Basic Information

Strain Name
C57BL/6-Pdcd1tm1(PDCD1) Cd274tm1(CD274) Tnfrsf18tm1(TNFRSF18)/Bcgen
Stock Number
131108
Common Name
B-hPD-1/hPD-L1/hGITR mice
Background
C57BL/6
Aliases
PDCD1 also known as PD1; PD-1; CD279; SLEB2; hPD-1; hPD-l; hSLE1. CD274 also known as B7-H; B7H1; PDL1; PD-L1; hPD-L1; PDCD1L1; PDCD1LG1. TNFRSF18  also known as AITR; GITR; CD357; GITR-D; ENERGEN
Target Strategy
Exon 2 of mouse Pdcd1 (which encodes the IgV domain) was replaced by human PDCD1 exon 2 in B-hPD-1/hPD-L1/hGITR mice. Exon 3 of mouse Cd274 (which encodes the IgV domain) was replaced by human CD274 exon 3 in B-hPD-L1/hPD-L1/hGITR mice. Exons 1-4 of mouse Tnfrsf18 (which encodes the extracellular domain) was replaced by human TNFRSF18 exons 1-4 in B-hPD-1/hPD-L1/hGITR mice.

Details

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.