• 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

TL1A

• 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.

DR3

• 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 TL1A and DR3 gene expression in wild-type C57BL/6N mice and homozygous TL1A/DR3 humanized mice by RT-PCR. Lung and colon tissues were collected 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 C57BL/6N mice. Human TL1A and DR3 mRNA were exclusively detectable in homozygous B-hTL1A/hDR3 mice, but not in wild-type C57BL/6N mice.

Soluble TL1A expression analysis in TL1A/DR3 humanized mice by ELISA. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow cells isolated from wild-type C57BL/6N mice (+/+) and homozygous TL1A/DR3 humanized mice (H/H;H/H), which were stimulated with LPS in vitro. After stimulation, the supernatants were collected and the levels of soluble TL1A were measured using the species-specific human TL1A ELISA kit. Soluble human TL1A was exclusively detectable in homozygous TL1A/DR3 humanized mice but not wild-type C57BL/6N mice. Values are expressed as mean ± SEM. ND: not detectable.

Strain specific DR3 expression analysis 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 with anti-mouse DR3 antibody (Biolegend, 144407) and anti-human DR3 antibody (Biolegend, 307105) by flow cytometry. Mouse DR3 was detectable in CD4+ T cells of wild-type C57BL/6N mice, human DR3 was detectable in CD4+ T cells of homozygous DR3 humanized mice and TL1A/DR3 humanized mice.

Strain specific DR3 expression analysis 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 with anti-mouse DR3 antibody (Biolegend, 144407) and anti-human DR3 antibody (Biolegend, 307105) by flow cytometry. Mouse DR3 was detectable in Treg cells of wild-type C57BL/6N mice, human DR3 was detectable in Treg cells of homozygous DR3 humanized mice and TL1A/DR3 humanized mice.

Anti-human DR3 antibody binding assessment in homozygous TL1A/DR3 humanized mice by flow cytometry. Splenocytes and blood were collected from wild-type C57BL/6N mice(+/+) and homozygous L1A/DR3 humanized mice (H/H;H/H), and analyzed by flow cytometry with anti-human DR3 antibody (Ab1), which is offered by the client. Ab1 can bind to Treg cells of homozygous TL1A/DR3 humanized mice.

Ex vivo functional analysis in TL1A humanized and TL1A/DR3 humanized mice. Splenocytes were collected from wild-type C57BL/6 mice (+/+), homozygous TL1A humanized mice (H/H), and homozygous TL1A/DR3 humanized mice (H/H; H/H), then the production of mouse IFN-γ and mouse IL-17A in supernatants was assessed by ELISA after 72 h of incubation with mIL23 (10 ng/mL) and hTL1A (300 ng/mL) in vitro.

Activation of DR3 signaling pathway increased the expansion of Treg cells in wild-type C57BL/6N mice and homozygous TL1A/DR3 humanized mice. Wild-type C57BL/6N mice(+/+) and homozygous TL1A/DR3 humanized mice(H/H;H/H) were given a single intraperitoneal injection of DPBS, anti-mDR3 and anti-hDR3 antibodies 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, and the agonistic anti-human DR3 antibody Ab3 increased Treg cells in homozygous TL1A/DR3 humanized mice. These results contribute to a deeper understanding of DR3 activation in Treg cells modulation. 

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

Complete blood count (CBC) of TL1A/DR3 hHumanized mice​​​​​​. Values are expressed as mean ± SD.

Blood biochemical parameters of TL1A/DR3 humanized mice are shown. Values are expressed as mean ± SD.

Organs of female C57BL/6N (8 week old, n=10) and TL1A/DR3 humanized mice (8 week old, n=10).

Organs of male C57BL/6N (8 week old, n=9) and TL1A/DR3 humanized mice (8 week old, n=9).

Average weight of the main organs of female C57BL/6N and TL1A/DR3 humanized mice.

Average weight of the main organs of male C57BL/6N and TL1A/DR3 humanized mice.

Major organs from C57BL/6N and TL1A/DR3 humanized mice were collected at 8 weeks of age and analyzed by H&E staining (female, n = 3).

Major organs from C57BL/6N and TL1A/DR3 humanized mice were collected at 8 weeks of age and analyzed by H&E staining (male, n = 3).

Therapeutic efficacy of anti-human TL1A antibodies and anti-human DR3 antibodies on the TNBS-induced acute colitis model in TL1A/DR3 humanized mice. TNBS solution was instilled into the colon lumen of TL1A/DR3 humanized mice (female, 8-10 weeks-old, n=8). The control group (Sham) received intrarectal injections of 50% ethanol. The treatment groups received anti-human TL1A antibodies RVT-3101 and Tulisokibart (25 mpk, provided by WuXi AppTec), anti-human DR3 antibodies anti-DR3-1 and anti-DR3-2 (25 mpk, provided by the client). (A) Body weight change. (B) DAI score. (C) Colon Index. (D) Representative colon images. A TNBS-induced acute colitis model was established inTL1A/DR3 humanized mice. Administration of anti-TL1A antibodies and anti-human DR3 antibodies effectively improved TNBS-induced acute colitis. The results indicate that TL1A/DR3 humanized mice are a powerful tool for evaluating in vivo efficacy of anti-human TL1A antibodies and anti-human DR3 antibodies in preclinical inflammatory bowel disease (IBD) research. Values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, versus Vehicle, ANOVA.

Note: This experiment was conducted by WuXi AppTec using TL1A/DR3 humanized mice.

Therapeutic efficacy of anti-human TL1A antibody Tulisokibart in a T cells transfer induced colitis model in using TL1A humanized Rag2 knockout (KO) mice (B-hTL1A, Rag2 KO mice). CD4⁺CD45RB^low T and CD4⁺CD45RB^high T cells were isolated from the spleens of TL1A/DR3 humanized mice. TL1A humanized Rag2 KO mice in group G1 were injected with CD4⁺CD45RB^low T cells,  while TL1A humanized Rag2 KO mice in groups G2-G4 were injected with CD4⁺CD45RB^high T cells. Animals in group G3 were given prophylactic administration of 25 mg/kg of anti-human TL1A antibody Tulisokibart every two days, and animals in group G4 were given therapeutic administration of 25 mg/kg of anti-human TL1A antibody Tulisokibart every two days. (A) Body weight changes. (B) Disease activity index (DAI) score. (C) Colon index. (D) Representative colon images. Administration of anti-human TL1A antibody tulisokibart effectively improved T cells transfer induced colitis. Two-way ANOVA or one-way ANOVA was used for multiple comparisons, with each group compared to Vehicle. Values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001..

Note: This experiment was conducted by WuXi AppTec, T cell transfer induced chronic colitis in TL1A humanized Rag2 KO mice (Donor: TL1A/DR3 humanized mice).

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.