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.

RAG2: An essential chromatin-sensing cofactor in V(D)J recombination

• Gene Information: Recombination Activating 2 (RAG2) is a protein-coding gene located on chromosome 11p12, which  is involved in the initiation of V(D)J recombination during B and T cell development.
• Protein Expression: RAG2 exists exclusively in T and B cells at specific early developmental stages within the bone marrow and thymus. It is expressed and survives only during the G0/G1 phase, and the Rag2 protein is rapidly degraded as soon as the cell undergoes DNA replication.
• Signaling Pathway: RAG2 forms a DNA-cleaving complex with RAG1. In this complex, RAG1 provides the catalytic activity, RAG2 acts as a structural scaffold: its N-terminus binds tightly to DNA, and its C-terminal PHD finger anchors the complex to chromatin via trimethylated histone H3 (H3K4me3), which is the core component in the immune system that controls the assembly of diverse immune receptors.
• Therapeutic Inhibition: Complete deficiency of RAG2 leads to severe immunodeficiency characterized by a near-total absence of mature T and B cells, thereby precluding the development of autoimmunity. RAG2 mutations can cause Omenn syndrome, a severe combined immunodeficiency associated with autoimmune-like symptoms.

TL1A

• The exons 1-4 of mouse Tl1a gene that encode extracellular domain were replaced by human counterparts in B-hTL1A, Rag2 KO 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.

Rag2

• The exon 3 and 3’UTR region of mouse Rag2 were knocked out in B-hTL1A, Rag2 KO mice, resulting in a disruption of the Rag2 gene.

• Soluble human TL1A was exclusively detectable in homozygous B-hTL1A, Rag2 KO mice but not wild-type C57BL/6N mice.

Soluble TL1A expression analysis in B-hTL1A, Rag2 KO 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, Rag2 KO mice (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, Rag2 KO mice but not wild-type C57BL/6N mice. Values are expressed as mean ± SEM. ND: not detectable.

• The percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, and neutrophils in homozygous B-hTL1A, Rag2 KO mice were similar to those in B-Rag2 KO mice, and B-hTL1A, Rag2 KO mice presented a near-total absence of T and B cells.
• Humanization of TL1A does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes and peripheral blood were isolated from wild-type C57BL/6N mice, B-hTL1A mice, B-Rag2 KO mice, and B-hTL1A, Rag2 KO mice (female, n=3, 6-week-old). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hTL1A mice were comparable to those in C57BL/6N mice, and B-hTL1A, Rag2 KO mice presented a near-total absence of CD4+ T cells, CD8+ T cells, and Tregs.
• Humanization of TL1A does not affect normal T cell development, differentiation, or splenic distribution

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes and peripheral blood were isolated from wild-type C57BL/6N mice, B-hTL1A mice, B-Rag2 KO mice, and B-hTL1A, Rag2 KO mice (female, n=3, 6-week-old). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• Prophylactic and therapeutic administration of Tulisokibart 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 spleen 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 (provided by WuXi AppTec) every two days, and animals in group G4 were given therapeutic administration of 25 mg/kg of anti-human TL1A antibody Tulisokibart (provided by WuXi AppTec)  every two days. (A) Body weight change. (B) DAI score. (C) Colon index. (D) Colon photo. 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).