Targeting the PD-1: Biological Roles and Therapeutic Strategies 

• Gene Information: The PDCD1 gene encodes the PD-1 receptor to critically regulate T-cell tolerance and prevent autoimmunity. 
• Protein Expression: PD-1 is induced on activated immune cells (like T cells), while its ligands, PD-L1 and PD-L2, are often hijacked and overexpressed by tumor cells. 
• Signaling Pathway: Engagement with its ligands triggers intracellular SHP-2 phosphatase recruitment, shutting down TCR/CD28 signaling (PI3K/Akt) to arrest T-cell proliferation and cytokine release. 
• Therapeutic Inhibition: Therapeutic anti-PD-1/PD-L1 antibodies block this checkpoint brake, successfully reactivating tumor-reactive T cells to destroy cancer cells.

•  Gene Information: The IL12RB1 gene (located on chromosome 19p13.1) encodes the IL-12Rβ1 subunit, which is a shared chain also utilized by the IL-23 receptor. The IL12RB2 gene (located on chromosome 1p31.3) encodes the IL-12Rβ2 subunit, which confers strict specificity for the IL-12 cytokine and acts as the primary signal-transducing chain.
•  Protein Expression: While IL-12Rβ1 is constitutively expressed on various immune cells, IL-12Rβ2 expression is tightly regulated. It is largely undetectable on resting T cells but is strongly upregulated on activated NK cells and T cells following T-cell receptor (TCR) stimulation, serving as a critical checkpoint for Th1 commitment. 
• Signaling Pathway: The binding of the IL-12 cytokine (p35/p40) brings IL-12Rβ1 and IL-12Rβ2 together, activating their associated kinases: TYK2 (linked to IL-12Rβ1) and JAK2 (linked to IL-12Rβ2). This specific pairing predominantly phosphorylates STAT4, which translocases to the nucleus to drive the robust production of IFN-γ and secure TH1 differentiation. 
• Therapeutic Inhibition: Targeting shared components (e.g., via Ustekinumab) provides dual blockade of IL-12/IL-23 signaling, offering deep clinical relief in diseases like psoriasis while opening new avenues for cancer immunotherapy.

• Gene Information: The IL2 and IL15 genes encode structurally related four-α-helix bundle cytokines that orchestrate lymphocyte homeostasis and immune responses.
•  Protein Expression: While IL-2 is a secreted cytokine produced mainly by activated T cells, IL-15 is predominantly membrane-bound via trans-presentation by dendritic cells, with both sharing the common βγ receptor subunits but utilizing distinct private αchains (CD25 vs CD215). 
• Signaling Pathway: Both cytokines signal through the shared IL-2/15Rβγ complex to activate the downstream JAK1/JAK3-STAT5, PI3K/Akt, and MAPK pathways, driving the proliferation and survival of T cells and NK cells. 
• Therapeutic Inhibition: Targeted blockade of IL-2/IL-15 signaling pathways (via antibodies against the shared β subunit or specific α chains) serves as a potent therapeutic strategy to alleviate chronic inflammation, autoimmune diseases, and T-cell-driven malignancies.

PD-1

• Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice. 

IL12RB1

• The exons 1-14 of mouse Il12rb1 gene that encode signal peptide and extracellular domain were replaced by human counterparts. The genomic region of mouse Il12rb1 gene that encodes transmembrane domain and cytoplasmic portion was retained.

• The promoter, 5’ UTR and 3’ UTR region of the mouse gene were also retained. The human IL12RB1 expression was driven by endogenous mouse Il12rb1 promoter, while mouse Il12rb1 gene transcription and translation will be disrupted.

IL12RB2

• A chimeric CDS that encodes mouse Il12rb2 signal peptide, human IL12RB2 extracellular domain, mouse Il12rb2 transmembrane and cytoplasmic domain, followed by mouse 3’ UTR-STOP was inserted after exon 2 of mouse Il12rb2.

• The chimeric IL12RB2 protein expression will be driven by endogenous mouse Il12rb2 promoter. 

IL2RA

• The exons 2-6 of mouse Il2ra gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice. The genomic region of mouse Il2ra gene that encodes cytoplasmic portion is retained. The promoter, 5’ UTR and 3’ UTR region of the mouse gene are also retained. The chimeric IL2RA expression is driven by endogenous mouse Il2ra promoter, while mouse Il2ra gene transcription and translation will be disrupted.

Strain specific PD-1 expression analysis in homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice (female, 8-week-old, n = 1) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h, and analyzed by flow cytometry with species-specific anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908).

Strain specific IL2RA expression analysis in homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice (female, 8-week-old, n = 1) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h, and analyzed by flow cytometry with species-specific anti-mouse IL2RA antibody (Biolegend, 102008) and anti-human IL2RA antibody (Biolegend, 302610).

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes and thymus were isolated from male C57BL/6JNifdc and B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice (female, n=3, 8-week-old). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, blood, and lymph nodes were isolated from C57BL/6JNifdc and B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad/hIL2RA mice (female, n=3, 8-week-old). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.