Basic Information
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Targeting strategy
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Gene targeting strategy for B-hTSLP/hTSLPR mice plus. 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.
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Protein expression analysis of TSLPR in spleen
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Mouse and human TSLPR expression analysis in splenocytes. Splenocytes were collected from wild type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. TSLPR expressed on cDC, pDC and non DCs were analyzed by flow cytometry with species-specific anti-TSLPR antibody. Mouse TSLPR were detectable on cDC, pDC and non DCs of wild type C57BL/6 mice, but not on the cells of TSLPR humanized mice. Human TSLPR was detectable on cDC, pDC and non DCs of B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus, but not on the cells of wild type C57BL/6 mice.
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Protein expression analysis of TSLPR in bone marrow
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Mouse and human TSLPR expression analysis in bone marrow. Bone marrow cells were collected from wild type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. TSLPR expressed on bone marrow cells were analyzed by flow cytometry with species-specific anti-TSLPR antibody. Mouse TSLPR were detectable on cDC, pDC and non DCs in wild type C57BL/6 mice, but not on the cells of TSLPR humanized mice. Human TSLPR was detectable on cDC, pDC and non DCs of B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus, but not on the cells of wild type C57BL/6 mice.
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Protein expression analysis of hTSLPR in cDC1 from bone marrow
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Human TSLPR expression analysis in cDC1 from bone marrow. Bone marrow were collected from wild type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. Human TSLPR expressed on cDC1 were analyzed by flow cytometry with species-specific antibodies. Human TSLPR was highly expressed on the cDC1 in B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus.
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Functional analysis
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Mouse TARC was respectively induced with human TSLP and mouse TSLP in wild type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. Dendritic cells were respectively induced with FLT3L from bone marrow of the three strains and stimulated with human TSLP or mouse TSLP in vitro. Concentration of mouse TARC secreted from DCs was assayed by ELISA. Mouse TARC was successfully induced with human TSLP, but not mouse TSLP in homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. The level of mTARC in B-hTSLP/hTSLPR mice plus was higher than that in B-hTSLP/hTSLPR mice plus. Meanwhile mouse TARC was successfully induced with mouse TSLP, but not human TSLP in wild-type C57BL/6 mice. Results indicated that TSLP and TSLPR are not cross-reactive between mouse and human. Human TSLP can activate the dendritic cells of B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus.
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In vivo efficacy of anti-human TSLP antibody
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Analysis of immune cells in BALF by FACS.
Male B-hTSLP/hTSLPR mice plus were immunized with OVA to induce mouse asthma model (n=6). Anti-human TSLP antibody (Tezepelumab, synthesized in house) was given during the sensitization phase. BALF was collected at the end of the experiment to detect infiltrated inflammatory cells in lung tissue. The results showed that the proportion of eosinophils in OVA induced but untreated group (G2) was significantly higher than that in the non-induced group (G1), while the proportion of these cells in the treated group (G3) decreased significantly when compared with the G2 group. The number of CD45+ cells and eosinophils in the G3 group also tended to decline. Data indicated that anti-human TSLP antibodies could effectively reduce the number and proportion of eosinophils in B-hTSLP/hTSLPR mice plus induced with OVA.
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In vivo efficacy of anti-human TSLP antibody
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OVA specific IgE production in serum of mouse asthma model
Serum was collected at the study endpoint. IgE levels responded to OVA-specific antibody were analyzed. The results showed that the levels of IgE in mice treated with tezepelumab (in house) was lower than that in untreated mice.
Data to be released:
Concentration of total IgE in serum; HE staining of lung tissue
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Summary
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Protein expression analysis:
- Mouse TSLPR was detectable on cDC, pDC and non DCs from spleen and bone marrow of wild type C57BL/6 mice.
- Human TSLPR was detectable on cDC, pDC and non DCs from spleen and bone marrow of B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus.
Functional analysis
- TSLP and TSLPR are not cross-reactive between mouse and human.
- Human TSLP can activate the dendritic cells of B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. Mouse TSLP can activate the dendritic cells of wild-type C57BL/6 mice.
In vivo efficacy：
Anti-human TSLP antibody was efficacious in reducing eosinophils and IgE induced by OVA in B-hTSLP/hTSLPR mice plus.
