B-hPD1/hLAG3/hTIM3 mice

Basic Information

Strain Name
C57BL/6-Pdcd1tm1(PDCD1) Havcr2tm1(HAVCR2) Lag3tm1(LAG3)/Bcgen
Common Name
B-hPD-1/hLAG3/hTIM3 mice
Background
C57BL/6
Catalog number
130999
Related Genes
PD-1 (Programmed death-1) ; LAG3 (lymphocyte-activation gene 3) HAVCR2 (hepatitis A virus cellular receptor 2)

Description

PD-1 (Programmed death-1) is mainly expressed on the surface of T cells and primary B cells. The two PD-1 ligands, PD-L1 and PD-L2, are widely expressed on antigen-presenting cells (APCs). PD-L1 expression is favorable for tumorigenesis and growth, for induction of anti-tumor T cell apoptosis, and for escaping responses by the immune system. Inhibition of PD-1 binding to its ligand can result in tumor cells that are exposed to the killing version of the immune system, and thus is a target for cancer treatments. LAG3 (Lymphocyte activation gene 3, CD223) is a lymphocyte activation gene and a member of the Ig family. It is mainly expressed in activated T cells, NK cells, B cells and plasmacytoid DCs. LAG3 is a negative regulator of immunity and mainly binds to MHC class Ⅱ molecules to regulate the function of dendritic cells. The expression of LAG3 is associated with the negative immunoregulatory function of specific T cells. Inhibition of LAG3 function enhances the antitumor effect of specific CD8+ T cells, therefore LAG3 is a potential target for tumor immunotherapy. T-cell immunoglobulin domain and mucin domain-3 (TIM3) is an activation-induced inhibitory molecule involved in immune tolerance and T-cell exhaustion in chronic viral infection and cancers. TIM3 maturation and cell surface expression is facilitated by forming a heterodimeric interaction with CD66a. Co-blockade of CD66a and TIM3 enhanced anti-tumor immune responses, and eliminated tumors in mouse colorectal cancer models.

Targeting strategy

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.

Details

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.

Back to top