hIL2/hIL2RA/hIL2RB/hIL2RG

Fig1. Detection of hIL-2 expression by ELISA in the hIL-2 knockin mice (Wild type and heterozygous hIL-2 KI mice were administrated with concanavalin).

Fig2. Intracellular staining of IL-2 by FACS post stimulation with anti-mCD3/anti-mCD28 (In collaboration with CrownBio).

Fig3. hlL-2RA is highly expressed on activated Tregs in spleen in the IL-2RA huamnzied mouse (In collaboration with CrownBio).

Fig4. hlL-2RA is highly expressed on activated NK cells in spleen in the IL-2RA humanized mouse (In collaboration with CrownBio).

Fig5. Frequency of lymphocyte and myeloid subpopulations in the blood by flow cytometry. The frequencies of T cells, CD4+ T, CD8+ T, B cells, dendritic cells, neutrophils, and monocytes in  HO hIL2/hIL2RA/IL2RB/IL2RG mice were similar to those in wild-type C57BL/6 mice, except that NK cells were reduced in the humanized mice. Blood was collected from WT C57BL/6 mice and HO hIL2/hIL2RA/IL2RB/IL2RG mice (8-week-old, n=6). Flow cytometry analysis of frequencies of (A) lymphocyte and (B) myeloid subpopulations in the blood. Bar indicated expressed as mean ± SEM.

Abbr.  HO, homozygous; WT, wild type.

Fig6. Frequency of lymphocyte and myeloid subpopulations in the spleen by flow cytometry. The frequencies of T cells, CD4+ T, CD8+ T, B cells, dendritic cells, neutrophils, monocytes, and macrophages in HO hIL2/hIL2RA/IL2RB/IL2RG mice were similar to those in wild-type C57BL/6 mice, except that NK cells were reduced in the humanized mice. Spleen was collected from WT C57BL/6 mice and HO hIL2/hIL2RA/IL2RB/IL2RG mice (8-week-old, n=6). Flow cytometry analysis of frequencies of (A) lymphocyte and (B) myeloid subpopulations in the spleen. Bar indicated expressed as mean ± SEM.

Abbr.  HO, homozygous; WT, wild type.

Fig7. Immunotype in hIL2/hIL2RA/hIL2RB/hIL2RG qKI mice in Blood.

Fig8. Immunotype in hIL2/hIL2RA/hIL2RB/hIL2RG qKI mice in Spleen.

Fig9. Detection of Signaling integrity in hIL2/hIL2RA/hIL2RB/hIL2RG qKI mice.

Note. Splenocytes of C57BL/6 mice were treated with anti-mCD3 and mIL-2(10 ng/ml) for 72h. Splenocytes of hIL2/hIL2RA/hIL2RB/hIL2RG qKI mice were treated with anti-mCD3 and hIL-2(10 ng/ml) for 72h.

Fig.10 Human IL-2 induced the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in the C57BL/6 WT mouse (n=2, 6 weeks old), hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 14 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 17 weeks old). 

Splenocytes were collected from the spleen, treated with different concentrations of human IL-2 for 15 min and analyzed by flow cytometry. As shown in the figure, human IL-2 induced pSTAT5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg.

Fig.11 Mouse IL-2 induced the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in the C57BL/6 WT mouse (n=2, 6 weeks old), hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 14 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 17 weeks old).

Splenocytes were collected from the spleen, treated with different concentrations of mouse IL-2 for 15 min and analyzed by flow cytometry. As shown in the figure, mouse IL-2 induced pSTAT5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg. However, the activation of mouse IL-2 is weaker than that of human IL-2 in hIL2RA/hIL2RB/hIL2RG knockin mice and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice.

Fig.12 Human IL-15 induced the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, NK cells and Treg in the C57BL/6 WT mouse (n=2, 6 weeks old), hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 14 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 17 weeks old). 

Splenocytes were collected from the spleen, treated with different concentrations of human IL-15 for 15 min, and analyzed by flow cytometry. As shown in the figure, human IL-15 induced pSTAT5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg.

Fig.13 Mouse IL-15 induced the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, NK cells and Treg in the C57BL/6 WT mouse (n=2, 6 weeks old), hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 14 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (n=2, 17 weeks old). 

Splenocytes were collected from the spleen, treated with different concentrations of mouse IL-15 for 15 min, and analyzed by flow cytometry. As shown in the figure, mouse IL-15 induced pSTAT5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in a dose dependent manner. However, the activation of mouse IL-15 is weaker than that of human IL-15 in all mice.

Fig.14 Analysis of the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, and Treg in human PBMC. 

PBMC were treated with different concentrations of human IL-2, human IL-15, mouse IL-2, and mouse IL-15 for 15 min, and analyzed by flow cytometry. As shown in the figure, human IL-2, human IL-15, and mouse IL-2 induced pSTAT5 in CD4+ T cells, CD8+ T cells, and Treg in a dose dependent manner. However, the activation of mouse IL-2 is weaker than that of human IL-2 and human IL-15.

Fig.15 Mouse IL-7 induced the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in the C57BL/6 WT mouse (6 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG mice (6 weeks old). Splenocytes were collected from the spleen, treated with different concentrations of mouse IL-7 for 15 min and analyzed by flow cytometry. As shown in the figure, mIL-7 induced comparable pSTAT5 levels in CD4+ T cells, CD8+ T cells, NK cells, and Tregs across WT and hIL2/hIL2RA/hIL2RB/hIL2RG knock-in mice.

Fig.16 Mouse IL-9 induced the phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in the C57BL/6 WT mouse (6 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (6 weeks old). Splenocytes were collected from the spleen, treated with different concentrations of mouse IL-9 for 15 min and analyzed by flow cytometry. As shown in the figure, mIL-9 induced comparable pSTAT5 levels in CD4+ T cells, CD8+ T cells, NK cells, and Tregs across WT and hIL2/hIL2RA/hIL2RB/hIL2RG knock-in mice.

Fig.17 Mouse IL-21 induced the phosphorylation of Stat3 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in the C57BL/6 WT mouse (8 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (8 weeks old). Splenocytes were collected from the spleen, treated with different concentrations of mouse IL-21 for 15 min and analyzed by flow cytometry. As shown in the figure, mIL-21 induced comparable pSTAT3 levels in CD4+ T cells, CD8+ T cells, NK cells, and Tregs across WT and hIL2/hIL2RA/hIL2RB/hIL2RG knock-in mice.

Fig.18 Mouse IL-4 induced the phosphorylation of Stat6 in CD4+ T cells, CD8+ T cells, NK cells, and Treg in the C57BL/6 WT mouse (9-10 weeks old) and hIL2/hIL2RA/hIL2RB/hIL2RG knockin mice (9 weeks old). Splenocytes were collected from the spleen, treated with different concentrations of mouse IL-4 for 15 min and analyzed by flow cytometry. As shown in the figure, mIL-4 induced comparable pSTAT6 levels in CD4+ T cells, CD8+ T cells, NK cells, and Tregs across WT and hIL2/hIL2RA/hIL2RB/hIL2RG knock-in mice.