Gene targeting strategy for B-hCD3EDG/h4-1BB mice.
The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human CD3E exons 2-9 in B-hCD3EDG/h4-1BB mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/h4-1BB mice, respectively. The exons 2-7 of mouse Tnfrsf9 gene that encode the extracellular domain were replaced by human TNFRSF9 exons 3-8 in B-hCD3EDG/h4-1BB mice.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/h4-1BB mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3EDG/h4-1BB mice (H/H;H/H;H/H;H/H), and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse CD3E was detectable in WT mice (+/+). Human CD3E was exclusively detectable in homozygous B-hCD3EDG/h4-1BB mice (H/H;H/H;H/H;H/H) but not in WT mice (+/+).

Strain specific 4-1BB expression analysis in homozygous B-hCD3EDG/h4-1BB mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3EDG/h4-1BB mice (H/H;H/H;H/H;H/H), and analyzed by flow cytometry with species-specific anti-4-1BB antibody. Mouse 4-1BB was detectable in WT mice (+/+). Human 4-1BB was exclusively detectable in homozygous B-hCD3EDG/h4-1BB mice (H/H;H/H;H/H;H/H) but not in WT mice (+/+).

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD3EDG/h4-1BB 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 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-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart 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-hCD3EDG/h4-1BB mice (n=3, 6 week-old). Flow cytometry analysis of the splenocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD3EDG/h4-1BB 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. Percent of T cells, B cells and NK cells in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart 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 female C57BL/6 and B-hCD3EDG/h4-1BB mice (n=3, 6 week-old). Flow cytometry analysis of the leukocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in lymph node. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3EDG/h4-1BB mice (n=3, 6 week-old). Flow cytometry analysis of the blood 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. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3EDG/h4-1BB mice (n=3, 6 week-old). Flow cytometry analysis of the leukocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in blood. Values are expressed as mean ± SEM.