• Gene Information: PD‑1 is encoded by the PDCD1 gene on chromosome 2q37.3, while PD‑L1 is encoded by CD274 on chromosome 9p24.1. Both are type I transmembrane proteins; PD‑1 belongs to the immunoglobulin superfamily and functions as an inhibitory receptor, whereas PD‑L1 is a member of the B7 family of co‑stimulatory molecules.
• Protein Expression: PD-1 is primarily expressed on activated T cells, B cells, and NK cells, whereas PD-L1 is expressed on antigen-presenting cells and many tumor cells, with expression often induced by IFN-γ.
• Signaling Pathway: Binding of PD-L1 to PD-1 recruits SHP2 phosphatase and suppresses TCR/CD28-mediated PI3K–AKT signaling, resulting in reduced T-cell proliferation, cytokine production, and cytotoxic activity.
• Therapeutic Inhibition: Anti-PD-1 and anti-PD-L1 antibodies block this inhibitory pathway, restoring T-cell function and enhancing anti-tumor immune responses.

CD80: a critical costimulatory molecule in immune regulation

• Gene Information: It encodes the B7-1 protein, a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily.
• Protein Expression: CD80 is primarily expressed on the surface of activated antigen-presenting cells, including dendritic cells, macrophages, and activated B cells. Its expression is usually low at baseline but is strongly upregulated by inflammatory signals, such as Toll-like receptor (TLR) activation and cytokines, acting as an "alert" signal during infection.
• Signaling Pathway: CD80 functions as a dual-role ligand in the immune system. It binds to the CD28 receptor on T cells to provide essential costimulatory signals that drive T cell activation, survival, and proliferation. Conversely, it also binds with high affinity to CTLA-4, an inhibitory receptor that dampens T cell responses to prevent hyperactivation and autoimmunity.
• Therapeutic Inhibition: Therapeutic inhibitors (eg. CTLA4 Ig) of CD80 function by physically blocking the CD80-CD28 protein-protein interaction. Without this crucial Signal 2, the T cell fails to fully activate, leading to a state of unresponsiveness (anergy) or immune tolerance. This reduces the proliferation of autoreactive T cells and decreases the production of inflammatory cytokines.

PD-1

• The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PDCD1 exon 2 in B-hPD-1/hPD-L1/hCD80 mice.

PD-L1

• The exon 3 of mouse Cd274 gene that encodes the IgV domain was replaced by human CD274 exon 3 in B-hPD-1/hPD-L1/hCD80 mice.

CD80

• The exons 1-4 of mouse CD80 gene that encode the extracellular domain were replaced by human CD80 exons 2-5 in B-hPD-1/hPD-L1/hCD80 mice.

The B-hPD-1/hPD-L1/hCD80 three knock-in model, was developed by breeding the B-hPD-1 mice, the B-hPD-L1 mice and the B-hCD80 mice

• Mouse PD-1 was detected exclusively in wild-type C57BL/6 mice
• Human PD-1 was detected in homozygous B-hPD-1/hPD-L1/hCD80 mice, but not in wild-type mice.

Strain specific PD-1 expression analysis in wild-type C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD80 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1/hCD80 mice (H/H, H/H, H/H) (female, 8-week-old, n=1) after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hours or not. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908) by flow cytometry.

• Mouse PD-L1 was detected exclusively in wild-type C57BL/6 mice
• Human PD-L1 was detected in homozygous B-hPD-1/hPD-L1/hCD80 mice, but not in wild-type mice.

Strain specific PD-L1 expression analysis in wild-type C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD80 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1/hCD80 mice (H/H, H/H, H/H) (female, 8-week-old, n=1) after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hours or not. Protein expression was analyzed with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) by flow cytometry.

• Mouse CD80 was detected exclusively in wild-type C57BL/6 mice
• Human CD80 was detected in homozygous B-hPD-1/hPD-L1/hCD80 mice, but not in wild-type mice.

Strain specific CD80 expression analysis in wild-type C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD80 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1/hCD80 mice (H/H, H/H, H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-human CD80 antibody (Biolegend, 375406) and anti-mouse CD80 antibody (Biolegend, 104708) by flow cytometry.

• Mouse CD80 was detected exclusively in wild-type C57BL/6 mice
• Human CD80 was detected in homozygous B-hPD-1/hPD-L1/hCD80 mice, but not in wild-type mice.

Strain specific CD80 expression analysis in wild-type C57BL/6 and homozygous B-hPD-1/hPD-L1/hCD80 mice by flow cytometry. Peritoneal exudative macrophages (PEMs) were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hPD-1 plus/hPD-L1/hCD80 mice (H/H, H/H, H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-human CD80 antibody (Biolegend, 375406) and anti-mouse CD80 antibody (Biolegend, 104708) by flow cytometry.

• The frequencies of T cells, B cells, NK cells, DCs, granulocyte, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hPD-1/hPD-L1/hCD80 mice were similar to those in C57BL/6 mice
• Humanization of PD-1, PD-L1 and CD80 does not change the frequency or distribution of these cell types in spleen.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hCD80 mice (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. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

• The frequencies of T cells, B cells, NK cells, DCs, granulocyte, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hPD-1/hPD-L1/hCD80 mice were similar to those in C57BL/6 mice
• Humanization of PD-1, PD-L1 and CD80 does not change the frequency or distribution of these cell types in blood.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hCD80 mice (n=3, 8-week-old). A. Flow cytometry analysis of the blood cells were performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

• The frequencies of T cells, B cells, NK cells, DCs, granulocyte, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hPD-1/hPD-L1/hCD80 mice were similar to those in C57BL/6 mice
• Humanization of PD-1, PD-L1 and CD80 does not change the frequency or distribution of these cell types in lymph nodes.

Frequency of leukocyte subpopulations in lymph nodes by flow cytometry. Lymph nodes were isolated from wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hCD80 mice (n=3, 8-week-old). A. Flow cytometry analysis of the lymph nodes were performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.