Gene targeting strategy for B-hIL4/hIL4RA/hTSLP/hTSLPR plus 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 mice plus. 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 mouse exon 2.The targeted mice will express the chimeric TSLPR protein, while mouse TSLPR will no longer express in B-hTSLP/hTSLPR mice plus. The exons 1-4 of mouse Il4 gene that encode the full length coding sequence were replaced by human IL4 exons 1-4 in B-hIL4/hIL4RA mice. The exons 4-7 of mouse Il4ra gene that encode the extracellular region coding sequences were replaced by human IL4RA exons 4-7 in B-hIL4/hIL4RA mice. B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice was developed by cross-mating the B-hTSLP/hTSLPR mice plus and the B-hIL4/hIL4RA mice together.

Strain specific IL4 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (n=3, 8-week-old, male) and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice(n=3, 8-week-old, male) that stimulated with anti-mouse CD3ε  (7.5 μg/mice, i.p.) in vivo for 4 hrs, and analyzed by ELISA with species-specific IL4 ELISA kit (mIL4 kit: BioLegend 431104; hIL4 kit:BioLegend 430304). Mouse IL4 was only detectable in wild-type C57BL/6JNifdc  mice. Human IL4 was only detectable in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice. Values are expressed as mean ± SEM.

Strain specific IL4RA expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice by flow cytometry. Splenocytes were stimulated with 2 μg/mL anti-mouse CD3ε (BioXcell, BE0001) and 5 μg/mL anti-mouse CD28 (BioXcell, BE0015) in vitro for 3 days. Protein expression was analyzed with anti-human IL4RA antibody (BD, 552178) by flow cytometry. Mouse IL4RA was only detectable in wild-type C57BL/6JNifdc mice. Human IL4RA was only detectable in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice.

Strain specific TSLP expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice by ELISA. Ear was collected from wild-type C57BL/6JNifdc mice and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice (male, n=3, 8-week-old) topically applied MC903 (0.2mmol/L, 0.2mL) in ear for 7 days. Expression level of mouse and human TSLP were analyzed by ELISA (mouse TSLP ELISA kit: BioLegend, 434107; human TSLP ELISA kit: BioLegend, 434207). Mouse TSLP was only detectable in wild-type C57BL/6JNifdc mice. Human TSLP was only detectable in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice. Values are expressed as mean ± SEM.

Strain specific TSLPR expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice by flow cytometry. To generate DCs in vitro, bone marrow cells were isolated and induced with 200 ng/mL FLT3L (acrobiosystems, FLL-H5218) for 6 days. Purified  DCs were stimulated with 1 μg/mL LPS(sigma, L4391). Protein expression was analyzed with anti-mouse TSLPR antibody (Biolegend, 151805) and anti-human TSLPR antibody (Biolegend, 322805) by flow cytometry. Mouse TSLPR was only detectable in wild-type C57BL/6JNifdc mice. Human TSLPR was only detectable in homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice . Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 8-week-old) and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice(male, n=3, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells,neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 8-week-old) and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice(male, n=3, 8-week-old). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph nodes by flow cytometry. Lymph nodes cells were isolated from wild-type C57BL/6JNifdc mice (male, n=3, 8-week-old) and homozygous B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice(male, n=3, 8-week-old). A. Flow cytometry analysis of the lymph nodes cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice were similar to those in C57BL/6JNifdc mice. 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 immune cells in BALF by flow cytometry. B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice (female, 6-8 week-old) were OVA etc inducer asthma mouse model. Anti-human IL4RA antibody combined with anti-human TSLP antibody were intraperitoneal injection. Broncheoalveolar fluid (BALF) was collected at the end of the experiment to detect inflammatory cell infiltration in lung tissue. The results showed that the number and frequency of eosinophils induced in the treated modeling group (G2) was significantly higher than that in the un-modeling group (G1), while the number and frequencies of these cells in the treated groups (G4-G6) decreased significantly when compared with the treated modeling group (G2). Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001. Note：The antibody is provided by RegeneCore Biotech Co., Ltd

Total IgE in serum was significantly reduced in the mouse asthma model treated with Anti-human IL4RA antibody combined with anti-human TSLP antibody . Serum was collected at the study endpoint. IgE levels was analyzed by ELISA. The result showed that the levels of total IgE treated groups (G4-G6) were significantly lower than treated modeling group (G2). Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

H&E staining of asthma model in B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that compared to the un-modeling group (G1), the modeling group (G2) showed significant  increased in inflammatory infiltration and mucus secretion in lung tissue. The treated groups (G5-6) showed significant reduction  in inflammatory infiltration and eosinophils  in lung tissue. Red arrow: inflammatory cells; Red triangle : mucus. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

PAS staining of asthma  model in B-hIL4/hIL4RA/hTSLP/hTSLPR plus mice Lung tissues were collected at the study endpoint and analyzed with PAS staining. The results showed that compared to the un-modeling group (G1), the modeling group (G2) showed a downward trend significant reduction in PAS positive signals in lung tissue. The treated groups (G5-6) showed a downward trend in PAS positive signals in lung tissue. Black arrows indicate PAS positive signals. Values are expressed as mean ± SEM.