Protein expression analysis

Strain specific OX40 expression analysis in homozygous B-hOX40/h4-1BB mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hOX40/h4-1BB (H/H) mice stimulated with or without anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-hOX40 antibody. Mouse OX40 was detectable in T cells of WT mice but not homozygous B-hOX40/h4-1BB mice. Human OX40 was exclusively detectable in T cells of homozygous B-hOX40/h4-1BB mice but not WT mice.

Strain specific 4-1BB expression analysis in homozygous B-hOX40/h4-1BB mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hOX40/h4-1BB (H/H) mice stimulated with or without anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-h4-1BB antibody. Mouse 4-1BB was detectable in T cells of WT mice but not homozygous B-hOX40/h4-1BB mice. Human 4-1BB was exclusively detectable in T cells of homozygous B-hOX40/h4-1BB mice but not WT mice.

Phenotypic analysis of spleen leukocytes cell subpopulations in B-hOX40/h4-1BB mice

Analysis of spleen leukocyte subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hOX40/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, B, NK, Monocyte, DC and macrophage cells in homozygous B-hOX40/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40 and h4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of spleen T cell subpopulations in B-hOX40/h4-1BB mice

Analysis of spleen T cell subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hOX40/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 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, CD4, and Treg cells in homozygous B-hOX40/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40 and h4-1BB 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 cell subpopulations in B-hOX40/h4-1BB mice

Analysis of lymph node leukocyte subpopulations by FACS

Lymph node were isolated from female C57BL/6 and B-hOX40/h4-1BB mice (n=3, 6-week-old). Flow cytometry analysis of the lymph node cells 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, B, NK, Monocyte, DC and macrophage cells in homozygous B-hOX40/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40 and h4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node.

Analysis of lymph node T cell subpopulations in B-hOX40/h4-1BB mice

Analysis of lymph node T cell subpopulations by FACS

Lymph node cells were isolated from female C57BL/6 and B-hOX40/h4-1BB mice (n=3, 6-week-old). Flow cytometry analysis of the lymph node cells was performed to assess lymph node cell subpopulations. 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, CD4, and Treg cells in homozygous B-hOX40/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40 and h4-1BB 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.