Basic Information
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Details
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PD-1 expression analysis
Splenocytes were collected from wild-type mice (+/+) and homozygous B-hPD-1/hPD-L1/hGITR mice (H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-1 antibody. Mouse PD-1 was detectable in WT mice but not in homozygous B-hPD-1/hPD-L1/hGITR mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1/hPD-L1/hGITR mice but not in WT mice.
PD-L1 expression analysis
Splenocytes were collected from wild-type mice (+/+) and homozygous B-hPD-1/hPD-L1/hGITR mice (H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-L1 antibody. Mouse PD-L1 was detectable in WT mice but not in homozygous B-hPD-1/hPD-L1/hGITR mice. Human PD-L1 was exclusively detectable in homozygous B-hPD-1/hPD-L1/hGITR mice but not in WT mice.
GITR expression analysis
Splenocytes were collected from wild-type mice (+/+) and homozygous B-hPD-1/hPD-L1/hGITR mice (H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-GITR antibody. Mouse GITR was detectable in WT mice but not in homozygous B-hPD-1/hPD-L1/hGITR mice. Human GITR was exclusively detectable in homozygous B-hPD-1/hPD-L1/hGITR mice but not in WT mice.
Analysis of leukocyte subpopulations in B-hPD-1/hPD-L1/hGITR mice
Splenocytes and blood were isolated from female C57BL/6 and B-hPD-1/hPD-L1/hGITR mice (n=3, 6-week-old). Single live cells were gated for the CD45+ population, and lineages were assessed using the markers TCRB, CD19, NK1.1, CD11c, CD11b, GR-1 and F4/80. 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 C57BL/6 mice, demonstrating that humanization of PD-1/PD-L1/GITR does not change the overall development, differentiation or distribution of these cell types. Values are expressed as mean ± SEM.
Analysis of T cell subpopulations in B-hPD-1/hPD-L1/hGITR mice
Splenocytes, blood, and lymph nodes were isolated from female C57BL/6 and B-hPD-1/hPD-L1/hGITR mice (n=3, 6-week-old). Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. 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/PD-L1/GITR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes. Values are expressed as mean ± SEM.
