• TL1A binds to death receptor 3 (DR3) to activate downstream signaling pathways that regulate immune cell proliferation, activation, apoptosis, and cytokine/chemokine production. Soluble decoy receptor 3 (DcR3) can bind TL1A and modulate TL1A–DR3 signaling, and may also regulate apoptosis, inflammation, and tissue damage through interactions with LIGHT and FasL.
• TL1A/DR3 humanized mice were generated by replacing the extracellular domain of mouse Tl1a with its human TL1A counterpart and replacing the full-length mouse Dr3 gene with human DR3. Mouse Tl1a and Dr3 mRNA were detectable in wild-type C57BL/6N mice, whereas human TL1A and DR3 mRNA were detectable in homozygous TL1A/DR3 humanized mice. Human TL1A and DR3 protein expression was confirmed in homozygous TL1A/DR3 humanized mice.
• DR3 pathway activation increased Treg expansion in both wild-type and TL1A/DR3 humanized mice, supporting preserved pathway responsiveness after humanization.
• This model can be applied to pharmacodynamic and safety assessment of therapeutics targeting the human TL1A–DR3 pathway.

Key Advantages

• Dual humanization of TL1A and DR3 enables evaluation of the human TL1A–DR3 signaling pathway in vivo.
• Physiological expression of humanized TL1A is maintained under endogenous regulatory elements.
• Human DR3 is expressed on Treg cells, supporting translational immunology studies.
• Functional DR3 signaling is preserved, enabling Treg expansion assays and mechanism-of-action studies.
• Validated in TNBS-induced acute colitis, supporting inflammatory bowel disease and autoimmune disease research.
• Suitable for pharmacodynamics, efficacy testing, safety evaluation, and antibody validation of anti-human TL1A and DR3 therapeutics.
• Provides a translational platform for evaluating next-generation biologics targeting TL1A/DR3 signaling.

Validation

• Molecular Validation: Mouse Tl1a and Dr3 mRNA were detectable in wild-type C57BL/6N mice, whereas human TL1A and DR3 mRNA were exclusively detectable in homozygous TL1A/DR3 humanized mice. Human TL1A protein expression was confirmed by ELISA in bone marrow-derived dendritic cells stimulated with LPS. Human DR3 protein was specifically detected on Treg cells in spleen and blood by flow cytometry.
• Functional Validation: Activation of the DR3 signaling pathway markedly expanded Treg cell populations in TL1A/DR3 humanized mice following treatment with agonistic anti-human DR3 antibody. These findings confirm that the humanized TL1A–DR3 signaling axis remains biologically functional in vivo.
• Antibody Validation in vivo: Anti-human DR3 antibody specifically bound Treg cells in TL1A/DR3 humanized mice but not in wild-type mice, demonstrating the suitability of this model for in vivo therapeutic antibody evaluation.
• Disease Model Validation: TNBS-induced acute colitis was successfully established in TL1A/DR3 humanized mice. Treatment with anti-human TL1A antibodies (RVT-3101 and Tulisokibart) and anti-human DR3 antibodies significantly improved disease severity, confirming the translational relevance of this model for inflammatory bowel disease research.

Applications

• Antibody and Biologic Validation Targeting TL1A or DR3
• Inflammatory Bowel Disease and Colitis Research
• Autoimmune and Chronic Inflammatory Disease Studies
• Treg Activation and Immune Regulation 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 sequence in TL1A/DR3 humanized mice. The transmembrane domain, cytoplasmic region, promoter, 5' UTR, and 3' UTR of the mouse Tl1a locus were retained. Chimeric TL1A expression was driven by the endogenous mouse Tl1a promoter, while endogenous mouse Tl1a transcription and translation were disrupted.

The exons 1–10 of the mouse Dr3 gene encoding the full-length protein, including promoter, 5' UTR, and 3' UTR regions, were replaced with the corresponding human DR3 genomic sequence in TL1A/DR3 humanized mice. Humanized DR3 expression was driven by the human DR3 promoter, while endogenous mouse Dr3 transcription and translation were disrupted.

Strain-specific analysis of human TL1A and DR3 mRNA expression was performed by RT-PCR in wild-type C57BL/6N mice and homozygous TL1A/DR3 humanized mice. RNA from spleen, lung, and colon tissues was isolated from wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H, H/H). Mouse Tl1a and Dr3 mRNA were detectable in wild-type mice, whereas human TL1A and DR3 mRNA were exclusively detectable in homozygous TL1A/DR3 humanized mice, but not in wild-type controls.

Strain-specific human TNFRSF25 expression was analyzed by flow cytometry in wild-type C57BL/6N mice, homozygous DR3 humanized mice, and TL1A/DR3 humanized mice. Splenocytes were collected from wild-type C57BL/6N mice (+/+), homozygous DR3 humanized mice (H/H), and TL1A/DR3 humanized mice (H/H;H/H), followed by in vivo stimulation with anti-mouse CD3ε antibody and anti-mouse CD28 antibody for 24 h. Protein expression was analyzed using anti-mouse TNFRSF25 antibody (BioLegend, 144407) and anti-human TNFRSF25 antibody (BioLegend, 307105). Mouse TNFRSF25 was detectable in Treg cells of wild-type mice, whereas human TNFRSF25 was detectable in Treg cells of homozygous DR3 humanized mice and TL1A/DR3 humanized mice.

Strain-specific TL1A expression was analyzed in homozygous TL1A/DR3 humanized mice by ELISA. Bone marrow-derived dendritic cells were isolated from wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H, H/H), followed by stimulation with 1 μg/mL LPS in vitro for 24 h. Cell supernatants were collected and analyzed using anti-human TL1A ELISA kit (R&D, DY1319-05). Human TL1A protein was exclusively detectable in homozygous TL1A/DR3 humanized mice, but not in wild-type mice.

Strain-specific DR3 expression was analyzed by flow cytometry in spleens from wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H, H/H). Splenocytes were stained using anti-mouse DR3 antibody (BioLegend, 144407) and anti-human DR3 antibody (BioLegend, 307105). Mouse DR3 was detectable on Treg cells of wild-type mice, whereas human DR3 was exclusively detectable on Treg cells of homozygous TL1A/DR3 humanized mice.

Blood samples were collected from wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H, H/H). Flow cytometry was performed using anti-mouse DR3 antibody (BioLegend, 144407) and anti-human DR3 antibody (BioLegend, 307105). Mouse DR3 was detectable on Treg cells of wild-type mice, whereas human DR3 was exclusively detectable on Treg cells of homozygous TL1A/DR3 humanized mice.

Binding activity of anti-humanized DR3 antibody was evaluated in homozygous TL1A/DR3 humanized mice by flow cytometry. Splenocytes and blood samples were collected from wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H, H/H). Cells were analyzed using anti-human DR3 antibody Ab1 provided by the client. Ab1 specifically bound Treg cells from homozygous TL1A/DR3 humanized mice.

Strain-specific TNFRSF25 expression was analyzed in wild-type C57BL/6N mice, homozygous DR3 humanized mice, and TL1A/DR3 humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice (+/+), homozygous DR3 humanized mice (H/H), and TL1A/DR3 humanized mice (H/H;H/H) stimulated with anti-mouse CD3ε antibody and anti-mouse CD28 antibody in vivo for 24 h. Protein expression was analyzed using anti-mouse TNFRSF25 antibody (BioLegend, 144407) and anti-human TNFRSF25 antibody (BioLegend, 307105) by flow cytometry. Mouse TNFRSF25 was detectable in CD4+ T cells of wild-type C57BL/6N mice, whereas human TNFRSF25 was detectable in CD4+ T cells of homozygous DR3 humanized mice and TL1A/DR3 humanized mice.

Activation of the DR3 signaling pathway increased the expansion of Treg cells in wild-type C57BL/6N mice and TL1A/DR3 humanized mice. Wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H,H/H) received a single intraperitoneal injection of DPBS, anti-mDR3 antibody, or anti-hDR3 antibody on day 0. Splenocytes and blood cells were collected on day 4 for flow cytometry analysis. The agonistic anti-mouse DR3 antibody Ab2 expanded Treg cell populations in wild-type C57BL/6N mice, whereas the agonistic anti-human DR3 antibody Ab3 increased Treg cells in TL1A/DR3 humanized mice. These findings demonstrate functional activation of the humanized DR3 signaling pathway and contribute to a deeper understanding of DR3-mediated Treg cell modulation in TL1A/DR3 humanized mice.

Note: This experiment was conducted by the client using TL1A/DR3 humanized mice.

TNBS solution was instilled into the colon lumen of TL1A/DR3 humanized mice (female, 8–10 weeks old, n=10). The control group (Sham) received intrarectal injections of 50% ethanol. Treatment groups received anti-human TL1A antibodies RVT-3101 and Tulisokibart (25 mpk, provided by WuXi AppTec), as well as anti-human DR3 antibodies anti-DR3-1 and anti-DR3-2 (25 mpk, provided by the client). Body weight and disease activity index (DAI) scores were recorded daily. On day 5, mice were sacrificed and colon length and colon weight were measured.

(A) Body weight change
(B) DAI score
(C) Colon index
(D) Colon morphology

A TNBS-induced acute colitis model was successfully established in TL1A/DR3 humanized mice. Treatment with anti-human TL1A antibodies and anti-human DR3 antibodies significantly improved TNBS-induced colitis severity. These results demonstrate that TL1A/DR3 humanized mice provide a robust preclinical platform for evaluating the in vivo efficacy of anti-human TL1A and anti-human DR3 therapeutics in inflammatory bowel disease research. Values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus Vehicle, ANOVA.

Q1: What are TL1A/DR3 humanized mice?

A1 TL1A/DR3 humanized mice express human TL1A and DR3 in place of the corresponding murine genes, enabling translational evaluation of the human TL1A–DR3 pathway.

Q2: Why is the TL1A–DR3 pathway important?

A2 The TL1A–DR3 signaling axis plays a critical role in intestinal inflammation, fibrosis, T cell activation, and autoimmune disease progression.

Q3: Can TL1A/DR3 humanized mice be used for antibody validation?

A3 Yes. These mice are highly suitable for in vivo validation of anti-human TL1A therapeutic antibodies.

Q4: Do TL1A/DR3 humanized mice maintain normal immune homeostasis?

A4 Yes. Immune cell distributions in spleen, blood, and lymphoid tissues remain comparable to wild-type controls.

Q5: What disease areas can be studied using this model?

A5 This model supports research in IBD, Crohn’s disease, ulcerative colitis, fibrosis, autoimmune disease, and inflammatory signaling.