Protein expression analysis

Strain specific IL21R expression analysis in homozygous B-hIL21R mice by flow cytometry. Splenocytes were collected from WT (+/+) and homozygous B-hIL21R mice (H/H) , and analyzed by flow cytometry with species-specific anti-IL21R antibody. Mouse IL21R was detectable in WT mice. Human IL21R was exclusively detectable in homozygous B-hIL21R mice but not in WT mice.

mRNA expression analysis

Strain specific analysis of IL21R gene expression in wild-type C57BL/6 mice and B-hIL21R mice by RT-PCR. Mouse Il21r mRNA was detectable in splenocytes of wild-type C57BL/6 mice (+/+) but not in homozygous B-hIL21R mice (H/H). Human IL21R mRNA was detectable only in homozygous B-hIL21R mice but not in wild-type mice.

Functional analysis

Mouse pSTAT3 was induced with mouse IL21 and human IL21 in homozygous B-hIL21R mice analyzed by flow cytometry. Splenocytes were collected from homozygous B-hIL21R mice (H/H), and stimulated with culture medium, mIL21 or hIL21. The induction of STAT5 phosphorylation on CD8+ T cells with the indicated stimulators was assayed by flow cytometry. Results indicated that STAT5 phosphorylation was successfully induced with mouse IL21 or human IL21 in homozygous B-hIL21R mice.

Analysis of leukocytes cell subpopulation in spleen

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL21R 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. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL21R mice were similar to those in the C57BL/6 mice, demonstrating that IL21R humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL21R 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 TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hIL21R mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL21R in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in lymph node

Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hIL21R 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 the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL21R mice were similar to those in the C57BL/6 mice, demonstrating that IL21R humanized does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from lymph nodes of female C57BL/6 and B-hIL21R 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+ T cells, CD8+ T cells, and Tregs in homozygous B-hIL21R mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL21R in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hIL21R mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells 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+ T cells, CD8+ T cells, and Tregs in homozygous B-hIL21R mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL21R in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

In vivo efficacy of human IL21-Fc

Intratumoral injection of IL21-Fc (in house) is superior to intraperitoneal injection for tumor control. (A) Human IL21-Fc inhibited MC38 tumor growth in B-hIL21R mice. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous B-hIL21R mice (female, 7-week-old, n=6). Mice were grouped when tumor volume reached approximately 80 mm³, at which time they were treated intraperitoneally (i.p.) or intratumorally (i.t.) with human IL21-Fc. (B) Body weight changes during treatment. As shown in panel A, local delivery of IL21-Fc was more efficacious than systematic delivery in controlling tumor growth in B-hIL21R mice, demonstrating that the B-hIL21R mice provide a powerful preclinical model for in vivo evaluation of IL21 cytokine or fusion protein drugs. Values are expressed as mean ± SEM.