IL12RB1/IL12RB2 has a distinct, specialized role in driving the Th1 (Type 1 helper T cell) immune response.

• 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.

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.

• Mouse IFNγ were increased after responsiveness to mIL12 in humanized mice and wild-type mice.
• Only hIL12 induced mouse IFNγ increase in humanized mice.

IL12 induced the IFNγ production in CD4+ T cells sorted from splenocytes. CD4+ T cells were sorted from the splenocytes of wild-type C57BL/6 mice and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice. The production of IFNγ in supernatants were assessed after incubation with mouse IL12 (mIL12) or human IL12 (hIL12) in combination with bead-associated 0.4 μg/mL anti-mCD3e and 0.8 μg/mL anti-mCD28 antibodies for 48 hours. Mouse IFNγ were increased after responsiveness to mIL12 in humanized mice and wild-type mice. While, only hIL12 induced mouse IFNγ increase in humanized mice.

• The frequencies of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hIL12RB1 plus/hIL12RB2 ad mice were similar to those in C57BL/6 mice.
• Humanization of IL12RB1 and IL12RB2 does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hIL36R mice (n=3, 6 week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL12RB1 plus/hIL12RB2 ad mice was similar to those in the C57BL/6 mice, demonstrating that introduction of hIL12RB1, hIL12RB2 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hIL12RB1 plus/hIL12RB2 ad mice were comparable to those in C57BL/6 mice.
• Humanization of IL12RB1 and IL12RB2 does not affect normal T cell development, differentiation, or splenic distribution.

Analysis of T cell subpopulations by FACS. Splenocytes, peripheral blood and lymph nodes were isolated from female C57BL/6 and B-hIL36R mice (n=3, 6 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hIL12RB1 plus/hIL12RB2 ad mice was similar to those in the C57BL/6 mice, demonstrating that introduction of hIL12RB1, hIL12RB2 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes. Values are expressed as mean ± SEM.

• Human IL-12 induces severe target-specific toxicity, evidenced by significant body weight loss and hepatosplenomegaly.
• Pathological responses observed in B-hIL12RB1 plus/hIL12RB2 ad mice are entirely target-mediated and free from non-specific cross-reactivity.

In vivo toxicity evaluation of hIL-12 in B-hIL12RB1 plus/hIL12RB2 ad mice and wild-type mice. Evaluation of body weight changes, spleen indices, and liver indices in both B-hIL12RB1 plus/hIL12RB2 ad humanized mice and wild-type C57BL/6J mice following hIL-12 administration. Repeated i.v. administration of hIL-12 induced progressive body weight loss and a highly significant increase in both spleen and liver weight indices in B-hIL12RB1 plus/hIL12RB2 ad humanized mice (G2 vs. G1), whereas no significant changes in body weight or organ indices were observed in wild-type C57BL/6J mice (G4 vs. G3) due to strict species specificity. Values are expressed as mean ± SEM.

• B-hIL12RB1 plus/hIL12RB2 ad mouse serves as a highly sensitive and reliable model for evaluating target-mediated systemic toxicities of human IL-12-targeted therapeutics.

hIL-12 induces target-specific hepatotoxicity and systemic inflammation in B-hIL12RB1 plus/hIL12RB2 ad mice. Following a 6-day i.v. administration of hIL-12 (0.3 mg/kg), humanized mice (G2) exhibited significantly elevated serum ALT and AST levels, along with a massive surge in serum mouse IFN-γ compared to PBS controls (G1). Concordantly, H&E staining (200×) revealed marked pathological damage and inflammatory cell infiltration (black arrows) in the spleen and liver of G2 mice. In contrast, hIL-12-treated wild-type C57BL/6J mice (G4) showed no robust IFN-γ release or severe histological abnormalities. These findings successfully demonstrate that the B-hIL12RB1 plus/hIL12RB2 ad mouse serves as a highly sensitive and reliable model for evaluating target-mediated systemic toxicities of human IL-12-targeted therapeutics.

Experimental scheme for in vivo toxicity study in B-hIL12RB1 plus/hIL12RB2 ad mice. Six-week-old female B-hIL12RB1 plus/hIL12RB2 ad mice (G1/G2) and wild-type C57BL/6J mice (G3/G4) were administered with either vehicle control (PBS) or hIL-12 (0.3 mg/kg) via intravenous (i.v.) injection daily for 6 consecutive days (QDx6D). At 24 hours post-final dosing, whole-body weight changes were recorded, and livers and spleens were harvested to calculate organ indices (% organ weight/body weight). Data are presented as mean ± SEM (n=6 mice per group).

• Human IL-12 showed inhibitory effects in a dose-dependent manner to decrease the tumor volume.

Antitumor activity of  human IL-12 in B-hIL12RB1 plus/hIL12RB2 ad mice. (A) Human IL12 inhibited MC38 tumor growth in B-hIL12RB1 plus/hIL12RB2 ad mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (female, 6-8 weeks-old, n=8). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were intravenous injection with hIL-12 in the panel. (B) Body weight changes during treatment. As shown in panel A, the human IL-12 showed inhibitory effects in a dose-dependent manner. Values are expressed as mean ± SEM.