalbumin (OVA) and other agents intranasally to establish an asthma model. An anti-human IL33 antibody (itepekimab analog) and an anti-human TSLP antibody (tezepelumab analog), both produced in-house, were administered via intraperitoneal injection (n = 8).

• CD45⁺ cell and eosinophil counts in bronchoalveolar lavage fluid (BALF) were significantly reduced in the anti-human IL33 antibody (itepekimab analog) monotherapy and itepekimab plus tezepelumab combination therapy groups compared with the OVA-induced PBS-treated group.

Analysis of inflammatory immune cells in bronchoalveolar lavage fluid (BALF) following anti-human IL33 and TSLP antibody treatment in IL33/TSLP/TSLPR humanized mice. Male IL33/TSLP/TSLPR humanized mice (11 weeks old, n = 8) were immunized with ovalbumin (OVA) and other agents to induce an asthma model. Mice received intraperitoneal injections of an anti-human IL33 antibody (itepekimab analog, produced in-house) and an anti-human TSLP antibody (tezepelumab analog, produced in-house). (A–B) CD45⁺ cell and eosinophil counts in BALF were significantly reduced in the itepekimab monotherapy and itepekimab plus tezepelumab combination therapy groups compared with the OVA-induced PBS-treated group. Values are expressed as mean ± SEM. Statistical significance was determined using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

• Itepekimab, tezepelumab or the combination therapy group of itepekimab and tezepelumab treated groups showed a significant reduction compared with untreated mice.

ELISA analysis of serum total IgE levels following anti-human IL33 and TSLP antibody treatment in an OVA-induced asthma model using IL33/TSLP/TSLPR humanized mice. Male IL33/TSLP/TSLPR humanized mice (11 weeks old, n = 8) were immunized with ovalbumin (OVA) and other agents to induce an asthma model. Mice received intraperitoneal injections of an anti-human IL33 antibody (itepekimab analog, produced in-house), an anti-human TSLP antibody (tezepelumab analog, produced in-house), or their combination. Serum samples were collected at the study endpoint, and total mouse IgE levels were measured by ELISA. Treatment with itepekimab, tezepelumab, or their combination significantly reduced serum total IgE levels compared with untreated mice. Values are expressed as mean ± SEM. Statistical significance was determined using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

• Itepekimab and the combination therapy group of Itepekimab and Tezepelumab in inflammatory infiltration and mucus secretion in lung tissue was lower than that in untreated mice

Histopathological analysis of lung inflammation and mucus secretion in an OVA-induced asthma model using IL33/TSLP/TSLPR humanized mice by H&E staining. Lung tissues were collected at the study endpoint and analyzed by hematoxylin and eosin (H&E) staining. Treatment with an anti-human IL33 antibody (itepekimab analog), either alone or in combination with an anti-human TSLP antibody (tezepelumab analog), reduced inflammatory cell infiltration and mucus secretion in lung tissues compared with untreated mice. These results demonstrate the utility of IL33/TSLP/TSLPR humanized mice as a preclinical model for evaluating the in vivo therapeutic efficacy of anti-human IL33 and TSLP antibodies in asthma research. Values are expressed as mean ± SEM. Statistical significance was determined using an unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.