• TL1A binds to death receptor 3 (DR3) and regulates immune signaling pathways involved in effector cell proliferation, activation, apoptosis, and cytokine production. Soluble decoy receptor 3 (DcR3) can bind TL1A and modulate TL1A–DR3-mediated inflammatory responses.
• IL-23 is a heterodimeric cytokine composed of IL23A (p19) and IL12B (p40) subunits that is primarily produced by macrophages and dendritic cells. IL-23 signaling regulates downstream inflammatory cytokines and plays a critical role in chronic intestinal inflammation and autoimmune disease progression.
• Integrin α4β7 is a heterodimer composed of ITGA4 and ITGB7 subunits that mediates lymphocyte adhesion and gut homing through interactions with MAdCAM-1, VCAM-1, and fibronectin. The α4β7 pathway is an important therapeutic target in inflammatory bowel disease and intestinal immune regulation.
• TL1A/IL23A/IL12B/α4β7 humanized mice were generated by replacing the extracellular or full-length coding regions of mouse Tl1a, Il23a, Il12b, Itga4, and Itgb7 genes with their human counterparts while retaining endogenous mouse regulatory elements. Human TL1A, IL23A, IL12B, ITGA4, and ITGB7 proteins were specifically expressed in homozygous TL1A/IL23A/IL12B/α4β7 humanized mice.
• This model provides a translational preclinical platform for evaluating anti-human TL1A, IL23, and α4β7 therapeutics in inflammatory bowel disease, autoimmune disease, and mucosal immunology research.

Key Advantages

• Simultaneous humanization of TL1A, IL23A, IL12B, ITGA4, and ITGB7 enables evaluation of multiple inflammatory pathways in a single model.
• Humanized α4β7 expression supports translational studies of lymphocyte trafficking and intestinal immune cell homing.
• Functional TL1A and IL23 signaling pathways enable mechanistic studies of inflammatory cytokine networks.
• Maintains normal immune cell development and leukocyte distribution across the spleen, blood, and lymph nodes.
• Suitable for the evaluation of anti-human TL1A, IL23, and α4β7 antibodies in vivo.
• Highly relevant for inflammatory bowel disease, Crohn’s disease, ulcerative colitis, and autoimmune disease research.

Validation

• Molecular Validation: Human TL1A, IL23A, IL12B, ITGA4, and ITGB7 genes were successfully humanized while endogenous mouse regulatory regions were retained to preserve physiological relevant expression patterns.
• Protein Expression Validation: Human TL1A protein was detectable in bone marrow-derived dendritic cells from TL1A/IL23A/IL12B/α4β7 humanized mice following LPS stimulation, but not in WT controls. Human IL23 protein was detected in homozygous TL1A/IL23A/IL12B/α4β7 humanized mice, whereas mouse IL23 was detected in wild-type controls.
• Flow Cytometry Validation: Human ITGA4 and ITGB7 proteins were detected in splenocytes from TL1A/IL23A/IL12B/α4β7 humanized mice but not in wild-type C57BL/6JNifdc mice.
• Functional Validation: Combined stimulation with TL1A and IL23 induced downstream cytokine production, including IFN-γ, IL17A, and IL22, in TL1A/IL23A/IL12B/α4β7 humanized mice, confirming preservation of functional inflammatory signaling pathways.
• Immune Homeostasis Validation: Frequencies of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and Tregs in spleen, blood, and lymph nodes remained comparable between TL1A/IL23A/IL12B/α4β7 humanized mice and wild-type controls.

Applications

• Antibody and Biologic Validation Targeting TL1A, IL23, or α4β7
• Inflammatory Bowel Disease and Colitis Research
• Autoimmune and Chronic Inflammatory Disease Studies
• Lymphocyte Homing and Gut Immunology Research
• Preclinical Efficacy and Pharmacodynamic Evaluation of Human-Specific Therapeutics

The exons 1–4 of the mouse Tl1a gene encoding the extracellular domain were replaced with the corresponding human TL1A sequences in TL1A/IL23A/IL12B/α4β7 humanized mice. The genomic regions encoding the transmembrane and cytoplasmic domains were retained. The endogenous mouse Tl1a promoter, 5′ UTR, and 3′ UTR regions were preserved to maintain physiologically relevant expression patterns. Human TL1A expression is driven by the endogenous mouse Tl1a promoter, while endogenous mouse Tl1a transcription and translation are disrupted.

The exons 1–4 of the mouse Il23a gene encoding the full-length protein (ATG to STOP codon) were replaced with the corresponding human IL23A sequences in TL1A/IL23A/IL12B/α4β7 humanized mice. The endogenous mouse Il23a promoter, 5′UTR, and 3′UTR regions were retained. Human IL23A expression is controlled by the endogenous mouse Il23a promoter, while endogenous mouse Il23a expression is disrupted.

The exons 2–8 of the mouse Il12b gene encoding the full-length protein, including the 3′ UTR region, were replaced with the human IL12B counterpart in TL1A/IL23A/IL12B/α4β7 humanized mice. The endogenous mouse Il12b promoter and 5′ UTR regions were retained to preserve physiological relevant regulation. Human IL12B expression is driven by the endogenous mouse Il12b promoter, while endogenous mouse Il12b transcription and translation are disrupted.

The exons 2–27 of the mouse Itga4 gene encoding the extracellular domain were replaced with the corresponding human ITGA4 sequences in TL1A/IL23A/IL12B/α4β7 humanized mice. The endogenous mouse Itga4 promoter, 5′ UTR, and 3′ UTR regions were preserved. Human ITGA4 expression is regulated by the endogenous mouse Itga4 promoter, while endogenous mouse Itga4 expression is disrupted.

The exons 2–14 of the mouse Itgb7 gene encoding the extracellular domain were replaced with the corresponding human ITGB7 sequences in TL1A/IL23A/IL12B/α4β7 humanized mice. The endogenous mouse Itgb7 promoter, 5′ UTR, and 3′ UTR regions were retained to preserve physiological relevant expression. Human ITGB7 expression is driven by the endogenous mouse Itgb7 promoter, while endogenous mouse Itgb7 transcription and translation are disrupted.

Note: TL1A/IL23A/IL12B/α4β7 humanized mice were generated by crossing TL1A/IL23A/IL12B humanized mice (Cat#113038) with TL1A/α4β7 humanized mice (Cat#113037).

Strain-specific human TL1A expression was analyzed in TL1A/IL23A/IL12B/α4β7 humanized mice by ELISA. Bone marrow-derived dendritic cells were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (H/H;H/H;H/H;H/H;H/H) (male, 6-week-old, n=3), stimulated with 1 μg/mL LPS in vitro for 24 h, and supernatants were analyzed using anti-human TL1A ELISA kit (R&D, DY1319-05). Human TL1A was detectable in homozygous TL1A/IL23A/IL12B/α4β7 humanized mice but not in wild-type mice. ND: not detectable.

Mouse IL23 and human IL23 expression were analyzed by ELISA in TL1A/IL23A/IL12B/α4β7 humanized mice. Bone marrow-derived dendritic cells were generated from wild-type C57BL/6JNifdc mice (+/+) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (H/H;H/H;H/H;H/H;H/H) (male, 6-week-old, n=3) and stimulated with 1 μg/mL LPS in vitro for 24 h. Supernatants were collected and analyzed using mouse and human IL23 ELISA kits (R&D, M2300; R&D, D2300B). Mouse IL23 was detectable in wild-type mice, whereas human IL23 was detectable in homozygous TL1A/IL23A/IL12B/α4β7 humanized mice. Values are expressed as mean ± SEM. ND: not detectable.

Strain-specific human ITGA4 and ITGB7 expression analysis was performed in wild-type C57BL/6JNifdc mice and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (H/H;H/H;H/H;H/H;H/H). Protein expression was analyzed using anti-mouse ITGA4 antibody (BioLegend, 103705), anti-mouse LPAM-1/α4β7 antibody (BioLegend, 120607), anti-human ITGA4 antibody (BioLegend, 304307), and anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were detectable in homozygous TL1A/IL23A/IL12B/α4β7 humanized mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse LPAM-1/α4β7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse LPAM-1/α4β7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse LPAM-1/α4β7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse LPAM-1/α4β7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

Ex vivo functional analysis was performed in TL1A/IL23A/IL12B/α4β7 humanized mice. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (H/H;H/H;H/H;H/H;H/H). Production of mouse IFN-γ, IL17A, and IL22 in supernatants was assessed after 72 h stimulation with mIL23 (10 ng/mL), mTL1A (30, 300 ng/mL), hIL23 (10 ng/mL), and hTL1A (30, 300 ng/mL) in vitro.

Combined stimulation of TL1A and IL23 promoted downstream cytokine production in both wild-type and TL1A/IL23A/IL12B/α4β7 humanized mice. These results indicate that both mouse and human TL1A can bind mouse DR3, and both mouse and human IL23 can bind the mouse IL23 receptor. Values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus Control, Student's t-test.

Splenocytes were isolated from wild-type C57BL/6JNifdc mice (female, n=3, 8-week-old) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (female, n=3, 8-week-old).
A. Flow cytometry analysis of splenocytes was performed to assess leukocyte subpopulations.
B. Frequencies of T cell subpopulations were analyzed.

Frequencies of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells, and Tregs in TL1A/IL23A/IL12B/α4β7 humanized mice were comparable to wild-type controls, demonstrating that humanization of TL1A, IL23A, IL12B, ITGA4, and ITGB7 does not alter leukocyte distribution in the spleen. Values are expressed as mean ± SEM.

Blood cells were isolated from wild-type C57BL/6JNifdc mice (female, n=3, 8-week-old) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (female, n=3, 8-week-old).
A. Flow cytometry analysis of blood cells was performed to assess leukocyte subpopulations.
B. Frequencies of T cell subpopulations were analyzed.
Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells, and Tregs in TL1A/IL23A/IL12B/α4β7 humanized mice were comparable to wild-type controls, demonstrating that humanization of TL1A, IL23A, IL12B, ITGA4, and ITGB7 does not alter immune cell distribution in blood. Values are expressed as mean ± SEM.

Lymph node cells were isolated from wild-type C57BL/6JNifdc mice (female, n=3, 8-week-old) and homozygous TL1A/IL23A/IL12B/α4β7 humanized mice (female, n=3, 8-week-old).
A. Flow cytometry analysis of lymph node cells was performed to assess leukocyte subpopulations.
B. Frequencies of T cell subpopulations were analyzed.

Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells, and Tregs in TL1A/IL23A/IL12B/α4β7 humanized mice were comparable to wild-type controls, demonstrating that humanization of TL1A, IL23A, IL12B, ITGA4, and ITGB7 does not alter immune cell distribution in lymph nodes. Values are expressed as mean ± SEM.

Q1: What are TL1A/IL23A/IL12B/α4β7 humanized mice?

A1: TL1A/IL23A/IL12B/α4β7 humanized mice are genetically engineered mice expressing human TL1A, IL23A, IL12B, ITGA4, and ITGB7, enabling translational evaluation of inflammatory and gut-homing pathways in vivo.

Q2: What diseases can be studied using TL1A/IL23A/IL12B/α4β7 humanized mice?

A2: These mice are highly suitable for inflammatory bowel disease, Crohn’s disease, ulcerative colitis, autoimmune disease, and chronic inflammatory disease research.

Q3: Can this model be used for antibody validation?

A3: Yes. TL1A/IL23A/IL12B/α4β7 humanized mice support in vivo evaluation of anti-human TL1A, IL23, and α4β7 antibodies and biologics.

Q4: Does humanization alter immune cell development?

A4: No. Frequencies of major leukocyte and T cell populations in spleen, blood, and lymph nodes remain comparable to wild-type mice.

Q5: What makes this model unique?

A5: This model simultaneously humanizes multiple clinically relevant inflammatory pathways involved in intestinal inflammation and lymphocyte trafficking, enabling comprehensive translational drug evaluation.