Protein expression analysis

Strain specific CTLA4 expression analysis in homozygous B-hCTLA4 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCTLA4 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CTLA4 antibody. Mouse CTLA4 was detectable in WT mice. Human CTLA4 was exclusively detectable in homozygous B-hCTLA4 but not WT mice.

Analysis of spleen leukocytes cell subpopulations in B-hCTLA4 mice

Analysis of spleen leukocyte subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hCTLA4 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-hCTLA4 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCTLA4 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 by FACS
Splenocytes were isolated from female C57BL/6 and B-hCTLA4 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-hCTLA4 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCTLA4 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.

Complete blood count (CBC). Blood from female C57BL/6 and B-hCTLA4 mice (n=3, 6 week-old) was collected and analyzed for CBC. There was no differences among any measurement between C57BL/6 and B-hCTLA4 mice, indicating that introduction of hCTLA4 in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood chemistry tests of B-hCTLA4 mice. Serum from the C57BL/6 and B-hCTLA4 mice (n=3, 6 week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hCTLA4 mice, indicating that introduction of hCTLA4 in place of its mouse counterpart does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human CTLA4 antibody