B-hPD-1/hPD-L1/hSIRPA/hCD47,Tg(hLILRB2/hLILRB3) mice obtained by mating B-hPD-1/hPD-L1, Tg(hLILRB2/hLILRB3) mice (111954) and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice (140577).

B-hPD-1/hPD-L1/hSIRPA/hCD47,Tg(hLILRB2/hLILRB3) mice obtained by mating B-hPD-1/hPD-L1, Tg(hLILRB2/hLILRB3) mice (111954) and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice (140577).

• The exon 2 of mouse Pd-1 gene that encode the IgV domain was replaced by human PD-1 exon 2.
• The exon 3 of mouse Pdl1 gene that encode the IgV domain was replaced by human PD-L1 exon 3.
• The exon 2 of mouse Sirpα gene that encode the IgV domain was replaced by human SIRPα exon 2.
• The exon 2 of mouse Cd47 gene that encode the IgV domain was replaced by human CD47 exon 2.
• Bacterial artificial chromosome (BAC) clones for both the human LILRB2 and LILRB2 genes were integrated into the mouse genome randomly.

Strain specific PD-1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908). Mouse PD-1 was only detectable in wild-type mice. Human PD-1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Strain specific PD-1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs, and analyzed by flow cytometry with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908). Mouse PD-1 was only detectable in wild-type mice. Human PD-1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.

Strain specific PD-L1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706). Mouse PD-L1 was only detectable in wild-type mice. Human PD-L1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Strain specific PD-L1 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs, and analyzed by flow cytometry with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706). Mouse PD-L1 was only detectable in wild-type mice. Human PD-L1 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.

Strain specific CD47 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human CD47 antibody (Biolegend, 323108). Mouse CD47 was only detectable in wild-type mice. Human CD47 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Strain specific CD47 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs, and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human CD47 antibody (Biolegend, 323108). Mouse CD47 was only detectable in wild-type mice. Human CD47 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.

Strain specific CD47 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Red blood cells were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human CD47 antibody (Biolegend, 323108). Mouse CD47 was only detectable in wild-type mice. Human CD47 was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.

Strain specific SIRPA expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with cross-recognized both human and mouse SIRPa antibody (Biolegend, 144011) and anti-human SIRPa antibody (Biolegend, 323810). Human SIRPa was only detectable in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, but not in wild-type mice.

Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was not detectable in T cells of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in T cells of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was not detectable in B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in B cells of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was only detectable in macrophages of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in macrophages of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Strain specific LILRB2 and LILRB3 expression analysis in wild-type mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice by flow cytometry. Blood cells were collected from wild-type C57BL/6N mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old), and analyzed by flow cytometry with anti-human LILRB2 antibody (Biolegend, 338708) and anti-human LILRB3 antibody (R&D, FAB1806P-100). Human LILRB2 was only detectable in monocytes of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice. Human LILRB3 was only detectable in monocytes of B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice, but not in wild-type mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6N mice, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice, B-Tg(hLILRB2/hLILRB3) mice and B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD4+ T cells, CD8a+ T cells and Tregs in B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice were similar to those in C57BL/6N mice, demonstrating that humanization of hPD-1, hPD-L1, hSIRPA, hCD47 and the transgene of LILRB2 and LILRB3 do not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM. The frequency of leukocyte subpopulations in lymph node of B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice were also comparable to wild-type C57BL/6N mice (Data not shown).

Subcutaneous homograft tumor growth of B-hPD-L1 plus/hCD47 MC38 cells. B-hPD-L1 plus/hCD47 MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1/hPD-L1/hSIRPA/hCD47, Tg(hLILRB2/hLILRB3) mice (female, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². As shown in panel A, B-hPD-L1 plus/hCD47 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.