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.

IL23: A key inflammation cytokine in the inflammatory response driven by Th17 cells

• Gene Information:  IL-23 is a heterodimeric cytokine composed of p19 and p40 subunits, and is encoded by interleukin-23 subunit alpha (IL23A) and  interleukin-12 subunit beta (IL12B) respectively. IL23A is located on chromosome 12q13.3, IL12B is located on chromosome 5q33.3.
• Protein Expression: Mature, biologically active IL-23 protein is secreted only when a cell expresses both the p19 and p40 subunits and assembles them correctly. IL-23 is mainly produced by activated antigen-presenting cells, such as macrophages and dendritic cells, as well as damaged keratinocytes or intestinal epithelial cells.
• Signaling Pathway: IL23 binds to the IL23R/IL12RB1 receptor complex to trigger JAK-STAT, p38 MAPK, and NF-κB signaling. This initiates the transcription of downstream pro-inflammatory factors, such as IL-17A, IL-17F, IL-22 and RORγt, thereby promoting the differentiation and maintenance of pathogenic Th17 cells and exacerbating chronic tissue inflammation.
• Therapeutic Inhibition: Blocking the interaction between IL23 and IL23 receptor can reduce the severity of autoimmune diseases, such as the IBD model and psoriasis.

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/hIL23A/hIL12B, 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.

IL23A

• The exons 1-4 of mouse Il23a gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice.
• The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human IL23A expression was driven by endogenous mouse Il23a promoter, while mouse Il23a gene transcription and translation will be disrupted.

IL12B

• The exons 2-8 of mouse Il12b gene that encode the whole molecule (ATG to STOP codon), including 3’UTR were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice.
• The promoter and 5’UTR region of the mouse gene were retained. The human IL12B expression was driven by endogenous mouse Il12b promoter, while mouse Il12b gene transcription and translation will be disrupted.

Rag2

• The exon 3 and 3’UTR region of mouse Rag2 were knocked out in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice, resulting in a disruption of the Rag2 gene

• Soluble human TL1A was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice but not wild-type C57BL/6JNifdc mice

Soluble TL1A expression analysis in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice by ELISA. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (H/H;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/hIL23A/hIL12B, Rag2 KO mice but not wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM. ND: not detectable.

• Human IL23 was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice but not wild-type C57BL/6JNifdc mice.

Mouse IL-23 and human IL-23 expression analysis in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice by ELISA. Bone marrow derived dendritic cells were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+), and homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (H/H;H/H;H/H;-/-), which were stimulated with LPS in vitro. After stimulation, the supernatants were collected and the levels of mouse and human IL23 were analyzed by ELISA(R&D, M2300; R&D, D2300B). Mouse IL23 was only detectable in wild-type C57BL/6JNifdc mice. Human IL23 was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO 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/hIL23A/hIL12B, Rag2 KO mice were similar to those in B-Rag2 KO mice, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice presented a near-total absence of T and B cells.
• Humanization of TL1A, IL23A, and IL12B 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/6JNifdc mice, B-Rag2 KO mice, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (female, n=3, 8-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/hIL23A/hIL12B, Rag2 KO mice were comparable to those in B-Rag2 KO mice, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice presented a near-total absence of CD4+ T cells, CD8+ T cells, and Tregs.
• Humanization of TL1A, IL23A, and IL12B 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/6JNifdc mice, B-Rag2 KO mice, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (female, n=3, 8-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.

• Administration of RVT3101 and Risankizumab efficiently improved T cells transfer induced colitis, and their combination provided better efficacy.

The therapeutic efficacy of RVT3101 and Risankizumab on T cells transfer induced colitis model in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. CD4+CD45RB^low T and CD4+CD45RB^high T cells were isolated from the spleen of B-hIL23A/hIL12B/hIL23R/hIL12RB1 plus mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 were injected with CD4+CD45RB^low T cells, while B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in groups G2-G5 were injected with CD4+CD45RB^high T cells. The treatment groups received anti-TL1A antibody RVT3101 (8 mpk, provided by WuXi AppTec), anti-human IL23p19 antibody Risankizumab (8 mpk, providedby WuXi AppTec) alone or in combination every three 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/hIL23A/hIL12B, Rag2 KO mice (Donor: B-hIL23A/hIL12B/hIL23R/hIL12RB1 plus mice).

• Administration of RVT3101 and Risankizumab efficiently reduced inflammatory infiltration and epithelial damage in colon tissuecompared with untreated controls, and their combination provided better efficacy.

The therapeutic efficacy of RVT3101 and Risankizumab on T cells transfer induced colitis model in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. CD4+CD45RB^low T and CD4+CD45RB^high T cells were isolated from the spleen of B-hIL23A/hIL12B/hIL23R/hIL12RB1 plus mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 were injected with CD4+CD45RB^low T cells, while B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in groups G2-G5 were injected with CD4+CD45RB^high T cells. The treatment groups received anti-TL1A antibody RVT3101 (8 mpk, provided by WuXi AppTec), anti-human IL23p19 antibody Risankizumab (8 mpk, providedby WuXi AppTec) alone or in combination every three days. (A) H&E staining of colon tissue. (B) Pathological score. 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/hIL23A/hIL12B, Rag2 KO mice (Donor: B-hIL23A/hIL12B/hIL23R/hIL12RB1 plus mice).

Experimental schedule for anti-mouse CD40 antibody induced colitis and in vivo efficacy of anti-human IL23p19 antibody in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 received PBS, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G2-G3 received anti-mouse CD40 antibody. Animals in group G3 were given 25 mg/kg of anti-human IL23p19 antibody Risankizumab analog (in house) every two days.

• Administration of anti-human IL23p19 antibody Risankizumab analog (in house) effectively improved anti-CD40 antibody induced colitis.

The therapeutic efficacy of anti-human IL23p19 antibody on anti-mouse CD40 antibody induced colitis model in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 received PBS, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G2-G3 received anti-CD40 antibody. Body weight and DAI score were recorded daily. On day 5, the mice were sacrificed, and colon length and weight were recorded. (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 G2. Values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

• Administration of anti-human IL23p19 antibody Risankizumab analog (in house) effectively reduced inflammatory infiltration in colon tissue.

The therapeutic efficacy of anti-human IL23p19 antibody on anti-mouse CD40 antibody induced colitis model in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 received PBS, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G2-G3 received anti-CD40 antibody. On day 5, the mice were sacrificed, colon tissues were collected at the study endpoint and analyzed by H&E staining. The red arrow represented the infiltration of inflammatory cells.

Experimental schedule for anti-mouse CD40 antibody induced colitis and in vivo efficacy of anti-human IL23p40 antibody in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 received PBS, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G2-G3 received anti-mouse CD40 antibody. Animals in group G3 were given 25 mg/kg of anti-human IL23p40 antibody Ustekinumab analog (in house) every two days.

• Administration of anti-human IL23p40 antibody Ustekinumab analog (in house) effectively improved anti-CD40 antibody induced colitis.

The therapeutic efficacy of anti-human IL23p40 antibody on anti-mouse CD40 antibody induced colitis model in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 received PBS, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G2-G3 received anti-CD40 antibody. Body weight and DAI score were recorded daily. On day 5, the mice were sacrificed, and colon length and weight were recorded. (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 G2. Values are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

• Administration of anti-human IL23p40 antibody Ustekinumab analog (in house) effectively reduced inflammatory infiltration in colon tissue.

The therapeutic efficacy of anti-human IL23p40 antibody on anti-mouse CD40 antibody induced colitis model in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G1 received PBS, and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice in group G2-G3 received anti-CD40 antibody. On day 5, the mice were sacrificed, colon tissues were collected at the study endpoint and analyzed by H&E staining. The red arrow represented the infiltration of inflammatory cells.