Strain specific IL2RB and IL2RG expression analysis in heterozygous and homozygous B-hIL2RB/hIL2RG mice by flow cytometry. Splenocytes were collected from wild-type mice and B-hIL2RB/hIL2RG mice, and analyzed by flow cytometry with species-specific antibody. Human IL2RB was detectable in heterozygous (H/+;H/+) and homozygous (H/H;H/H) B-hIL2RB/hIL2RG mice. Human IL2RG was too low to detectable in heterozygous mice, and it was unable to judge the IL2RG expression in NK cells, because NK cells were diminished in homozygous B-hIL2RB/hIL2RG mice.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL2RB/hIL2RG mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. T cells, B cells and NK cells were significantly different between homozygous B-hIL2RB/hIL2RG mice and C57BL/6 mice.

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL2RB/hIL2RG mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hIL2RB/hIL2RG mice mice were similar to those in the C57BL/6 mice. Values are expressed as mean ± SEM.

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hIL2RB/hIL2RG mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of T cells, B cells, NK cells, in homozygous B-hIL2RB/hIL2RG mice were similar to those in the C57BL/6 mice. Values are expressed as mean ± SEM.

Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hIL2RB/hIL2RG mice (n=3, 6- week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells in homozygous B-hIL2RB/hIL2RG mice were similar to those in the C57BL/6 mice. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hIL2RB/hIL2RG mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of T cells, B cells and NK cells were significantly different between homozygous B-hIL2RB/hIL2RG mice and C57BL/6 mice.

Analysis of blood T subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hIL2RB/hIL2RG mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+, CD8+, Tregs in homozygous B-hIL2RB/hIL2RG mice mice were similar to those in the C57BL/6 mice. Values are expressed as mean ± SEM.

Intracellular pSTAT5 analysis in B-hIL2RB/hIL2RG mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice, homozygous and heterozygous B-hIL2RB/hIL2RG mice, and stimulated with IL2 or complex of IL15/IL15RA in vitro for 30 min. Then analyzed by flow cytometry with anti-pSTAT5 antibodies. The results show that human IL2 induced a low phosphorylation level of STAT5 in C57BL/6 mice compared with humanized mice. As for the IL15/IL15RA complex, both mouse and human complex can induce the pSTAT5, but the mouse complex results low in homozygous mice. Above all, the IL2RB and IL2RG humanization did not change the signaling pathway.

Antitumor activity of IL2 analogs (provided by the client) in B-hIL2RB/hIL2RG mice. (A) MC38 tumor growth. (B) Body weight changes during treatment. The tumor growth curve of individual mice in PBS group (C) and IL2 analogs treatment group (D). Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hIL2RB/hIL2RG mice (female, 8 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with IL2 analogs (red arrows). As shown in panel, IL2 analogs were efficacious in controlling tumor growth but causing some toxic, resulting decrease of body weight and some mice dying. B-hIL2RB/hIL2RG mice provide a powerful preclinical model for in vivo evaluation of IL2 therapy molecules. Values are expressed as mean ± SEM.