TL1A: A key inflammation cytokine in chronic intestinal inflammation and fibrosis-related diseases

• Gene Information: TNF Superfamily Member 15 (TNFSF15, also known as TL1A) is a protein-coding gene located on chromosome 9q32. This cytokine is a ligand for receptor TNFRSF25 (also known as DR3) and  TNFRSF6B (also known as DcR3).
• Protein Expression: TL1A is expressed in various immune cells (such as monocytes, macrophages, dendritic cells, and T cells) as well as in non-immune cells (such as synovial fibroblasts and endothelial cells). TL1A is a type II transmembrane protein that exists in either membrane-bound (mTL1A) or soluble (sTL1A) forms.
• Signaling Pathway: TL1A competes with Death Receptor 3 (DR3) for binding, providing stimulus signals for downstream signaling pathways, thereby regulating the proliferation, activation, apoptosis of effector cells, and the production of cytokines and chemokines.
• Therapeutic Inhibition: Blocking the interaction between TL1A and DR3 can reduce the severity of autoimmune diseases, such as the IBD model.

TL1A

• The exons 1-4 of mouse Tl1a gene that encode extracellular domain were replaced by human counterparts in B-hTL1A/hDR3 mice..
• The genomic region of mouse Tl1a 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 TL1A expression was driven by endogenous mouse Tl1a promoter, while mouse Tl1a gene transcription and translation will be disrupted.

DR3

• The exons 1-10 of mouse DR3 gene that encode the whole molecule (ATG to STOP codon), including promoter, 5’UTR and 3’UTR were replaced by human counterparts in B-hTL1A/hDR3 mice.
• The human DR3 expression was driven by human DR3 promoter, while mouse DR3 gene transcription and translation will be disrupted.

• Human TL1A and DR3 mRNA were specifically and correctly expressed in B-hTL1A/hDR3 mice.

Strain specific analysis of TL1A and DR3 gene expression in in wild-type C57BL/6N mice and homozygous B-hTL1A/hDR3 mice by RT-PCR. Lung and colon tissues were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hTL1A/hDR3 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 human TL1A was exclusively detectable in homozygous B-hTL1A/hDR3 mice but not wild-type C57BL/6N mice.

Soluble TL1A expression analysis in B-hTL1A/hDR3 mice by ELISA. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow from wild-type C57BL/6N mice (+/+) and homozygous B-hTL1A/hDR3 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 B-hTL1A/hDR3 mice but not wild-type C57BL/6N mice. Values are expressed as mean ± SEM. ND: not detectable.

• Human DR3 was detectable in CD4+ T cells of homozygous B-hDR3 mice and B-hTL1A/hDR3 mice.

Strain specific DR3 expression analysis in wild-type C57BL/6N mice, homozygous B-hDR3 mice and B-hTL1A/hDR3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice (+/+), homozygous B-hDR3 mice (H/H) and B-hTL1A/hDR3 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 B-hDR3 mice and B-hTL1A/hDR3 mice.

• Human DR3 was detectable in Treg cells of homozygous B-hDR3 mice and B-hTL1A/hDR3 mice.

Strain specific DR3 expression analysis in wild-type C57BL/6N mice, homozygous B-hDR3 mice and B-hTL1A/hDR3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice (+/+), homozygous B-hDR3 mice (H/H) and B-hTL1A/hDR3 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 B-hDR3 mice and B-hTL1A/hDR3 mice.

• Anti-human DR3 antibody can bind to Treg cells of homozygous B-hTL1A/hDR3 mice.

Anti-human DR3 antibody binding assessment in homozygous B-hTL1A/hDR3 mice by flow cytometry. Splenocytes and blood were collected from wild-type C57BL/6N mice(+/+) and homozygous B-hTL1A/hDR3 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 B-hTL1A/hDR3 mice.

• The synergistic stimulation of human TL1A and mouse IL23 could promote the production of downstream cytokines in wild-type C57BL/6 mice, homozygous B-hTL1A mice and homozygous B-hTL1A/hDR3 mice.

Ex vivo functional analysis in B-hTL1A mice and B-hTL1A/hDR3 mice. Splenocytes were collected from wild-type C57BL/6 mice (+/+), homozygous B-hTL1A mice (H/H) and homozygous B-hTL1A/hDR3 mice (H/H;H/H), then the production of mouse IFN-γ and mouse IL-17A in supernatants were assessed by ELISA after 72 h of incubation with mIL23 (10 ng/mL) and hTL1A (300 ng/mL) in vitro.

• The agonistic anti-human DR3 antibody could increase Treg cells in homozygous B-hTL1A/hDR3 mice.

Activation of DR3 signaling pathway increased the expansion of Treg cells in wild-type C57BL/6N mice and homozygous B-hTL1A/hDR3 mice. Wild-type C57BL/6N mice(+/+) and homozygous B-hTL1A/hDR3 mice(H/H;H/H) were given 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 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 B-hTL1A/hDR3 mice. These results contribute to a deeper understanding of DR3 activation in Treg cells modulation. Note: This experiment was conducted by the client using B-hTL1A/hDR3 mice.

• Anti-TL1A antibodies and anti-human DR3 antibodies treatment efficiently improved TNBS-induced acute colitis.

The therapeutic efficacy of anti-TL1A antibodies and anti-human DR3 antibodies on the TNBS-induced acute colitis model in B-hTL1A/hDR3 mice. TNBS solution was instilled into the colon lumen of B-hTL1A/hDR3 mice (female, 8-10 weeks-old, n=8). The control group (Sham) received intrarectal injections of 50% ethanol. The treatment groups received anti-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) Colon photo. A TNBS-induced acute colitis model was established in B-hTL1A/hDR3 mice. Administration of anti-TL1A antibodies and anti-human DR3 antibodies effectively improved TNBS-induced acute colitis. The results indicate that B-hTL1A/hDR3 mice are a powerful tool for evaluating in vivo efficacy of anti-TL1A antibodies and anti-human DR3 antibodies. 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 B-hTL1A/hDR3 mice.

• Prophylactic and  therapeutic administration of anti-human TL1A antibody efficiently improved T cells transfer induced colitis.

The therapeutic efficacy of Tulisokibart on T cells transfer induced colitis model in B-hTL1A, Rag2 KO mice. CD4+CD45RBlow T and CD4+CD45RBhigh T cells were isolated from the spleens of B-hTL1A/hDR3 mice. B-hTL1A, Rag2 KO mice in group G1 were injected with CD4+CD45RBlow T cells,  while B-hTL1A, Rag2 KO mice in groups G2-G4 were injected with CD4+CD45RBhigh 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 change. (B) DAI score. (C) Colon index. (D) Colon photo. Administration of anti-human TL1A antibody 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 B-hTL1A, Rag2 KO mice (Donor: B-hTL1A/hDR3 mice).