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 inflammatory bowel disease (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.

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.

TL1A

• The exons 1-4 of mouse Tl1a gene that encode extracellular domain were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B/hα4β7 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/hα4β7 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/hα4β7 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.

ITGA4

• The exons 2-27 of mouse Itga4 gene that encode the extracellular domain were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B/hα4β7 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/hIL23A/hIL12B/hα4β7 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.

Note: B-hTL1A/hIL23A/hIL12B/hα4β7 mice were obtained by mating B-hTL1A/hIL23A/hIL12B mice (113038) and B-hTL1A/hα4β7 mice (113037).

• Soluble human TL1A was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Soluble TL1A expression analysis in B-hTL1A/hIL23A/hIL12B/hα4β7 mice by ELISA. Bone marrow derived dendritic cells (BMDC) were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H), which were stimulated with LPS in vitro. After stimulation, the supernatants were collected and the level of soluble TL1A was analyzed by ELISA. Soluble human TL1A was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice, but not in 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/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Mouse IL23 and human IL23 expression analysis in B-hTL1A/hIL23A/hIL12B/hα4β7 mice by ELISA. BMDCs were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;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/hα4β7 mice. Values are expressed as mean ± SEM. ND: not detectable. 

• Human ITGA4 and ITGB7 were exclusively detectable in T cells of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in T cells of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in T cells of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in B cells of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in B cells of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in B cells of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in DCs of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in DCs of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in DCs of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in monocytes of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in monocytes of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in monocytes of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice.

• Human ITGA4 and ITGB7 were exclusively detectable in macrophages of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice but not wild-type C57BL/6JNifdc mice.

Strain specific ITGA4 and ITGB7 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTL1A/hIL23A/hIL12B/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in macrophages of wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in macrophages of homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice.

• The synergistic stimulation of TL1A and IL23 could promote the production of downstream cytokines in wild-type C57BL/6 mice and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice.
• Both mTL1A and hTL1A could bind to mouse DR3.

Ex vivo functional analysis in B-hTL1A/hIL23A/hIL12B/hα4β7 mice. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H), then the production of mouse IFN-γ, mouse IL17A, and mouse IL22 in supernatants were assessed after 72 h of incubation with mIL23 (10 ng/mL), mTL1A (30, 300 ng/mL), hIL23 (10 ng/mL) and hTL1A (30, 300 ng/mL) in vitro.

• Both mouse MAdCAM-1 and human MAdCAM-1 could bind to mouse α4β7, and both mouse MAdCAM-1 and human MAdCAM-1 could bind to human α4β7. 

Assessment of MAdCAM-1 binding in C57BL/6 and B-hTL1A/hIL23A/hIL12B/hα4β7 mice. Splenocytes were collected from homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice (H/H;H/H;H/H;H/H;H/H), and were incubated with human MAdCAM-1 protein (3, 10, 30 μg/mL) or mouse MAdCAM-1 protein (3, 10, 30 μg/mL) in vitro for 1 h. The binding of MAdCAM-1 to α4β7 were detected by flow cytometry.

• The percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, and neutrophils in homozygous B-hTL1A/hIL23A/hIL12B/hα4β7 mice were similar to those in C57BL/6JNifdc mice.
• Humanization of TL1A, IL23A, IL12B, ITGA4, and ITGB7 does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6JNifdc and B-hTL1A/hIL23A/hIL12B/hα4β7 mice (female, 8-week-old, n = 3). 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/hα4β7 mice were comparable to those in C57BL/6JNifdc mice.
• Humanization of TL1A, IL23A, IL12B, ITGA4, and ITGB7 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, peripheral blood, and lymph nodes were isolated from female C57BL/6JNifdc and B-hTL1A/hIL23A/hIL12B/hα4β7 mice (female, 8-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.