Gene targeting strategy for B-hPD -1/hBTLA/hHVEM mice. The exon 2 of mouse PD-1 gene that encodes the IgV domain was replaced by human PD-1 exon 2 in B-hPD-1 mice. The exon 2 of mouse Btla gene that encodes the extracellular domain was replaced by human BTLA exon 2 in B-hBTLA/hHVEM mice. The exons 1~6 of mouse Tnfrsf14 gene that encode the extracellular domain were replaced by human TNFRSF14 exons 1~6 in B-hBTLA/hHVEM mice.

B-hPD-1/hBTLA/hHVEM mice were obtained by mating B-hPD-1/hBTLA mice and B-hBTLA/hHVEM mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (female, n=3, 7-week-old) and homozygous B-hPD-1/hBTLA/hHVEM mice (female, 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. 

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood were isolated from wild-type C57BL/6 mice (female, n=3, 7-week-old) and homozygous B-hPD-1/hBTLA/hHVEM mice (female, n=3, 7-week-old). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. 

Frequency of leukocyte subpopulations in blood by flow cytometry. Lymph nodes cells were isolated from wild-type C57BL/6 mice (female, n=3, 7-week-old) and homozygous B-hPD-1/hBTLA/hHVEM mice (female, n=3, 7-week-old). A. Flow cytometry analysis of the lymph nodes cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations.

Strain specific BTLA expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1/hBTLA/hHVEM mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hBTLA/hHVEM mice (H/H;H/H;H/H). Protein expression was analyzed with anti-mouse BTLA antibody (Biolegend, 134809) and anti-human BTLA antibody (Biolegend, 344509) by flow cytometry. Mouse BTLA was only detectable in wild-type C57BL/6 mice. Human BTLA was exclusively detectable in homozygous B-hPD-1/hBTLA/hHVEM mice, but not in wild-type C57BL/6 mice.

Strain specific HVEM expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1/hBTLA/hHVEM mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hBTLA/hHVEM mice (H/H;H/H;H/H). Protein expression was analyzed with anti-mouse HVEM antibody (Biolegend, 136305) and anti-human HVEM antibody (Biolegend, 318806) by flow cytometry. Mouse HVEM was only detectable in wild-type C57BL/6 mice. Human HVEM was exclusively detectable in homozygous B-hPD-1/hBTLA/hHVEM mice, but not in wild-type C57BL/6 mice.

Strain specific HVEM expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1/hBTLA/hHVEM mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hBTLA/hHVEM mice (H/H;H/H;H/H) stimulated without or with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329904) by flow cytometry. Mouse PD-1 was only detectable in wild-type C57BL/6 mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1/hBTLA/hHVEM mice, but not in wild-type C57BL/6 mice.