Strain specific PD-1 and PD-L1 expression analysis in wild-type BALB/c mice and homozygous B-hPD-1/hPD-L1 mice(C) by flow cytometry.  Splenocytes were collected from wild-type BALB/c mice (+/+) and homozygous B-hPD-1/hPD-L1 mice(C) (H/H) stimulated with anti-mCD3ɛ in vivo. Mouse PD-1 and PD-L1 were only detectable in wild-type BALB/c mice. Human PD-1 and PD-L1 were only detectable in homozygous B-hPD-1/hPD-L1 mice(C), but not in wild-type BALB/c mice.

Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from female BALB/c and homozygous B-hPD-1/hPD-L1 mice(C) (n=3, 7-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, DCs, granulocytes, monocytes and macrophages in homozygous B-hPD-1/hPD-L1 mice(C) were similar to those in the BALB/c mice. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations by flow cytometry. Splenocytes were isolated from female BALB/c and homozygous B-hPD-1/hPD-L1 mice(C) (n=3, 7-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. The percent of CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hPD-1/hPD-L1 mice(C) was similar to those in the BALB/c mice, demonstrating that introduction of hPD-1/hPD-L1 in place of its mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of lymph node leukocyte subpopulations by flow cytometry. Leukocytes were isolated from female BALB/c and B-hPD-1/hPD-L1 mice(C) (n=3, 7- week-old) lymph nodes. 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. The percent of T cells, B cells and NK cells in homozygous B-hPD-1/hPD-L1 mice(C) were similar to those in the BALB/c mice, demonstrating that introduction of hPD-1/hPD-L1 in place of its mouse counterparts does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in lymph nodes

Analysis of lymph node T cell subpopulations by flow cytometry. Lymph node were isolated from female BALB/c and homozygous B-hPD-1/hPD-L1 mice(C) (n=3, 7-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 CD3+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T, CD8+ T cells and Tregs in homozygous B-hPD-1/hPD-L1 mice(C) were similar to those in the BALB/c mice, demonstrating that introduction of hPD-1/hPD-L1 in place of its mouse counterparts 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 leukocyte subpopulations by flow cytometry. Blood were isolated from female BALB/c and homozygous B-hPD-1/hPD-L1 mice(C) (n=3, 7-week-old). Flow cytometry analysis of the blood 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-hPD-1/hPD-L1 mice(C) were similar to those in the BALB/c mice, demonstrating that hPD-1/hPD-L1 humanized 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 flow cytometry. Blood were isolated from female BALB/c and homozygous B-hPD-1/hPD-L1 mice(C) (n=3, 7-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 CD3+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T, CD8+ T cells and Tregs in homozygous B-hPD-1/hPD-L1 mice(C) were similar to those in the BALB/c mice, demonstrating that introduction of hPD-1/hPD-L1 in place of its mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

Antitumor activity of combination of pembrolizumab analog (in house) and entinostat (ENT) in B-hPD-1/hPD-L1 mice(C). (A) Combination of pembrolizumab analog and ENT inhibited B-hPD-L1 CT26.WT tumor growth in B-hPD-1/hPD-L1 mice(C). Murine colon cancer B-hPD-L1 CT26.WT cells were subcutaneously implanted into homozygous B-hPD-1/hPD-L1 mice(C) (female, 7-week-old, n=6). Mice were grouped when tumor volume reached approximately 60-80 mm³, at which time they were treated with pembrolizumab analog and ENT with doses indicated in panel. (B) Body weight changes during treatment. As shown in panel A, combination of pembrolizumab analog and ENT was efficacious in controlling tumor growth in B-hPD-1/hPD-L1 mice(C), demonstrating that the B-hPD-1/hPD-L1 mice(C) provide a powerful preclinical model for in vivo evaluation of anti-human PD-1 antibodies and tumor microenvironment inhibitors. Values are expressed as mean ± SEM.