Gene targeting strategy for B-hIL33/hTSLP/hTSLPR mice.

• The exons 1-5 of mouse Tslp gene that encode the full-length protein were replaced by human TSLP exons 1-4 in B-hIL33/hTSLP/hTSLPR mice.

• The signal peptide, extracellular and transmembrane region of human TSLPR gene and the cytoplasmic region of mouse Tslpr gene were constructed into a chimeric CDS vector and inserted into the exon 2 of mouse Tslpr gene. The targeted mice will express the chimeric TSLPR protein, while mouse TSLPR will no longer express.

• The exons 2-8 of mouse Il33 gene were replaced by human IL33 exons 2-8 in B-hIL33/hTSLP/hTSLPR mice.

Strain specific IL33 and TSLP expression analysis in homozygous B-hIL33/hTSLP/hTSLPR mice by ELISA. Calcipotriol (MC903) was dissolved in ethanol and topically applied on ears of either wild type C57BL/6 mice (+/+) or homozygous B-hIL33/hTSLP/hTSLPR mice (H/H) for 7 days. n=3. Ear grinding supernatant from the two mice was analyzed by ELISA. Mouse IL33 and TSLP were only detectable in wild-type C57BL/6 mice. Human IL33 and TSLP were exclusively detectable in homozygous B-hIL33/hTSLP/hTSLPR mice but not in wild-type mice. ND: not detectable.

Strain specific TSLPR expression analysis in homozygous B-hIL33/hTSLP/hTSLPR mice by flow cytometry. Bone marrow was collected from wild type C57BL/6 mice (+/+) and homozygous B-hIL33/hTSLP/hTSLPR mice (H/H), Dendritic cells were induced from bone marrow cells in culture and analyzed by flow cytometry with species-specific anti-TSLPR antibody. Mouse TSLPR was only detectable in wild type C57BL/6 mice. Human TSLPR was only detectable in homozygous B-hIL33/hTSLP/hTSLPR mice but not in wild type mice.

Analysis of immune cells in BALF. B-hIL33/hTSLP/hTSLPR mice (male, 11-week-old, n=8) were immunized with OVA/hIL33/hTSLP to induce asthma. Anti-human  IL33 antibody (Itepekimab analog, synthesized in-house) and anti-human TSLP antiboday (Tezepelumab analog, synthesized in-house) were intraperitoneally injected from Day -1 and Day 6. (A&B) The number of CD45+ cells and eosinophils of BALF in the Itepekimab and the combination therapy group of Itepekimab and Tezepelumab treated groups decreased significantly compared with the OVA/hIL33/hTSLP-induced PBS treated group. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001. 

Analysis of mouse total IgE in serum. B-hIL33/hTSLP/hTSLPR mice (male, 11-week-old, n=8) were immunized with OVA/hIL33/hTSLP to induce asthma. Anti-human  IL33 antibody (Itepekimab analog, synthesized in-house) and anti-human TSLP antiboday (Tezepelumab analog, synthesized in-house) were intraperitoneally injected to B-hIL33/hTSLP/hTSLPR mice. Serum was collected at the study endpoint. IgE level was analyzed by ELISA. The results showed that the levels of total IgE in mice treated with Itepekimab, Tezepelumab or the combination therapy group of Itepekimab and Tezepelumab treated groups showed a significant reduction compared with untreated mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001. 

H&E staining of asthma-like model in B-hIL33/hTSLP/hTSLPR mice. Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that the group of mice treated with 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, indicating that B-hIL33/hTSLP/hTSLPR mice provide a powerful preclinical model for in vivo evaluation of anti-human IL33 antibodies and anti-human TSLP antibodies. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *P < 0.05, **P < 0.01, ***P < 0.001.