Protein expression analysis

Analysis of leukocytes subpopulation in B-hPD-1/hPD-L1/hGITR mice

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/hPD-L1/hGITR mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes were 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-hPD-1/hPD-L1/hGITR mice were similar to those in the C57BL/6 mice, demonstrating that PD-1/PD-L1/GITR humanized does not change the overall development, differentiation or distribution of these cell types in spleen. The same results were obtained in lymph nodes and blood (data not shown). Values are expressed as mean ± SEM.

Analysis of  spleen T cell subpopulations in B-hPD-1/hPD-L1/hGITR mice

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/hPD-L1/hGITR mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes ere 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. The percent of CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hPD-1/hPD-L1/hGITR mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1/hPD-L1/hGITR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. The same results were obtained in lymph nodes and blood (data not shown). Values are expressed as mean ± SEM.

Analysis of leukocytes subpopulation in B-hPD-1/hPD-L1/hGITR mice

Analysis of blood T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/hPD-L1/hGITR mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells were 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. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hPD-1/hPD-L1/hGITR mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1/hPD-L1/hGITR 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.

Combination therapy of anti-human PD-1 antibody and anti-human GITR antibody