Basic Information
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Details
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1. mRNA expression analysis
RT-PCR analysis of IL33 gene.
The hIL-33, but not mIl-33, mRNA was detectable in splenocytes of the homozygous B-hIL33 mice.
2. Protein expression analysis
Lung abrasive fluid from both wild type (WT) C57BL/6 and homozygous B-hIL33 mice were analyzed by ELISA.
When treated with LPS, mouse IL33 was detectable in the WT mice, while human IL33 was detectable in the homozygous B-hIL33 mice, albeit being at a lower level. ND, not detectable. n=3.
3. Functional analysis
3.1 Analysis of leukocyte subpopulation in spleen of humanized mice
Analysis of leukocyte subpopulation in spleen.
Splenocytes were isolated from C57BL/6 and B-hIL33 mice (n=3). Proportions of leukocytes subpopulation were determined by flow cytometry. As shown in panels here，the expression profile of leukocytes subpopulation in homozygous B-hIL33 mice is similar to that in the C57BL/6 mice, indicating that differentiation of B cells, T cells, NK cells, CD4+ T cells, CD8+ T cells, granulocytes, dendritic cells, macrophages and monocytes are not affected by the humanization of hIL33.
3.2 Analysis of T cell subpopulation in spleen of humanized mice
Analysis of T cell subpopulation in spleen.
Lymphocytes were isolated from spleen in C57BL/6 and B-hIL33 mice (n=3). Proportions of T cell subpopulation were determined by flow cytometry. As shown here, the expression profile of lymphocyte subpopulation in homozygous B-hIL33 mice is similar to that in the C57BL/6 mice, indicating that T cell differentiation is not affected by the humanization of hIL33.
3.3 Analysis of leukocyte subpopulation in blood of humanized mice
Analysis of leukocyte subpopulation in blood.
Blood were isolated from C57BL/6 and B-hIL33 mice (n=3). Proportions of leukocytes subpopulation were determined by flow cytometry. As shown in panels here, the expression profile of leukocytes subpopulation in homozygous B-hIL33 mice is similar to that in the C57BL/6 mice, indicating that differentiation of B cells, T cells, NK cells, CD4+ T cells, CD8+ T cells, granulocytes, dendritic cells, macrophages and monocytes are not affected by the humanization of hIL33.
3.4 Analysis of T cell subpopulation in blood of humanized mice
Analysis of T cell subpopulation in blood
Lymphocytes were isolated from blood in C57BL/6 and B-hIL33 mice (n=3). Proportions of T cell subpopulation were determined by flow cytometry. As shown here, the expression profile of lymphocyte subpopulation in homozygous B-hIL33 mice is similar to that in the C57BL/6 mice, indicating that T cell differentiation is not affected by the humanization of hIL33.
4. Antibody efficacy evaluation
4.1 Model schematic and antibody evaluation scheme
- Blue arrows: time for sensitization and challenge;
- Red arrows: time for drug administration
4.2 The proportion of BALF immune cells in acute mouse asthma model
BALF immune cell profiles in acute mouse asthma model
BALF Immune cells were isolated from B-hIL33 mice (n=5). The proportions of CD45+ cells, eosinophils were deterimined by flow cytometry in acute asthma mice treated with or without etokimab anolog. Treatment of etokimab analog almost completely abolished the inflammatory cells in homozygous B-hIL33 mice as opposed to in untreated mice.
4.3 The number of BALF immune cells in acute mouse asthma model
BALF immune cell profiles in acute mouse asthma model
BALF Immune cells were isolated from B-hIL33 mice (n=5). The number of eosinophils were deterimined by flow cytometry in acute asthma mice treated with or without etokimab anolog. Treatment of etokimab analog almost completely abolished the inflammatory cells in homozygous B-hIL33 mice as opposed to in untreated mice.
4.4 IgE production in mouse asthma model
IgE production
Serum was collected at the study endpoint. IgE levels responded to OVA-specific antibody were analyzed. The results show that the levels of IgE in mice treated with etokimab is much lower than that in untreated mice.
5.H&E stain in mouse asthma model
Airways from B-hIL33 mice exposed to PBS aerosols did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammatory infiltrates, as well as increase in the level of mucus secretion. A reduction in inflammatory infiltrates and mucus secretion was observed in mice treated with etokimab analog.
6. Summary
1. Gene expression: human IL33 was detectable in the homozygous B-hIL33 mice.
2. Protein expression: human IL33 was detectable in the homozygous B-hIL33 mice.
3. Analysis of leukocyte subpopulation: the expression profile of leukocytes subpopulation in homozygous B-hIL33 mice is similar to that in the C57BL/6 mice.
4. Antibody efficacy evaluation: OVA-induced acute asthma model has been successfully established as indicated by increased eosinophilia, IgE production. This model is further validated with etokimab, which greatly reduced the inflammatory cells and IgE.
5. B-hIL33 mice is a validated, valuable tool for in vivo efficacy evaluation of anti-human IL33 antibodies.
