• BTN3A1 and BTN3A2 are expressed by CD4+ and CD8+ T cells, with BTN3A1 being the predominant form expressed in NK cells. BTN2A1 and BTN3 have no homologous genes in mice, with only BTN2A2 having a homolog. BTN3A1-3 specific antibodies bind to BTN3A proteins and can remodel the anti-tumor effects of αβ T cells, while also restoring the cytotoxicity of γδ T lymphocytes against BTN3A1-expressing cancer cells, ultimately eliminating malignant tumor progression. Current research is primarily focused on BTN3A1, with most of the drugs being monoclonal antibodies for use in tumor immunotherapy.
• The BAC containing the full coding sequences of human BTN2A1, BTN2A2, BTN3A1, BTN3A2 and BTN3A3 gene by human BTN2A1, BTN2A2, BTN3A1, BTN3A2 and BTN3A3 promoter were inserted into mouse Gt(ROSA)26Sor locus in B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice.
• Human BTN3A was detectable on T cells, B cells，NK cells，DCs, monocytes and macrophages in homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice.
• BTN2A1 was exclusively detectable in heart, spleen, lung and kidney in homozygous mice. BTN2A2 was detectable in heart, liver and kidney in both wild-type and homozygous mice.

Gene targeting strategy for B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice. The BAC containing the full coding sequences of human BTN2A1, BTN2A2, BTN3A1, BTN3A2 and BTN3A3 gene by human BTN2A1, BTN2A2, BTN3A1, BTN3A2 and BTN3A3 promoter were inserted into mouse Gt(ROSA)26Sor locus in B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice.

Strain specific analysis of BTN3A and BTN2A gene expression in wild-type C57BL/6 mice and homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice by RT-PCR. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice (H/H). Human BTN3A1/2/3 mRNA and BTN2A1/2 mRNA were detectable in homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice but not in wild-type mice.

Strain specific BTN3A expression analysis in wild-type C57BL/6 mice and homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice (H/H), and analyzed by flow cytometry with anti-human BTN3A1/2/3 antibody (R&D, FAB7136P-025) . Human BTN3A1/2/3 was detectable on T cells, B cells，NK cells，DCs, monocytes and macrophages in homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice.

Strain specific BTN2A1 expression analysis in homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice by Western Blot. Various tissues were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice (H/H), and then analyzed by western blot with anti-BTN2A1 antibody (proteintech, 68265-1-Ig). BTN2A1 was exclusively detectable in heart, spleen, lung and kidney in homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice. M: marker.

Strain specific BTN2A2 expression analysis in homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice by Western Blot (JESS). Various tissues were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hBTN3A2/hBTN2A2/hBTN3A1/hBTN3A3/hBTN2A1 mice (H/H), and then analyzed by JESS with anti-BTN2A2 antibody (R&D, AF8645-SP). BTN2A2 was detectable in heart, liver and kidney in both wild-type and homozygous mice, as the antibody was cross-reactive between human and mouse.