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.

α4β7: A central pathway for directing lymphocyte migration to the gut and other mucosal sites during inflammation 

• Gene Information:  Integrin α4β7 is composed of a 150 kD (α4 or CD49d) and a 130 kD (β7) heterodimer, also known as CD49d/β7 or LPAM-1. ITGA4 is located on chromosome 2q31.3, ITGB7 is located on chromosome 12q13.13. Integrin α4β7 binds its ligand MAdCAM-1, and plays an important role in lymphocytes adhesion. 
• Protein Expression: The α4β7 integrin is a heterodimer, composed of an α4 subunit and a β7 subunit. α4β7 is mainly expressed in leukocytes, such as T cells, B cells, NK cells, macrophages, dendritic cells, monocytes, and others. 
• Signaling Pathway: Integrin α4β7 interacts with the cell surface adhesion molecules MAdCAM-1 which is normally expressed by the vascular endothelium of the gastrointestinal tract, which is a central pathway for directing lymphocyte migration to the gut and other mucosal sites during inflammation. 
• Therapeutic Inhibition: Blocking the binding of α4β7 to MAdCAM-1, such as with Vedolizumab, inhibits lymphocyte homing and infiltration into inflammatory tissues such as the intestine.

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/hα4β7, 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. 

ITGA4 

• The exons 2-27 of mouse Itga4 gene that encode the extracellular domain were replaced by human counterparts in B-hTL1A/hα4β7, Rag2 KO mice. The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. 
• The human ITGA4 expression was driven by endogenous mouse Itga4 promoter, while mouse Itga4 gene transcription and translation will be disrupted. 

ITGB7 

• The exons 2-14 of mouse Itgb7 gene that encode the extracellular domain were replaced by human counterparts in B-hTL1A/hα4β7, Rag2 KO mice. 
• The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human ITGB7 expression was driven by endogenous mouse Itgb7 promoter, while mouse Itgb7 gene transcription and translation will be disrupted. 

Rag2 

• The exon 3 and 3’UTR region of mouse Rag2 were knocked out in B-hTL1A/hα4β7, Rag2 KO mice, resulting in a disruption of the Rag2 gene.

• Soluble human TL1A was exclusively detectable in B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice, but not wild-type B-Rag2 KO mice.

Soluble TL1A expression analysis in B-hTL1A/hα4β7, Rag2 KO mice by ELISA. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow from wild-type B-Rag2 KO mice (-/-), homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), and homozygous B-hTL1A/hα4β7, 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/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice. Values are expressed as mean ± SEM. ND: not detectable.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice.

Strain specific ITGA4 and ITGB7 expression analysis in homozygous B-hTL1A/hα4β7, Rag2 KO mice by flow cytometry. Splenocytes were collected from wild-type B-Rag2 KO mice (-/-), homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), and homozygous B-hTL1A/hα4β7, Rag2 KO mice (H/H;H/H;H/H;-/-). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307), and anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type B-Rag2 KO mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice, but not in wild-type B-Rag2 KO mice.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice.

Strain specific ITGA4 and ITGB7 expression analysis in homozygous B-hTL1A/hα4β7, Rag2 KO mice by flow cytometry. Splenocytes were collected from wild-type B-Rag2 KO mice (-/-), homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), and homozygous B-hTL1A/hα4β7, Rag2 KO mice (H/H;H/H;H/H;-/-). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307), and anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type B-Rag2 KO mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice, but not in wild-type B-Rag2 KO mice.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice.

Strain specific ITGA4 and ITGB7 expression analysis in homozygous B-hTL1A/hα4β7, Rag2 KO mice by flow cytometry. Splenocytes were collected from wild-type B-Rag2 KO mice (-/-), homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), and homozygous B-hTL1A/hα4β7, Rag2 KO mice (H/H;H/H;H/H;-/-). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307), and anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type B-Rag2 KO mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice, but not in wild-type B-Rag2 KO mice.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice.

Strain specific ITGA4 and ITGB7 expression analysis in homozygous B-hTL1A/hα4β7, Rag2 KO mice by flow cytometry. Splenocytes were collected from wild-type B-Rag2 KO mice (-/-), homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), and homozygous B-hTL1A/hα4β7, Rag2 KO mice (H/H;H/H;H/H;-/-). Protein expression was analyzed with anti-mouse ITGA4 antibody (Biolegend, 103705), anti-mouse ITGB7 antibody (Biolegend, 120607), anti-human ITGA4 antibody (Biolegend, 304307), and anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type B-Rag2 KO mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice, but not in wild-type B-Rag2 KO mice.

• Human α4β7 were exclusively detectable in homozygous B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice.

Strain specific α4β7 expression in homozygous B-hTL1A/hα4β7, Rag2 KO mice by flow cytometry. Splenocytes were collected from homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), wild-type B-Rag2 KO mice (-/-), and homozygous B-hTL1A/hα4β7, Rag2 KO mice (H/H;H/H;H/H;-/-), and protein expression was analyzed by flow cytometry with human α4β7-vedolizumab-hIgG1 (commercially purchased). Human α4β7 was only detectable in DCs and macrophages of homozygous B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice.

• Human α4β7 were exclusively detectable in homozygous B-hTL1A/hα4β7, Rag2 KO mice but not wild-type B-Rag2 KO mice.

Strain specific α4β7 expression in homozygous B-hTL1A/hα4β7, Rag2 KO mice by flow cytometry. Splenocytes were collected from homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H), wild-type B-Rag2 KO mice (-/-), and homozygous B-hTL1A/hα4β7, Rag2 KO mice (H/H;H/H;H/H;-/-), and protein expression was analyzed by flow cytometry with human α4β7-vedolizumab-hIgG1 (commercially purchased). Human α4β7 was only detectable in monocytes and NK cells of homozygous B-hTL1A/hα4β7 mice and B-hTL1A/hα4β7, Rag2 KO mice.

• The percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, and neutrophils in homozygous B-hTL1A/hα4β7, Rag2 KO mice were similar to those in B-Rag2 KO mice, and B-hTL1A/hα4β7, Rag2 KO mice presented a near-total absence of T and B cells. 
• Humanization of TL1A and α4β7 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 B-Rag2 KO mice, homozygous B-hTL1A/hα4β7 mice, and homozygous B-hTL1A/hα4β7, 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/hα4β7, Rag2 KO mice were comparable to those in B-Rag2 KO mice, and B-hTL1A/hα4β7, Rag2 KO mice presented a near-total absence of CD4+ T cells, CD8+ T cells, and Tregs. 
• Humanization of TL1A and α4β7 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 B-Rag2 KO mice, homozygous B-hTL1A/hα4β7 mice, and homozygous B-hTL1A/hα4β7, Rag2 KO mice (female, n=3, 8-week-old). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.