Gene targeting strategy for B-hTSLP/hTSLPR/hIL33/hST2 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-hTSLP/hTSLPR/hIL33/hST2 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 that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hTSLP/hTSLPR/hIL33/hST2 mice. The promoter 5’UTR and 3’UTR region of the mouse gene are retained. The human IL33 expression is driven by endogenous mouse IL33 promoter, while mouse Il33 gene transcription and translation will be disrupted. The exons 2-9 of mouse St2 gene that encode signal peptide, extracellular domain are replaced by human counterparts in B-hTSLP/hTSLPR/hIL33/hST2 mice. The genomic region of mouse St2 gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric ST2 expression is driven by endogenous mouse St2 promoter, while mouse St2 gene transcription and translation will be disrupted.
• The B-hTSLP/hTSLPR/hIL33/hST2 mice were obtained by breeding B-hTSLP/hTSLPR mice with B-hIL33/hST2 mice.

Strain specific ST2 expression analysis in homozygous B-hTSLP/hTSLPR/hIL33/hST2 mice by flow cytometry. Peritoneal lavage fluid was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR/hIL33/hST2 mice (H/H;H/H;H/H;H/H), and analyzed by flow cytometry with species-specific anti-mouse ST2 antibody (Biolegend, 145305) and anti-human ST2 antibody (Biolegend, 379403). Mouse ST2 was only detectable in wild-type C57BL/6JNifdc mice. Human ST2 was exclusively detectable in homozygous B-hTSLP/hTSLPR/hIL33/hST2 mice, but not in wild-type C57BL/6JNifdc mice.

Analysis of immune cells in BALF. B-hTSLP/hTSLPR/hIL33/hST2 mice (female, 8-week-old, n=5) were immunized with OVA etc. inducer to induce asthma. Anti-human  ST2 antibody (synthesized in-house) and anti-human TSLP antiboday (tezepelumab analog, synthesized in-house) were intraperitoneally injected to B-hTSLP/hTSLPR/hIL33/hST2 mice. (A&B) The number of CD45+ cells and eosinophils of BALF in the anti-human  ST2 antibody and the combination therapy group of anti-human  ST2 antibody and tezepelumab analog treated groups decreased significantly compared with the 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-hTSLP/hTSLPR/hIL33/hST2 mice (female, 8-week-old, n=5) were immunized with OVA etc. inducer to induce asthma. Anti-human ST2 antibody (synthesized in-house) and anti-human TSLP antiboday (Tezepelumab analog, synthesized in-house) were intraperitoneally injected to B-hTSLP/hTSLPR/hIL33/hST2 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 anti-human ST2 antibody, tezepelumab analog or the combination therapy group of anti-human ST2 antibody and tezepelumab analog 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-hTSLP/hTSLPR/hIL33/hST2 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 anti-human ST2 antibody and the combination therapy group of anti-human ST2 antibody and tezepelumab analog in inflammatory infiltration and mucus secretion in lung tissue was lower than that in untreated mice, indicating that B-hTSLP/hTSLPR/hIL33/hST2 mice provide a powerful preclinical model for in vivo evaluation of anti-human ST2 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.