B-hNKG2D mice plus

Basic Information

Targeting strategy

Gene targeting strategy for B-hNKG2D mice plus. The 5’UTR and exons 2~8 of mouse Nkg2d gene encoding the full-length were replaced by 5’UTR and exons 2~8 of human NKG2D in B-hNKG2D mice plus.

Protein expression analysis in CD4+T cells

Strain specific analysis of NKG2D expression in wild-type (WT) mice (+/+) and homozygous B-hNKG2D mice plus(H/H) by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hNKG2D mice plus (H/H), and analyzed by flow cytometry with species-specific anti-NKG2D antibody. mNKG2D and hNKG2D were not detectable in CD4+T cells of wild-type and homozygous B-hNKG2D mice plus.

Protein expression analysis in CD8+T cells

Strain specific analysis of NKG2D expression in wild-type (WT) mice (+/+) and homozygous B-hNKG2D mice plus(H/H) by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hNKG2D mice plus(H/H), and analyzed by flow cytometry with species-specific anti-NKG2D antibody. mNKG2D was not detectable in CD8+T cells of wild-type C57BL/6 mice, while hNKG2D was detectable in homozygous B-hNKG2D mice plus.

Protein expression analysis in NK cells

Strain specific analysis of NKG2D expression in wild-type (WT) mice (+/+) and homozygous B-hNKG2D mice plus(H/H) by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hNKG2D mice plus(H/H), and analyzed by flow cytometry with species-specific anti-NKG2D antibody. mNKG2D was detectable in NK cells of wild-type C57BL/6 mice and homozygous B-hNKG2D mice plus, while hNKG2D was only detectable in homozygous B-hNKG2D mice plus.

Protein expression analysis

Protein expression analysis in NK cells

Strain specific NKG2D expression analysis in wild-type C57BL/6JNifdc mice and homozygous NKG2D humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (female, n=3, 6-week-old), homozygous B-hNKG2D mice (female, n=3, 6-week-old) and homozygous B-hNKG2D mice plus (female, n=5, 6-week-old), and analyzed by flow cytometry with anti-mouse NKG2D (BioLegend, 115711) and anti-human NKG2D (eBioscience, 46-5878-42) antibody. NKG2D expression was analyzed in NK cells gated by NK1.1+. Mouse NKG2D was only detectable in wild-type C57BL/6JNifc mice. Human NKG2D was exclusively detectable in NKG2D humanized mice, and NKG2D expression levels in five B-hNKG2D mice plus was consistent, and was higher than that in B-hNKG2D mice.

Protein expression analysis in CD8+T cells

Strain specific NKG2D expression analysis in wild-type C57BL/6JNifdc mice and homozygous NKG2D humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (female, n=3, 6-week-old), homozygous B-hNKG2D mice (female, n=3, 6-week-old) and homozygous B-hNKG2D mice plus (female, n=5, 6-week-old), and analyzed by flow cytometry with anti-mouse NKG2D (BioLegend, 115711) and anti-human NKG2D (eBioscience, 46-5878-42) antibody. NKG2D expression was analyzed in CD8+ T cells gated from T cells. Mouse NKG2D was not detectable in CD8+ T cells from wild-type C57BL/6JNifc mice. Human NKG2D was exclusively detectable B-hNKG2D mice plus but not in B-hNKG2D mice.

Protein expression analysis in CD4+T cells

Strain specific NKG2D expression analysis in wild-type C57BL/6JNifdc mice and homozygous NKG2D humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (female, n=3, 6-week-old), homozygous B-hNKG2D mice (female, n=3, 6-week-old) and homozygous B-hNKG2D mice plus (female, n=5, 6-week-old), and analyzed by flow cytometry with anti-mouse NKG2D (BioLegend, 115711) and anti-human NKG2D (eBioscience, 46-5878-42) antibody. NKG2D expression was analyzed in CD4+ T cells gated from T cells. Both mouse NKG2D and human NKG2D were not detectable in CD4+ T cells.

Protein expression analysis in NK1.1, NKP46 and KLRG1

Protein expression analysis of NK1.1, NKP46 and KLRG1 in wild-type C57BL/6JNifdc mice and homozygous NKG2D humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (female, n=3, 6-week-old), homozygous B-hNKG2D mice (female, n=3, 6-week-old) and homozygous B-hNKG2D mice plus (female, n=5, 6-week-old), and analyzed by flow cytometry with anti-mouse NK1.1 (BioLegend, 108745), anti-mouse NKP46 (BioLegend, 137612) and anti-KLRG1 (BioLegend, 138416) antibody. mNKP46 and mKLRG1 expression were analyzed from NK1.1+ cell populations. NK1.1 and KLRG1 expression was consistent between NKG2D humanized mice and wild-type mice. But mNKP46 was not detectable in three of the five B-hNKG2D mice plus.

Protein expression analysis in NK and CD8+ T cells

Human NKG2D is ubiquitously expressed on NK cells(A) and CD8+ T cells(B) in B-hNKG2D mice plus females bearing MC38-hTAA tumors. Blood, spleen, bone marrow(BM), lung, liver and tumor were collected from homozygous B-hNKG2D mice plus (H/H), and analyzed by flow cytometry with species-specific anti-human NKG2D antibody. Note: This experiment was performed by the client using B-hNKG2D mice plus. All the other materials were provided by the client.

Analysis of leukocytes cell subpopulation in spleen

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6N mice (male, n=3, 7-week-old) and homozygous B-hNKG2D mice plus (male, n=3, 7-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hNKG2D mice plus were similar to those in C57BL/6N mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***p < 0.001.

Treatment with NK cell engagers demonstrated significant anti-tumor activity against MC38-hTAA tumors B-hNKG2D mice plus

Treatment with NK cell engagers demonstrated significant anti-tumor activity against MC38-hTAA tumors B-hNKG2D mice plus. Female B-hNKG2D mice plus(n=8 in each treatment group) were inoculated with 5×10⁵ MC38-hTAA tumor cells subcutaneously in the right flank. On day 0, 3 and 6 mice received 5 mg/kg of NKG2D/TAA intravenously (i.v). Mean tumor volume ± SEM presented. TGI: Tumor growth inhibition; TAA: tumor associated antigen; RSV: respiratory syncytial virus; SEM: standard error mean. Note: This experiment was performed by the client using B-hNKG2D mice plus. All the other materials were provided by the client.