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.

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/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.

ITGA4

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

• Human TL1A, ITGA4, and ITGB7 mRNA were specifically and correctly expressed in B-hTL1A/hα4β7 mice.

Species specific analysis of TL1A, ITGA4 and ITGB7 gene expression in wild-type C57BL/6JNifdc mice and homozygous B-hTL1A/hα4β7 mice by RT-PCR. Spleen, Colon and lung were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hα4β7 mice (H/H;H/H;H/H). Mouse Tl1a, Itga4 and Itgb7 mRNA were detectable only in wild-type C57BL/6JNifdc mice. Human TL1A, ITGA4, and ITGB7 mRNA were detectable only in homozygous B-hTL1A/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

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

Soluble TL1A expression analysis in B-hTL1A/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/hα4β7 mice (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/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM. ND: not detectable.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/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/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hα4β7 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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/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/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hα4β7 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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/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/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hα4β7 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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

• Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/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/hα4β7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hα4β7 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) anti-human ITGB7 antibody (Invitrogen, MA5-23541) by flow cytometry. Mouse ITGA4 and ITGB7 were only detectable in wild-type C57BL/6JNifdc mice. Human ITGA4 and ITGB7 were exclusively detectable in homozygous B-hTL1A/hα4β7 mice, but not in wild-type C57BL/6JNifdc mice.

• The percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, and neutrophils in homozygous B-hTL1A/hα4β7 mice were similar to those in C57BL/6JNifdc mice.
• Humanization of TL1A, 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/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/hα4β7 mice were comparable to those in C57BL/6JNifdc mice.
• Humanization of TL1A, 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/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.

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

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

Growth curve of wild-type C57BL/6JNifdc and B-hTL1A/hα4β7 mice. Eight-week-old mice were grouped by sex (10 males and 10 females). Body weight was measured on the same day of every week, until 13 weeks. The minimum and maximum body weights shown in the table were calculated from the mean ± SD. The growth curve of the B-hTL1A/hα4β7 mice was similar to the growth curve of C57BL/6JNifdc mice.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of B-hTL1A/hα4β7 mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of B-hTL1A/hα4β7 mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

Organs of female C57BL/6JNifdc (13-week-old, n=10) and B-hTL1A/hα4β7 mice (13-week-old, n=10).

• No abnormalities were observed.

Organs of male C57BL/6JNifdc (13-week-old, n=10) and B-hTL1A/hα4β7 mice (13-week-old, n=9).

• No abnormalities were observed.

Average weight of the main organs of female C57BL/6 and B-hTL1A/hα4β7 mice.

• No abnormalities were observed.

Average weight of the main organs of male C57BL/6 and B-hTL1A/hα4β7 mice.

• No obvious abnormalities were observed in all of the organs (brain, heart, lung, liver, spleen, kidney, thymus, stomach, small intestine, large intestine, bone marrow, lymph node, uterus and ovary).

Histopathological analysis of organs in female C57BL/6JNifdc and B-hTL1A/hα4β7 mice. Major organs from C57BL/6JNifdc and B-hTL1A/hα4β7 mice were collected at 13 weeks of age and analyzed by H&E staining (female, n = 3).

• No obvious abnormalities were found in all of the organs (brain, heart, lung, liver, spleen, kidney, thymus, stomach, small intestine, large intestine, bone marrow, lymph node, and testis).

Histopathological analysis of organs in male C57BL/6JNifdc and B-hTL1A/hα4β7 mice. Major organs from C57BL/6JNifdc and B-hTL1A/hα4β7 mice were collected at 13 weeks of age and analyzed by H&E staining (female, n = 3).

• RVT-3101 and Vedolizumab treatment efficiently improved TNBS-induced acute colitis

The therapeutic efficacy of anti-TL1A and anti-human α4β7 antibodies on the TNBS-induced acute colitis model in B-hTL1A/hα4β7 mice. TNBS solution was instilled into the colon lumen of B-hTL1A/hα4β7 mice (female, 8-10 weeks-old, n=8). The control group (Sham) received intrarectal injections of 50% ethanol. The treatment groups received anti-TL1A antibody RVT-3101 (10 mpk, provided by WuXi AppTec), anti-human α4β7 antibody Vedolizumab (10 mpk, provided by WuXi AppTec) alone or in combination. (A) Body weight change. (B) DAI score. (C) Colon Index. (D) Colon photo. An acute colitis disease model induced by TNBS was established in B-hTL1A/hα4β7 mice, and administration of anti-TL1A antibody RVT-3101 and anti-human α4β7 antibody Vedolizumab effectively improved TNBS-induced acute colitis, and their combination provided better efficacy. 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/hα4β7 mice.

• RVT-3101 and Vedolizumab treatment reduced the production of inflammatory cytokines.

Analysis of inflammatory cytokines on the TNBS-induced acute colitis model in B-hTL1A/hα4β7 mice. TNBS solution was instilled into the colon lumen of B-hTL1A/hα4β7 mice (female, 8-10 weeks-old, n=8). The control group (Sham) received intrarectal injections of 50% ethanol. The treatment groups received anti-TL1A antibody RVT-3101 (10 mpk, provided by WuXi AppTec), anti-human α4β7 antibody Vedolizumab (10 mpk, provided by WuXi AppTec) alone or in combination. (A) The concentrations of IL-1β, IL-6, IL-17A, IFN-γ, TNF-α in intestinal mucosa. (B) The concentrations of IL-1β, IL-6, IL-17A, IFN-γ, TNF-α in serum. Administration of anti-TL1A antibody RVT-3101 and anti-human α4β7 antibody Vedolizumab markedly reduced the production of inflammatory cytokines in TNBS-induced acute colitis mice, and their combination provided better efficacy. The results indicate that B-hTL1A/hα4β7 mice are a powerful tool for evaluating in vivo efficacy of the combination of anti-TL1A antibody and anti-human α4β7 antibody. 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/hα4β7 mice.