Basic Information
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Targeting strategy
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Gene targeting strategy for B-hPD1/hLAG3/hTIM3 mice. The exon 2 of mouse Pd-1 gene that encodes the extracellular domain was replaced by human PD-1 exon 2 in B-hPD1/hLAG3/hTIM3 mice. The exons 2~3 of mouse Lag3 gene that encode the extracellular domain were replaced by human LAG3 exons 2~3 in B-PD1/hLAG3/hTIM3 mice. The exon 2 of mouse Tim3 gene that encode the Ig-like V-type domain were replaced by human TIM3 exon 2 in B-hPD1/hLAG3/hTIM3 mice.
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Details
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Protein expression analysis in T cell-Spleen
Strain specific PD-1, LAG3 and TIM3 expression analysis in homozygous B-hPD-1/hLAG3/hTIM3 mice by flow cytometry. Splenocytes from both wild type (+/+) C57BL/6 and homozygous B-hPD-1/hLAG3/hTIM3 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry. Mouse PD-1+ and TIM3+ T cells were only detectable in the WT C57BL/6 mice, human PD-1+ and TIM3+ T cells were only detectable in the homozygous B-hPD-1/hLAG3/hTIM3 mice. Mouse LAG3 was detectable in WT mice. This anti-mouse LAG3 antibody also cross reacts with hLAG3.
Analysis of spleen leukocytes cell subpopulations in B-hPD-1/hLAG3/hTIM3 mice
Analysis of spleen leukocyte subpopulations by FACS Splenocytes were isolated from female C57BL/6 and B-hPD-1/hLAG3/hTIM3 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 cells, B cells, NK cells, monocytes, DCs, granulocytes and macrophages in homozygous B-hPD-1/hLAG3/hTIM3 mice ce were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1，hLAG3 and hTIM3 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
Analysis of spleen T cell subpopulations in B-hPD-1/hLAG3/hTIM3 mice
Analysis of spleen T cell subpopulations by FACS Splenocytes were isolated from female C57BL/6 and B-hPD-1/hLAG3/hTIM3 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-hPD-1/hLAG3/hTIM3 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1，hLAG3 and hTIM3 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.
